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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-241616806010.1186/1472-6750-5-24Methodology ArticleEffects of DNA mass on multiple displacement whole genome amplification and genotyping performance Bergen Andrew W [email protected] Ying [email protected] Kashif A [email protected] Robert A [email protected] Stephen J [email protected] Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA2 Core Genotyping Facility, National Cancer Institute, National Institutes of Health, Gaithersburg, MD, USA3 Intramural Research Support Program, SAIC-Frederick, NCI-FCRDC, Frederick, MD, USA4 Section on Genomic Variation, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA2005 16 9 2005 5 24 24 28 3 2005 16 9 2005 Copyright © 2005 Bergen 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
Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel) and N = 49 SNP (TaqMan®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.
Results
The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.
Conclusion
The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR® Identifiler® panel from wgaDNA equivalent to that of gDNA.
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Background
The potential for the molecular analysis of human genetic material has increased enormously with the availability of the human genome sequence, SNP identification efforts and the development of high-throughput genotyping platforms [1]. The expanding demand for single nucleotide polymorphism (SNP) genotyping is a consequence of the recognition that many SNPs will need to be analyzed to characterize the effects of genes on complex disorders [2], especially when performing whole genome association studies [3]. With notable exceptions [4], total DNA requirements for genotyping will increase as the number of loci investigated expands, despite increased efficiency of individual genotyping assays. Whole genome amplification (WGA) is an in vitro procedure to amplify a genomic DNA (gDNA) sample to generate amplified DNA (wgaDNA) for further molecular genetic analyses, and has been considered by some as a potential solution to the problem of limiting gDNA availability. While PCR-based methods of WGA have been under continuous development for over a decade [5,6], recent application of a highly processive φ29 DNA polymerase [7], has enabled multiple displacement amplification (MDA) WGA, an isothermal, hyperbranching amplification method, with a low level of locus or allelic bias [8]. Dean [8] and Lovmar [9] have evaluated the genotyping performance of MDA WGA using a range of genomic DNA inputs (0.3, 3, 30 and 300 ng, and 0.003, 0.03, 0.3 and 3 ng, respectively). Both authors focused attention in their evaluation of genotyping performance on genotyping wgaDNA derived from 3 ng of genomic DNA template. Lasken and Egholm [10] have recommended 10–100 ng of undegraded gDNA template in the MDA WGA reaction to avoid stochastic amplification. The present study has characterized the yield, composition and genotyping performance of wgaDNA produced from lymphoblast gDNA templates of 1, 10, 25, 50, 100 and 200 ng. Three DNA quantification methods, two genotyping methods, and adequate numbers of genotyping assays and DNA samples were used to detect significant differences in the yield, composition and genotyping performance of the wgaDNA produced from this range of gDNA inputs and to provide additional recommendations on the amounts of gDNA template to be used in the MDA WGA reaction.
Results
WGA reaction yield
The yield of H. sapiens PCR-amplifiable (hereafter "RT-PCR") DNA, ssDNA, dsDNA and total DNA in wgaDNA by gDNA input mass is presented in Figure 1. RT-PCR DNA yield increased significantly as gDNA input increased at each level (all p values ≤ 0.02), where the proportion of the total wgaDNA represented by the RT-PCR DNA increased from 20% to 46%, at 1 to 200 ng gDNA input into the WGA reaction, respectively. The yield of ssDNA decreased, and that of dsDNA increased, as the gDNA input into the WGA reaction was increased. The variability in wgaDNA yield by wgaDNA component was least for total DNA and dsDNA yield, greatest for RT-PCR yield, and intermediate for ssDNA yield.
Genetic profiling with AmpFlSTR® Identifiler® assay (N = 15 STR and AMEL)
gDNA exhibited STR genotype completion and concordance rates of 100%, which were significantly greater than the completion rate exhibited by wgaDNA produced from 1 ng gDNA input and the wgaDNA concordance produced from all gDNA inputs, respectively (Table 1). wgaDNA produced from 1 ng gDNA input exhibited significantly lower STR genotype completion and concordance rates than did wgaDNA produced from other gDNA inputs, while wgaDNA produced from 200 ng gDNA input exhibited STR genotyping completion and concordance rates similar, but not identical to gDNA. 98% of wgaDNA STR genotypes discordant with gDNA genotypes were homozygote genotypes, reflecting loss of heterozygosity. There was a trend for preferential loss of shorter alleles (129 short alleles/232 total alleles, p = 0.088), but only for wgaDNA produced from 1 ng of gDNA was this significant (90 short alleles/145 total alleles, p = 0.0037). Peak heights were significantly and negatively correlated with discordance for all gDNA inputs, and for 1, 10 and 100 ng gDNA inputs separately (Spearman r = -0.58, -0.64, -0.55 and -0.62, with p values <0.0001, = 0.008, = 0.025 and = 0.001, respectively, data not shown), and peak height ratios of concordant heterozygote wgaDNA genotypes (from wgaDNA produced from 1 and 50 ng gDNA inputs) were significantly higher than those from gDNA genotypes (Wilcoxon's p values ≤ 0.03, data not shown).
The rate of no amplification and discordant genotypes per STR locus was 0.8% and 4%, respectively. Five STR loci (TPOX, FGA, D7S820, D13S317 and D18S51) accounted for the majority of STR no amplification failures (82%) and discordant (56%) genotypes following WGA (Table 2). The discordance rate for AMEL genotypes for all wgaDNA strata was 0.15%, but was 0.73% for wgaDNA produced from 1 ng gDNA input (Table 2). Composite genotype quality (GQ) scores for gDNA heterozygote and homozygote concordant genotypes were significantly better (fewer genotypes in the poorer quality categories) than for concordant wgaDNA heterozygote genotypes at all gDNA input levels and for concordant wgaDNA homozygote genotypes produced from 1, 100 and 200 ng gDNA input levels, respectively (Table 3). wgaDNA heterozygote and homozygote concordant genotypes produced from 1 ng gDNA input exhibited significantly reduced GQ scores compared to wgaDNA heterozygote and homozygote concordant genotypes produced from all other gDNA input levels, except for wgaDNA homozygote concordant genotypes produced from 200 ng gDNA (Table 3). GQ scores of discordant homozygote wgaDNA genotypes were significantly worse than those for concordant homozygote wgaDNA genotypes at all gDNA input levels except 50 ng (p = 0.02 for 25 ng gDNA input, all other p < 0.0001, data not shown).
SNP genotyping with the TaqMan® assay (N = 49 SNPs)
Results of genotyping using N = 49 TaqMan® SNP genotyping assays with 1, 4 and 20 ng of gDNA and wgaDNA using 1, 10, 25, 50, 100 and 200 ng of gDNA input into the WGA reaction are summarized in Table 4. We observed a TaqMan® SNP genotype completion rate of >99.55%, an undetermined rate of <0.45%, zero discordant genotypes and zero "no amplification failures" in 7938 attempted TaqMan® SNP genotypes using gDNA template in the TaqMan® SNP assay. No significant differences in genotyping performance between gDNA template inputs into the TaqMan® SNP assay were observed (Table 4). In pairwise tests, gDNA exhibited a significantly higher TaqMan® SNP genotype completion rate due to significantly decreased undetermined TaqMan® SNP genotypes, compared to wgaDNA produced from 1 ng of gDNA input for all wgaDNA template inputs into TaqMan® SNP genotyping, and when compared to wgaDNA produced from 50 and 100 ng of gDNA input when using 1 or 4 ng of wgaDNA template input into the TaqMan® SNP assay. Over all gDNA and wgaDNA strata, we observed significantly reduced SNP genotyping performance when using 1 ng of gDNA or wgaDNA in TaqMan® SNP genotyping assays with respect to completion rate, due to a significant increase in the undetermined genotype rate (Table 4). However, genotype concordance rates were not significantly different among the three DNA (gDNA or wgaDNA) input levels into the TaqMan® SNP assay, although there was a significant decrease in the concordance rate of 1 ng wgaDNA produced from 1 ng gDNA into the TaqMan® SNP assay, when compared to 1 ng wgaDNA produced from 10, 50 and 100 ng of gDNA input (Table 4).
Predictors of wgaDNA SNP genotyping performance
We were interested to identify parameters from the Core Genotyping Facility's standard DNA sample handling protocol that might be predictive of the SNP genotyping performance of wgaDNA. We performed exploratory correlation analysis among measures of wgaDNA yield (RT-PCR, ssDNA, dsDNA, total DNA, ratio of RT-PCR to dsDNA) and genotyping performance (concordance and completion rates for AmpFlSTR® Identifiler™ and TaqMan® SNP assay genotyping) within gDNA input strata. Measures of wgaDNA yield (especially the ratio of RT-PCR to dsDNA and total DNA) and genotyping performance were observed to be highly correlated with one another (92%, 3% and 5% of 162 pairwise correlations were statistically significant, trending and non-significant, respectively). We then performed linear regression analysis with the dependent variables "SNP completion rate" and "SNP concordance rate", in order to identify WGA reaction, wgaDNA yield and STR genotyping performance factors that are significantly associated with wgaDNA SNP genotyping performance. Independent variables included: gDNA input, STR completion rate, concordance rate, GQ score, peak height, and RT-PCR wgaDNA yield. "STR completion rate" was a highly significant factor in both SNP rate models (p < 0.0001), and "STR concordance rate" and "GQ score" were significant factors in the SNP concordance rate model (p = 0.0008 and 0.045, respectively). The variable "gDNA input" into the WGA reaction was significant only in those models incorporating the 1 ng gDNA input strata.
WGA yield and genotyping performance with no template control (NTC) samples
No template control (NTC) input samples, i.e., where no gDNA was used in the WGA reaction, yielded substantial amounts of wgaDNA, similar in quantity to the total wgaDNA obtained with gDNA inputs, but with a substantially higher proportion of ssDNA than with gDNA inputs (Table 5). The wgaDNA produced from the NTC samples in the 1, 50 and 100 ng gDNA input strata exhibited mean RT-PCR results that were greater than zero (Table 5). We observed N = 35 STR peaks with a signal strength > 50 RFUs that fell within the expected base-pair range of an AmpFlSTR® Identifiler™ locus allele from the wgaDNA produced from the NTC gDNA and wgaDNA samples for an overall false positive STR genotyping rate of 4.2% (Table 5). While these false positive STR peaks fulfill the criteria for valid AmpFlSTR® Identifiler™ STR alleles, they are characterized by low heterozygosity (2 observed versus 26 expected heterozygote genotypes), moderate signal strength (median amplitude = 357 RFUs), and representation of 12 out of 15 AmpFlSTR® Identifiler™ STR loci. The 50 and 100 ng gDNA input strata (Table 5) and three STR loci (D2S1338, D8S1179 and FGA)account for the majority (66% and 51%, respectively) of the wgaDNA false positive STR genotypes produced from the NTC samples.
In N = 7056 TaqMan SNP genotype attempts with wgaDNA produced from the NTC samples, 80%, 14.5% and 5.5% of the resulting datapoints were incorporated into the no amplification (NTC) cluster, into a genotype cluster ("false positive SNP genotypes"), and into the undetermined genotype space of the two color TaqMan® SNP genotyping assay plot, respectively (Table 6). The number of false positive and undetermined SNP genotypes from the wgaDNA produced from the NTC samples increased significantly with increasing amounts of wgaDNA input into the TaqMan® SNP assay (Table 6). The majority (96.4%) of these false positive SNP genotypes from NTC samples were homozygotes (Table 6), significantly more allele 2 alleles were observed than allele 1 alleles (Table 6), and all N = 49 TaqMan® SNP assays exhibited false positive SNP genotypes (data not shown). wgaDNA NTC samples from the gDNA input strata of 1, 50 and 100 ng exhibited significantly greater numbers of false positive and undetermined SNP genotypes than did the wgaDNA NTC samples from the gDNA input strata of 10, 25 and 200 ng (Table 6).
Discussion
wgaDNA may not be suitable for STR genotyping
wgaDNA STR genotyping completion rates reach that of gDNA at the 10 ng gDNA input level into WGA. However, the wgaDNA STR concordance rate is significantly worse than that of gDNA, even with 200 ng of gDNA input into the WGA reaction (Table 1). Thus, the use of MDA wgaDNA for accurate STR genotyping will require larger amounts of input gDNA into the WGA than have been recommended in the past [8,10]. In the absence of sufficient gDNA template for MDA WGA, investigators face the tradeoff of no data, or data with increased loss of heterozygosity, such as that observed with MDA wgaDNA produced from low mass gDNA templates [11,12]. Development of laboratory and data analysis protocols optimized for STR genotyping of MDA wgaDNA may be required before MDA wgaDNA can be routinely used for STR genotyping. Thus, it might be prudent to adjust genotype analysis algorithms before application of the AmpFlSTR® Identifiler™ panel to wgaDNA for forensic purposes, as has been recommended for the analysis of STR profiles from highly limited unamplified gDNA template [13], or to utilize analysis methods that incorporate STR genotyping error, as has been recommended for the analysis of STR linkage scan data [14].
wgaDNA is a suitable template for SNP genotyping
wgaDNA produced from ≥ 10 ng of gDNA input into the WGA reaction exhibits robust wgaDNA TaqMan® SNP assay genotyping performance rates, similar to that of gDNA TaqMan® SNP assay genotyping performance rates. 1 ng of wgaDNA template into the TaqMan® SNP assay exhibits significantly reduced TaqMan® SNP assay genotyping performance compared to both 4 and 20 ng wgaDNA templates into the TaqMan® SNP assay. 4 ng wgaDNA template into the TaqMan® SNP assay exhibits a significantly increased no amplification rate over both 1 and 20 ng wgaDNA templates, although no amplification rates are very low (all <0.01%) for all three wgaDNA template inputs into the TaqMan® SNP assay. These results suggest that optimal TaqMan® SNP assay genotyping performance, i.e., minimal wgaDNA TaqMan® no amplification and undetermined genotyping rates, should be expected for wgaDNA inputs greater than 4 ng.
False positive NTC sample SNP genotypes
A non-zero RT-PCR yield and significantly increased numbers of observed false positive genotypes in wgaDNA from NTC samples in the 1, 50 and 100 ng gDNA input strata are consistent with human gDNA contamination of these gDNA input strata. However, we also observed significantly more false positive and undetermined SNP assay genotypes in each of the 10, 25 and 200 ng gDNA input strata (the apparently uncontaminated strata) than in the gDNA strata (all p < 0.0001), concordant with the hypothesis that a portion of the NTC TaqMan® genotypes may be due to degradation of TaqMan® SNP assay reagents. Thus, contamination of NTC samples with gDNA and TaqMan® SNP assay probe oligonucleotide degradation during the genotyping of wgaDNA are both associated with false-positive TaqMan® SNP assay genotypes.
Limitations
This study is distinguished by the use of multiple assays to estimate wgaDNA yield and composition, the use of STR and SNP genotyping assays that have been validated by sequencing the same DNA samples used in this study, and the use of an adequate number of samples and assays to provide statistical power to detect small differences in the genotyping performance of wgaDNA and gDNA, when using 1–200 ng of gDNA as template in the WGA reaction. Nevertheless, there are limitations, with respect to generalizing to all gDNA templates, MDA protocols and genotyping methods, respectively.
The gDNA used in this study was extracted from lymphoblasts and samples from most studies are unlikely to be of such high quality. Using a model system to evaluate the effect of significant gDNA degradation on the WGA reaction, it has been shown that MDA wgaDNA produced from irradiated gDNA exhibits significantly reduced yield and genotyping performance compared to MDA wgaDNA produced from unirradiated gDNA [15]. The yield and genotyping differences observed in wgaDNA produced from high quality (this study) and low quality [15] gDNA samples suggest that those gDNA samples with DNA extraction, storage and usage histories that have reduced concentrations of high molecular weight DNA in the sample are likely to exhibit less than optimal MDA wgaDNA yield and genotyping performance.
While only one commercially available MDA WGA protocol was used in this study, we have evaluated two MDA WGA protocols on gDNA extracted from multiple tissue types, and no systematic significant differences in genotyping performance between the two MDA WGA protocols was observed [16]. STR and SNP genotyping performance of MDA wgaDNA derived from 4 ng of gDNA input in that study is seen to be intermediate between the genotyping performance of MDA wgaDNA produced from 1 ng and 10 ng in this study. Alternative WGA technologies that can prepare wgaDNA of acceptable quality from gDNA with reduced complexity or concentration may be required for some degraded gDNA samples. For example, PCR-based methods that reduce genome complexity before amplification are one approach [6,17], and methods that combines genome circularization with φ29 DNA polymerase are another [18].
Finally, we applied two commonly used genotyping methods to evaluate the genotyping performance of wgaDNA in this study. Different genotyping technologies may be better suited to produce optimal genotyping performance with wgaDNA than the two we evaluated. For STRs, genotyping panels designed for linkage scanning usually employ lower levels of multiplexing and use larger amounts of DNA template than do STR panels designed for forensic analysis, such as the AmpFlSTR® Identifiler™. E.g., reported MDA wgaDNA STR genotype discordance rates using linkage scan STR panels [19,20] and forensic STR panels [15,16,21] range from ~0% to ~6% and the average rate of the five studies cited (2.0%) is similar to the rates observed in this study. For SNPs, those genotyping technologies with redundant data sampling for SNP genotype determination, such as minisequencing [22], the Golden Gate™ assay [23] or the GeneChip® variant detection array [24], may be more resistant to SNP genotype failure when genotyping wgaDNA [9,25,26] than those SNP genotyping technologies with single data point genotype determination [27]. However, in a recent direct comparison of Golden Gate™, TaqMan® and Invader™ SNP assays, with gDNA extracted from lymphoblasts using an organic extraction method and MDA wgaDNA produced from 20 ng of this gDNA, the Golden Gate™ assay exhibited a higher exclusion rate of DNA samples, and a higher completion rate and lower concordance rate on the remaining samples, than exhibited by the TaqMan® and Invader™ SNP assays [28]. For all three SNP genotyping technologies evaluated, the genotyping performance of gDNA was observed to be significantly better than that of MDA wgaDNA [28].
Conclusion
We have evaluated the yield, composition and genotyping performance of wgaDNA based on a range of high-quality lymphoblastoid gDNA templates between 1 and 200 ng in order to provide empirical data on the performance of MDA WGA technology. A detailed analysis of the observed genotyping failures has been performed to facilitate an understanding of the reduction in genotyping performance likely to be observed when genotyping wgaDNA produced from a range of gDNA inputs. Increasing gDNA input from 1 – 200 ng in the MDA WGA reaction improves the yield of H. sapiens PCR-amplifiable DNA and improves the genotyping performance of the AmpFlSTR® Identifiler® assay. More than 100 ng of high quality gDNA template into the MDA WGA reaction is required in order to observe MDA wgaDNA AmpFlSTR® Identifiler® STR genotyping performance similar to that observed with gDNA. At least 10 ng of high quality lymphoblastoid gDNA template into the WGA reaction is required to observe optimal TaqMan® SNP genotyping performance from MDA wgaDNA.
Methods
gDNA samples
N = 22 lymphoblast genomic DNA (gDNA) samples were obtained directly from the Coriell Cell Repository (Camden, NJ); these samples were from individuals within the SNP500 Cancer dataset [29]. Each gDNA was quantified by UV spectroscopy, the PicoGreen® assay (Molecular Probes, Eugene, OR), and a Real-Time (RT) TaqMan® assay specific to human DNA [30]. Five of twenty-two Coriell Cell Repository lymphoblast gDNA samples were replicated for a total of N = 27 lymphoblast gDNA samples subjected to WGA and post-WGA analysis in order to increase statistical power to detect genotyping error.
Whole genome amplification
1, 10, 25, 50, 100 and 200 ng of each gDNA sample was used as template and amplified according to the GenomiPhi™ WGA protocol (1X). The 200 ng gDNA template sample was amplified separately after evaluation of the genotyping performance of the wgaDNA produced from 1 – 100 ng gDNA. Each gDNA sample was subjected to the WGA protocol once; four no gDNA template controls (NTC) reactions were included at each gDNA input level. wgaDNA was quantified with OD260, PicoGreen® and RT-PCR, as was performed for gDNA. The concentrations of ssDNA, dsDNA, total DNA and human-specific PCR amplifiable (RT-PCR) DNA in the wgaDNA samples were estimated as described [16].
AmpFlSTR® Identifiler® assay
300 pg of dsDNA (both gDNA and wgaDNA, as determined by PicoGreen®) was used as template DNA for AmpFlSTR® Identifiler® assay (Applied Biosystems Inc., Foster City, CA), and scoring of alleles, assignment of Genotype Quality scores and calculation of genotype failure rates were performed as described [16]. Peak height ratio distributions at a signal strength threshold of = 50 RFUs were evaluated for normality and differences between assigned and observed GQ score category distributions evaluated using Wilcoxon's rank sum test and contingency table analyses.
TaqMan® SNP genotype assays
N = 49 TaqMan® (Applied Biosystems Inc., Foster City, CA) genotyping assays from the publicly available SNP500 Cancer Database portfolio [29] were chosen as described [16]. 1.0, 4.0 and 20.0 ng of dsDNA (both gDNA and wgaDNA, as determined by PicoGreen®) was used as template for genotyping using the N = 49 TaqMan® assays. Reaction and cycling conditions, control samples, fluorescence detection and genotype cluster assignment were performed as described [16]. SNP genotype completion, undetermined genotype, no amplification, and discordance rates were calculated, with the wgaDNA discordance rate calculated to be the number of instances in which a wgaDNA SNP genotype differed from the scored gDNA SNP genotype. Differences in rates were evaluated using contingency table analyses.
Data management and analysis
Data was managed using a Sapphire Laboratory Information Management System (LabVantage, New Brunswick, NJ), exported in Microsoft Excel (Redmond, WA) and statistical analyses (descriptive statistics and tests of normality, distribution and correlation) were performed using SAS (Cary, NC) software. Tests of proportion, correlation, etc., are considered significant at a Type I error level of 0.05, with additional information on p values provided if appropriate.
Authors' contributions
AWB conceived of the study and drafted the manuscript. AWB, YQ, KAH and RAW participated in the experimental design. KAH designed and performed the amplification, quantification and genotyping experiments. YQ performed the statistical analysis. AWB, YQ, KAH and RAW participated in the interpretation of the data, and YQ, KAH, RW and SJC helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors acknowledge the contribution of Michael B. Beerman to data management and Cynthia Glaser, Ian Barrow and Jessica Hartwell to genotyping. This research was supported by the Intramural Research Program of the NCI.
Figures and Tables
Figure 1 Yield of DNA components of wgaDNA by gDNA input into WGA. Mean ("+"), Median (middle bar), lower and upper quartile (lower and upper end of box), and minimum and maximum of BRCA1 locus equivalents, ssDNA, dsDNA and total DNA.
Table 1 STR genotyping performance
gDNA Input (ng) Completed1 % Completion No Amplification GQ<0.252 Concordant % Concordance Discordant Genotypes % Discordance
gDNA 864 100.03 0 13 851 100.04 0 0.0
1 821 95.0 43 70 638 80.1 150 19.0
10 862 99.8 2 9 830 97.1 25 2.9
25 863 99.9 1 22 823 97.7 19 2.3
50 863 99.9 1 15 836 98.6 12 1.4
100 861 99.7 3 11 826 96.8 27 3.2
200 864 100.0 0 0 858 99.3 6 0.7
1N = 27 gDNA or wgaDNA samples were genotyped in duplicate using the AmpFlSTR® Identifiler® assay for N = 864 attempted genotypes/sample. 2A Genotype Quality Score (GQ) of <0.25 indicates a STR genotype with a Genotype Quality Score beloew the calling threshold of GeneMapper v3.0 software. 3gDNA exhibited significantly greater STR genotype completion rate than did 1 ng gDNA input (p < 0.001). 4gDNA exhibited significantly greater STR genotype concordance rates compared to wgaDNA (p < 0.0001, except for 50 ng gDNA input, with p = 0.001, and 200 ng gDNA input, p = 0.03).
Table 2 STR genotyping failures by locus
gDNA Input (ng) Failure Type Genotype Failures1 L12 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 L15 L16
gDNA No Amp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Disc. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 No Amp. 43 5 0 0 3 0 0 7 0 0 3 5 0 15 1 1 3
Disc. 150 11 3 9 10 4 5 13 9 2 5 22 9 24 7 11 6
10 No Amp. 2 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
Disc. 25 2 0 0 5 1 0 2 0 1 0 7 1 5 0 0 1
25 No Amp. 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Disc. 19 4 1 1 0 0 2 2 0 0 0 2 0 3 2 1 1
50 No Amp. 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Disc. 12 0 0 1 3 0 0 1 2 0 0 0 0 2 1 1 1
100 No Amp. 3 0 0 0 1 0 0 0 0 0 0 0 1 1 0 0 0
Disc. 27 5 5 0 2 0 1 2 1 6 0 0 2 1 2 0 0
200 No Amp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Disc. 6 1 1 0 2 0 0 0 0 0 0 2 0 0 0 0 0
No Amp. Total 23 10 11 22 5 8 20 12 9 5 33 12 35 12 13 9
Discordant Total 6 0 0 5 0 0 7 0 0 3 5 1 18 1 1 3
1The number of attempted genotypes for all DNA inputs is N = 864. No amplification (No Amp.) genotypes reduce the number of genotypes available for concordance analysis. 2Loci 1–16 = TPOX, D2S1338, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S2317, D16S539, D18S51, D19S433, D21S11, AMEL.
Table 3 Genotype Quality (GQ) scores of concordant STR genotypes
gDNA Input (ng) GQ1Category, Heterozygotes
I II III Total
gDNA 5 53 580 6382
1 43 137 256 4363
10 27 140 451 618
25 26 126 463 615
50 21 130 474 625
100 25 110 482 617
200 78 31 537 646
Total 225 727 3243 4195
gDNA Input (ng) GQ Category, Homozygotes
I II III Total
gDNA 0 9 204 2134
1 9 25 168 2025
10 2 8 202 212
25 1 8 199 208
50 4 11 196 211
100 8 10 191 209
200 8 20 184 212
Total 32 91 1344 1467
1Category I = GQ scores, originally ≥ 0.25 and <0.40, that have been successfully edited; Category II = GQ scores ≥ 0.40 and ≤ 0.50; Category III = GQ scores ≥ 0.79 and ≤ 0.90. 2GQ score distribution, heterozygote genotypes, gDNA versus all wgaDNA, p < 0.0001. 3GQ score distribution, heterozygote genotypes, wgaDNA produced from 1 ng gDNA input versus other gDNA inputs, p < 0.0001.4GQ score distribution, homozygote genotypes, gDNA versus wgaDNA produced from 1, 100, 200 ng gDNA input, p < 0.0001, 0.01, 0.0001, respectively. 5GQ score distribution, homozygote genotypes, wgaDNA produced from 1 ng gDNA input versus 10, 25, 50, 100 ng gDNA inputs, p = 0.0003, 0.0001, 0.009, 0.02, respectively.
Table 4 SNP genotyping performance
gDNA Input (ng) Completed Genotypes1 % Completion Undeter. Genotypes % Undeter. No Amp. % No Amp. Concordant Genotypes % Concordance Discordant Genotypes % Discordance
1 ng gDNA or wgaDNA input into TaqMan® SNP genotype assay2,3
gDNA 2636 99.62 10 0.38 0 0 2636 100.00 0 0.00
1 25524 96.45 945 3.55 0 0 2546 99.76 68 0.24
10 2627 99.28 19 0.72 0 0 2627 100.00 0 0.00
25 2636 99.62 10 0.38 0 0 2635 99.96 1 0.04
50 2614 98.79 316 1.17 1 0.04 2614 100.00 0 0.00
100 2620 99.02 256 0.94 1 0.04 2620 100.00 0 0.00
200 2623 98.07 21 0.79 2 0.08 2622 99.96 1 0.04
4 ng gDNA or wgaDNA into TaqMan® SNP genotype assay4
gDNA 2634 99.55 12 0.45 0 0.00 2634 100.00 0 0.00
1 25894 97.85 555 2.08 2 0.08 2586 99.88 3 0.12
10 2628 99.32 17 0.64 1 0.04 2627 99.96 1 0.04
25 2629 99.36 14 0.53 3 0.11 2629 100.00 0 0.00
50 2621 99.06 235 0.87 2 0.08 2619 99.92 2 0.08
100 2619 98.98 205 0.76 77 0.26 2617 99.92 2 0.08
200 2635 99.4 10 0.38 1 0.04 2632 99.89 3 0.11
20 ng gDNA or wgaDNA into TaqMan® SNP genotype assay
gDNA 2637 99.66 9 0.34 0 0 2637 100.00 0 0.00
1 25694 97.09 775 2.91 0 0 2566 99.88 3 0.12
10 2633 99.51 13 0.49 0 0 2633 100.00 0 0.00
25 2639 99.74 7 0.26 0 0 2638 99.96 1 0.04
50 2633 99.51 13 0.49 0 0 2632 99.96 1 0.04
100 2630 99.40 16 0.60 0 0 2629 99.96 1 0.04
200 2635 99.55 11 0.42 0 0 2632 99.89 3 0.11
1There were N = 27 samples gentoyped in duplicate at N = 49 SNPs for N = 2,646 attempted genotypes/sample. 21 versus 4 ng gDNA or wgaDNA input, SNP genotyping rates: completion, p = 0.0178; undetermined, p = 0.0022; no amplification, p = 0.0139; concordance, p = n.s. 31 versus 20 ng gDNA or wgaDNA input, SNP genotyping rates: completion, p = 0.0004; undetermined, p = 0.0008; no amplification, p = n.s.; concordance, p = n.s. 44 versus 20 ng gDNA or wgaDNA input, SNP genotyping rates: completion, p = n.s.; undetermined, p = n.s.; no amplification, p = 0.0002; concordance, p = n.s. 5gDNA versus wgaDNA produced from 1 ng gDNA input, SNP genotype completion rates, p < 0.0001. 6gDNA versus wgaDNA produced from 50 and 100 ng gDNA input, p ≤ 0.05. 7gDNA versus wgaDNA produced from 100 ng gDNA input, p = 0.02. 8wgaDNA produced from 1 ng versus gDNA and wgaDNA produced from 10, 50 and 100 ng of gDNA input, p ≤ 0.014.
Table 5 Yield and STR genotypes from NTC samples
gDNA Input (ng) Yield
N Mean RT-PCR (ng) Median ssDNA (%) Median total DNA (ng)
gDNA - - - -
1 4 135 51.5 12230
10 4 0 50.8 11771
25 4 0 55 12340
50 4 24 45 12395
100 4 220 48.6 12686
200 4 0 54 14879
STR
gDNA Input (ng) N False Positive Rate (%)1 Mean Height Allele 1 Mean Height Allele 2
gDNA 64 6 93 93
1 128 5 2185 2199
10 128 0 - -
25 128 0 - -
50 128 92 375 393
100 128 92 571 571
200 128 2 2360 2360
1The number of "attempted" AmpFlSTR® Identifiler™ panel genotypes using gDNA NTCs and wgaDNA from NTC samples is 832 [two NTC DNA samples genotyped for gDNA in duplicate (64 possible genotype bins), and four wgaDNA samples from NTCs for each gDNA input level genotyped in duplicate (768 possible genotype bins)]. 2There were significantly more false positive STR genotypes in the wgaDNA produced from 50 ng and 100 ng gDNA inputs compared to the wgaDNA produced from 200 ng gDNA input (p = 0.01 and 0.02).
Table 6 SNP genotypes from NTC samples
gDNA Input (ng) Allele 1 Allele 2 Both No Amp. Undeter. Total
1 ng gDNA or wgaDNA input into TaqMan® SNP genotype assay1
gDNA 0 2 0 385 5 392
13 27 29 2 315 19 392
10 6 12 1 362 11 392
25 6 10 0 364 12 392
503 38 49 5 266 34 392
1003 26 48 2 289 27 392
200 18 20 0 351 3 392
Total 121 1702 10 2332 111 2744
4 ng gDNA or wgaDNA input into TaqMan® SNP genotype assay1
gDNA 1 2 0 381 8 392
13 39 36 2 292 23 392
10 7 7 0 360 18 392
25 6 18 1 349 18 392
503 50 43 0 275 24 392
1003 26 43 2 274 47 392
200 26 38 3 316 9 392
Total 155 1872 8 2247 147 2744
20 ng gDNA or wgaDNA input into TaqMan® SNP genotype assay1
gDNA 0 1 0 383 8 392
13 29 37 2 297 27 392
10 11 15 1 346 19 392
25 8 9 0 364 11 392
503 37 54 4 263 34 392
1003 25 55 2 277 33 392
200 38 42 10 284 18 392
Total 148 2132 19 2214 150 2744
1A significant increase in the number of false positive and undetermined SNP genotypes is observed with increasing amounts of wgaDNA template: 11.0%, 12.8% and 13.8% for 1, 4 and 20 ng wgaDNA input, respectively, p = 0.045, 1 versus 4 ng wgaDNA input, and p = 0.0014, 1 versus 20 ng wgaDNA input, p = 0.0046 test for trend, 1 vs. 4 vs 20 ng wgaDNA input, for false positive genotypes; 4.0%, 5.34% and 5.47%, respectively, p = 0.026, 1 versus 4 ng, and p = 0.0159, 1 versus 20 ng, p = 0.0153 for trend, for undetermined genotypes. 2Significantly more allele 2 NTC TaqMan® SNP assay alleles were observed than allele 1 NTC TaqMan® assay alleles (p = 0.006, 0.091 and 0.001 for 1, 4 and 20 ng gDNA and wgaDNA input into the TaqMan® SNP assays, respectively), where the fluorescent label was 6-Fam for allele 1, and Vic for allele 2, in all the TaqMan® SNP assays in this study. 3The gDNA input strata of 1, 50 and 100 ng exhibited significantly greater numbers of false positive and undetermined SNP genotypes than did the gDNA input strata of 10, 25 and 200 ng (p < 0.0001).
==== Refs
Collins FS Morgan M Patrinos A The Human Genome Project: lessons from large-scale biology Science 2003 300 286 290 12690187 10.1126/science.1084564
Risch N Merikangas K The future of genetic studies of complex human diseases Science 1996 273 1516 1517 8801636
Carlson CS Eberle MA Kruglyak L Nickerson DA Mapping complex disease loci in whole-genome association studies Nature 2004 429 446 452 15164069 10.1038/nature02623
Matsuzaki H Loi H Dong S Tsai YY Fang J Law J Di X Liu WM Yang G Liu G Huang J Kennedy GC Ryder TB Marcus GA Walsh PS Shriver MD Puck JM Jones KW Mei R Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array Genome Res 2004 14 414 425 14993208 10.1101/gr.2014904
Telenius H Carter NP Bebb CE Nordenskjold M Ponder BA Tunnacliffe A Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer Genomics 1992 13 718 725 1639399 10.1016/0888-7543(92)90147-K
Tanabe C Aoyagi K Sakiyama T Kohno T Yanagitani N Akimoto S Sakamoto M Sakamoto H Yokota J Ohki M Terada M Yoshida T Sasaki H Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA Genes Chromosomes Cancer 2003 38 168 176 12939744 10.1002/gcc.10269
Blanco L Bernad A Lazaro JM Martin G Garmendia C Salas M Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication J Biol Chem 1989 264 8935 8940 2498321
Dean FB Hosono S Fang L Wu X Faruqi AF Bray-Ward P Sun Z Zong Q Du Y Du J Driscoll M Song W Kingsmore SF Egholm M Lasken RS Comprehensive human genome amplification using multiple displacement amplification Proc Natl Acad Sci U S A 2002 99 5261 5266 11959976 10.1073/pnas.082089499
Lovmar L Fredriksson M Liljedahl U Sigurdsson S Syvanen AC Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA Nucleic Acids Res 2003 31 e129 14576329 10.1093/nar/gng129
Lasken RS Egholm M Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens Trends Biotechnol 2003 21 531 535 14624861 10.1016/j.tibtech.2003.09.010
Rook MS Delach SM Deyneko G Worlock A Wolfe JL Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping Am J Pathol 2004 164 23 33 14695315
Handyside AH Robinson MD Simpson RJ Omar MB Shaw MA Grudzinskas JG Rutherford A Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease Mol Hum Reprod 2004 10 767 772 15322224 10.1093/molehr/gah101
Whitaker JP Cotton EA Gill P A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis Forensic Sci Int 2001 123 215 223 11728750 10.1016/S0379-0738(01)00557-6
Sobel E Papp JC Lange K Detection and integration of genotyping errors in statistical genetics Am J Hum Genet 2002 70 496 508 11791215 10.1086/338920
Bergen AW Qi Y Haque KA Welch RA Garcia-Closas M Chanock SJ Vaught J Castle PE Effects of electron-beam irradiation on whole genome amplification Cancer Epidemiol Biomarkers Prev 2005 14 1016 1019 15824182 10.1158/1055-9965.EPI-04-0686
Bergen AW Haque KA Qi Y Beerman MB Garcia-Closas M Rothman N Chanock SJ Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlextrade mark whole genome amplified DNA generated from multiple DNA sources Hum Mutat 2005 26 262 270 16086324 10.1002/humu.20213
Langmore JP Rubicon Genomics, Inc Pharmacogenomics 2002 3 557 560 12164778 10.1517/14622416.3.4.557
Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation Genome Res 2004 14 2357 2366 15520297 10.1101/gr.2813404
Bark C Pettengill J Tsai YBP Golembieski J Gearhart J Stewart L Zilka M Doheny K Performance of whole genome amplified samples for microsatellite genotyping.: 2004/10/29. 2004 Bethesda, MD, The American Society of Human Genetics
Dickson PA Montgomery GW Henders A Campbell MJ Martin NG James MR Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan Nucleic Acids Res 2005 33 e119 16055919 10.1093/nar/gni126
Sun G Kaushal R Pal P Wolujewicz M Smelser D Cheng H Lu M Chakraborty R Jin L Deka R Whole-genome amplification: relative efficiencies of the current methods Leg Med (Tokyo) 2005 7 279 86 15990351
Pastinen T Raitio M Lindroos K Tainola P Peltonen L Syvanen AC A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays Genome Res 2000 10 1031 1042 10899152 10.1101/gr.10.7.1031
Fan JB Oliphant A Shen R Kermani BG Garcia F Gunderson KL Hansen M Steemers F Butler SL Deloukas P Galver L Hunt S McBride C Bibikova M Rubano T Chen J Wickham E Doucet D Chang W Campbell D Zhang B Kruglyak S Bentley D Haas J Rigault P Zhou L Stuelpnagel J Chee MS Highly parallel SNP genotyping Cold Spring Harb Symp Quant Biol 2003 68 69 78 15338605 10.1101/sqb.2003.68.69
Chee M Yang R Hubbell E Berno A Huang XC Stern D Winkler J Lockhart DJ Morris MS Fodor SP Accessing genetic information with high-density DNA arrays Science 1996 274 610 614 8849452 10.1126/science.274.5287.610
Barker DL Hansen MS Faruqi AF Giannola D Irsula OR Lasken RS Latterich M Makarov V Oliphant A Pinter JH Shen R Sleptsova I Ziehler W Lai E Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel Genome Res 2004 14 901 907 15123587 10.1101/gr.1949704
Paez JG Lin M Beroukhim R Lee JC Zhao X Richter DJ Gabriel S Herman P Sasaki H Altshuler D Li C Meyerson M Sellers WR Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification Nucleic Acids Res 2004 32 e71 15150323 10.1093/nar/gnh069
Tranah GJ Lescault PJ Hunter DJ De Vivo I Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies Biotechnol Lett 2003 25 1031 1036 12889810 10.1023/A:1024173909401
Pask R Rance HE Barratt BJ Nutland S Smyth DJ Sebastian M Twells RC Smith A Lam AC Smink LJ Walker NM Todd JA Investigating the utility of combining phi29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray genotyping BMC Biotechnol 2004 4 15 15279678 10.1186/1472-6750-4-15
Packer BR Yeager M Staats B Welch R Crenshaw A Kiley M Eckert A Beerman M Miller E Bergen A Rothman N Strausberg R Chanock SJ SNP500Cancer: a public resource for sequence validation and assay development for genetic variation in candidate genes Nucleic Acids Res 2004 32 Database issue D528 D532 14681474 10.1093/nar/gkh005
Haque KA Pfeiffer RM Beerman MB Struewing JP Chanock SJ Bergen AW Performance of high-throughput DNA quantification methods BMC Biotechnol 2003 3 20 14583097 10.1186/1472-6750-3-20
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-1241618803310.1186/1471-2407-5-124Research ArticleDifferential expression of MUC genes in endometrial and cervical tissues and tumors Hebbar Vidya [email protected] Gautam [email protected] Goverdhan P [email protected] Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK- 73190, USA2 Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ-08854, USA2005 27 9 2005 5 124 124 4 5 2005 27 9 2005 Copyright © 2005 Hebbar 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
Mucin glycoprotein's are major components of mucus and are considered an important class of tumor associated antigens. The objective of this study was to investigate the expression of human MUC genes (MUC1, MUC2, MUC5B, MUC5AC and MUC8) in human endometrium and cervix, and to compare and quantitate the expression of MUC genes in normal and cancerous tissues.
Methods
Slot blot techniques were used to study the MUC gene expression and quantitation.
Results
Of the five-mucin genes studied, MUC1, MUC5B and MUC8 showed high expression levels in the normal and cancerous endometrial and cervical tissues, MUC2 and MUC5AC showed considerably lower expression. Statistically, higher levels of MUC1, MUC5B and MUC8 were observed in endometrial adenocarcinomas compared to normal tissues. In contrast, only MUC1 levels increased with no significant changes in expression of MUC5B and MUC8 in cervical tumors over normal cervical tissues.
Conclusion
Endometrial tumors showed increased expression of MUC1, MUC5B and MUC8 over normal tissues. Only MUC1 appears to be increase, in cervical tumors. All the studied tissues showed high and consistent expression of MUC8 mRNA. Low to neglible levels of MUC2 and MUC5AC were observed in all studied endometrial and cervical tissues.
==== Body
Background
Mucins are high molecular weight glycoprotein components of mucus (> 250 kDa), which protect and lubricate the epithelial surfaces of the respiratory, gastrointestinal and reproductive tracts in the body [1]. Mucins are heavily glycosylated (40–80%) and the oligosaccharides are attached through O-glycosidic linkages to the hydroxyl group of serine and threonine in the protein backbone [2,3]. The striking feature of nearly all mucin genes isolated thus far is the presence of repeat sequences that are either tandem in nature as in the case of MUC1 [4] or slightly imperfect repeats as in the case of MUC8 [5]. In general, the repeats are found in the central portion of the protein backbone, which are flanked by unique regions. Mucins have been classified as either membrane-bound or secretory depending on the presence of a putative trans-membrane region.
Altered mucin secretions and/or MUC gene expression patterns have been implicated in several cancerous conditions, such as gastric carcinomas [6-10], colorectal carcinomas [11-13], breast cancers [14-16] esophageal carcinomas [17], pancreatic tumors [18-20] and lung adenocarcinomas [21-23]. Accordingly, studies have also been conducted to determine the expression of MUC genes in human reproductive tissues and to investigate their possible altered quantitative and/or qualitative expression in cancerous conditions. Serial analysis of gene expression of ovarian cell lines and tissues indicated that among other genes, MUC1 is up-regulated in cancerous conditions [24]. Another study also showed that the expression of MUC1 correlated with poor prognosis in ovarian carcinoma [25]. Normal endocervical epithelium was found to express MUC genes 1, 4, 5AC, 5B, and 6, with relatively weak expression of MUC2. Normal endocervical and vaginal epithelium expresses MUC genes 1 and 4 and endometrial epithelium expresses MUC1 and MUC6 [26,27]. In an earlier report, we demonstrated the antigenic similarities between respiratory and reproductive tract mucins using a variety of mucin antibodies [28]. In another study, we reported the antigenic cross reactivity of human tracheal mucin with male and female reproductive tissues expressing MUC8 mRNA[29]. While these reports, in general, indicate the manifestation of a variety of MUC genes in reproductive tissues, information regarding mucin gene expression in corresponding tumor tissues is still lacking.
According to The American Cancer Society's Cancer Facts and Figures, it is estimated that there will be 12,200 new cases of invasive cervical cancer (uterine cervix) and 40,100 new cases of endometrial cancer (uterine corpus) that will be diagnosed this year. Hence, a better understanding of MUC gene expression patterns in female reproductive malignancies would help enhance prognosis and therapy. To contribute towards this purpose, we investigated the expression of five mucin genes (MUC1, MUC2, MUC5AC, MUC5B and MUC8) in normal reproductive and cancerous tissues.
Methods
Human tissues
Normal and malignant endometrium and cervical tissues were obtained from Co-operative Human Tissue Network (CHTN, Birmingham, AL) and National Disease Research Interchange (NDRI, Philadelphia, PA). Malignant tissues with over 95 percent tumor content were selected for the study. The nomenclature adopted for the tissues was as follows: endometrial adenocarcinomas (EA), normal endometrium (EN), cervical carcinomas (CA), normal cervix (CX). These tissues were acquired on dry ice and kept frozen at -80°C until use. The classification of tumors was performed according to the International Federation of Gynecology and Obstetrics (FIGO) as indicated in Table 2.
The normal tissues EN (mean age: 41, median age: 43) and CX (mean age: 43, median age: 44) used in the study were obtained by hysterectomy for benign gynecological disorders. The menstrual cycle of all the patients with normal tissues were in early to late proliferative phases, except for EN3, EN4, EN10, CX3, CX4 and CX13 were in late secretory phase. The provided pathology reports of both normal and tumor tissues indicated no inflammation or infections.
Total RNA isolation
Total RNA was extracted from frozen tissues using the TRIzol reagent (Life Technologies) according to manufacturer's protocol. Briefly, endometrial and cervical tissues were ground using a mortar and pestle under liquid nitrogen. The ground tissue was transferred to tubes containing appropriate amounts of TRIzol reagent and homogenized using a Brinkmann homogenizer. The mixture was allowed to set for 5–10 min followed by the addition of appropriate amount of chloroform. This mixture was shaken vigorously and centrifuged at 8,000 × g for 30 min. The aqueous layer was collected and the RNA precipitated using isopropanol. The RNA pellet was washed using 70% alcohol and dissolved in RNase free water. To determine the quality of extracted RNA, the dissolved samples were elecrophoresed on 1% formaldehyde-agarose gels to check the integrity of 18s and 28s bands. Samples with OD 260/280 greater than 1.50 were used in the study.
Slot blot analyses
Total RNA (10 μg) extracted from tissues was blotted directly onto nylon membranes using a Hoefer PR 648 slot blot manifold (Amersham Biosciences, San Francisco, CA). The membranes were pre-hybridized in 5× SSPE, 5× Denhardt's solution, 0.5% SDS, 50% formamide and 40 μg/ml salmon sperm DNA for 12 h at 42°C. The cDNA probes (MUC1, MUC2, MUC5B, MUC5AC, MUC8 and β-actin) were labeled by the random priming technique using DECAprime II kit (Ambion, Austin, TX). Hybridization of 32P-labeled cDNA probes was carried out at 42°C for 14–16 h. After hybridization, membranes were washed twice in 2× SSC and 0.1% SDS for 10 min. at room temperature, followed by two additional washes for 20 min at 65°C in 1× SSC and 0.1% SDS solution. The membranes were subsequently exposed to Kodak X-Omat AR films at -70°C. The films were scanned using a Personal SI Densitometer (Molecular Dynamics, Sunnyvale, CA). Following exposure, the membranes were stripped with 0.5% SDS and re-probed with β-actin. Densitrometric units were calculated for each sample after normalization of the readings with corresponding densitrometric readings obtained using β-actin cDNA probe. The hybridization experiments on total RNA from the tissues was performed in triplicate for statistical analyses.
Source of mucin cDNA probes
The cDNA probes used in the study were designed to exclude the VNTR regions of the studied MUC genes. This step was deemed essential to avoid the differences in the numbers of tandem repeats commonly associated with mucins among different individuals. The cDNA probes for MUC1 (~500 bp) was kindly provided by Dr. Sandra Gendler [30], MUC2 (~450 bp) was a kind gift from Dr. James Gum [31], MUC5AC (~800 bp) and MUC5B (~340 bp) were generated in the laboratory using specific primers to the published sequences. Earlier, MUC8 cDNA probe (1.4 kb) sequence has been reported from our laboratory [5]. This sequence was used in this study to develop a non-repeat 195 bp MUC8 cDNA probe. A 1.1 kb cDNA probe specific for β-actin was used for the analyses of housekeeping gene.
Reverse Transcription and Polymerase Chain Reaction (RT-PCR)
Five micrograms of total RNA was reverse transcribed to cDNA by 200 units Superscript II reverse transcriptase (Life Technologies). The reaction mixture was then treated with 2U RNaseH at 37°C for 20 min and stored at -20°C. Twenty percent of the first strand cDNA was used for the PCR amplification. Primers and annealing temperatures used for RT-PCR are summarized in Table 1.
Statistical analyses
The data obtained from slot blot analyses of MUC genes in both normal and tumor tissues were subjected to parametric statistical analysis using SAS software (SAS Institute, Cary, NC). Two tailed unequal variance student t test was used to establish statistical significance. A p value of less than 0.05 was considered significant.
Results
Quantitation of mucin gene expression in endometrial tissues
As shown in Fig. 1MUC1 expression was significantly lower in the normal endometrium as compared to the endometrial adenocarcinomas (p = 0.001). Expression of MUC5B followed a similar pattern with higher expression observed in 8 out of 13 endometrial tumor tissues studied over normal tissues (Fig. 2). The differences in MUC5B levels between the cancerous and non-cancerous endometrial samples was significant (p = 0.006). On the other hand, expression of MUC8 was high in all endometrial tissues, with the expression in endometrial adenocarcinomas being significantly higher than the normal endometrium (p = 0.003) (Fig. 3). The box plot analyses showing the relationship between MUC gene expression in endometrial tumor and normal tissues are depicted in Fig. 7. No appreciable expression of MUC2 and MUC5AC in normal and tumor tissues was detected by slot blot analyses.
Quantitation of mucin gene expression in cervical tissues
Levels of expression of MUC1 in cervical carcinomas were significantly different from normal cervical tissue. (p = 0.002) (Fig. 4). While the expression of MUC5B, was higher than MUC1 in normal cervical tissues, no statistical significance was observed in MUC5B levels between normal and cancerous cervical tissues (p = 0.14) (Fig. 5). The expression MUC8 mRNA, were high in all cervical tissues with no statistical difference observed in expression between cancerous and non-cancerous tissues (p = 0.5). Also, box plots depicting MUC gene expression in cervical tumor and normal tissues are illustrated in Fig. 8.
Discussion
This study is primarily focused on the quantitating the expression of five mucin genes, namely, MUC1, MUC2, MUC5AC, MUC5B and MUC8 in normal human endometrial and cervical tissues and respective tumors. Studies in tumors have suggested that mucins are aberrantly expressed in cancerous conditions. In the present investigation we observed that of the five MUC genes studied, MUC1, MUC5B and MUC8 were expressed at higher levels than MUC2 and MUC5AC in endometrial and cervical tissues. Similar studies by other investigators on endocervical tissues have revealed MUCs 1, 4, 5AC, 5B and 6 were expressed at high levels with very low expression of MUCs 2, 3 and 7 [26]. However, a follow-up study to this work quantifying the expression of these MUC genes in normal endocervical epithelium revealed that MUC4 and MUC5B were predominantly expressed as compared to MUC6 and MUC5AC [32]. While acknowledging the variations in the analyzed tissue types, it appears that expression patterns of MUC2 and MUC5AC genes in this study are broadly confirmatory with earlier reports.
Human endometrial epithelium undergoes progesterone-modulated differentiation during menstrual cycle [33,34]. The qualitative and quantitative changes in the secretion of the endometrium are associated with the proliferation of glandular epithelium with increased golgi and secretory apparatus [35]. Accordingly, mucin secretions and MUC gene expression of the female reproductive tissues are dependant on the stage of the menstrual cycle[36]. Earlier studies have shown that MUC1 expression in endometrial tissues is at the highest in early to mid secretory phases [37]. To minimize the ambiguity of elevated MUC gene expression due to menstrual cycle, the tissues studied in the present investigation are from patients in either proliferative or late secretory phases. Mucin gene regulation and expression has been associated with the effects of immune cell derived inflammatory mediators [38] and bacterial endotoxins [39] on the secretory epithelium in various chronic diseases states of human body. However, in this study the pathology reports of the obtained tissues indicate no inflammation or infection thus minimizing the effect of these mediators on overall results of this study.
MUC1, a trans-membrane mucin, has been studied extensively in the female reproductive tract and is expressed in the endometrium. It plays an important role during implantation and maintenance of the embryo [40,41]. Our studies revealed high MUC1 levels in endometrial adenocarcinomas and cervical carcinomas when compared to the normal endometrial and cervical tissues. These results are in accord with studies where MUC1 up-regulation was reported in variety of carcinomas including pancreas[42], breast[43], stomach[44], colon rectum [45] and lung [46].
In addition, to altered gene regulation patterns in cancerous conditions, it is reported that alterations may exist in the carbohydrate structures attached to the protein backbone or in the protein backbone itself. In MUC1, alteration in glycosylation patterns leads to tumor-specific peptide epitopes that are exposed in cancerous cells [47]. In breast cancer tissue, an alternatively spliced form of MUC1, which is completely devoid of repeats was observed [14]. This variant of MUC1 was not expressed in adjacent normal breast tissue. Such studies and others indicate the importance of mucins as tumor markers for diagnostic as well as for therapeutic purposes. For example, in ovarian cancers the CA125 antigen is routinely used to monitor the progress of patients. Recently, investigators found that the CA125 antigen does indeed belong to the mucin family of genes and its core was identified and designated as MUC16 [48].
Our laboratory has previously reported a novel mucin gene MUC8 from human normal tracheal tissue [5] and here we have studied the expression of MUC8 in human normal as well as cancerous endometrial and cervical tissues. Our earlier studies demonstrated that MUC8 is expressed in the male and female reproductive tract [29]. The present work has led us to believe that MUC8 is a major mucin expressed in the female reproductive tract. Levels of MUC8 are significantly higher in the endometrial adenocarcinomas as compared to the normal endometrium. Although the expression of MUC8 was very high in the cervical tissues, no difference in expression was observed among the cancerous and non-cancerous tissues. In the context of MUC gene expression, this is the first study reporting differential expression of MUC8 and MUC5B in cervical and endometrial tissues.
One significant conclusion that can be drawn from this study is that mucin genes, MUC1, MUC5B and MUC8 are all up-regulated in endometrial adenocarcinomas. Furthermore, MUC2 and MUC5AC were found to be expressed at extremely low levels in the endometrial and cervical tissues studied. In conclusion, this study provides significant information on mucin genes in the female reproductive tract and attempts to understand if there are disease-related changes that mucin genes may undergo in endometrial and cervical carcinomas.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
VH and GD carried out gene expression studies and performed statistical analysis. GPS conceived and coordinated the study.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported, in part by NIH grant HL34012. All authors contributed equally towards this work. The authors thank Dr. Donald Harrison, College of Pharmacy, OUHSC, for his help in statistical analysis of the data.
Figures and Tables
Figure 1 Slot blot analyses of total RNA from endometrial normal (EN) and tumors (EA) tissues using mucin MUC1 cDNA probe. Each graph is representative of three experimental replicates. Data normalized using β-actin as described in Methods.
Figure 2 Slot blot analyses of total RNA from endometrial normal (EN) and tumors (EA) tissues using mucin MUC5B cDNA probe. The data was normalized using β-actin as described in Methods section.
Figure 3 Slot blot analyses of total RNA from endometrial normal (EN) and tumors (EA) tissues using mucin MUC8 cDNA probe. The data was normalized using β-actin. Note: Larger scale used, indicative of higher expression of MUC8.
Figure 4 Analyses of total RNA from cervical normal (CN) and tumors (CA) tissues using MUC1 cDNA probe. Data was normalized using β-actin cDNA probe.
Figure 5 Analyses of total RNA from cervical normal (CN) and tumors (CA) tissues using MUC5B cDNA probe. Data was normalized using β-actin as described in Methods section.
Figure 6 Analyses of total RNA from cervical normal (CN) and tumors (CA) using MUC8 cDNA probe. Data was normalized using β-actin as described in Methods section. Note: Larger scale used, indicative of higher expression of MUC8.
Figure 7 Box plots showing the relationship between MUC gene expression in endometrial tumors tissues (EA) and normal tissues (EN). (a) MUC1 (b) MUC5B (c) MUC8.
Figure 8 Box plots showing the relationship between MUC gene expression in cervical tumors tissues (CA) and normal tissues (CN). (a) MUC1 (b) MUC5B (c) MUC8.
Table 1 Primers, annealing temperatures and accession numbers for MUC genes
Gene Acc. # Primers A.temp
MUC5AC AF015521 S-GTGGAACCACGATGACAGC 60°C
AS-TCAGCACATAGCTGCAGTCG
MUC5B Y09788 S-TGCAATCAGCACTGTGACATTGAC 60°C
AS-TTCTCCAGGGTCCAGGTCTCATTC
MUC8 U14383 S-GTTCAGGTCTCCTGCCGG 57°C
AS-CGAGGCGCCATCATGGAC
Acc. # are obtained from GenBank provided by National Center for Biotechnology Information, Bethesda, MD.
Table 2 Tissue classification of endometrial (EA) and cervical (CA) carcinomas.
Tissue Age Grade Differentiation
EA1 44 II wd
EA2 47 II md
EA3 50 I wd
EA4 48 III pd
EA5 38 III pd
EA6 53 I-II md
EA7 45 III wd
EA8 54 I md
EA9 58 II md
EA10 44 III md
EA11 36 I wd
EA12 50 II md
EA13 31 I md
CA1 30 IB md
CA2 35 IB pd
CA3 48 IB pd
CA4 39 IB md
CA5 63 III pd
CA6 46 II wd
CA7 57 II md
CA8 40 IIB pd
CA9 33 I md
CA10 42 I pd
CA11 52 I-II wd
CA12 31 II pd
CA13 47 II md
pd-poorly differentiated, md-moderately differentiated and wd-well differentiated
==== Refs
Rose MC Mucins: structure, function, and role in pulmonary diseases Am J Physiol 1992 263 L413 29 1415719
Gum JRJ Mucin genes and the proteins they encode: structure, diversity, and regulation Am J Respir Cell Mol Biol 1992 7 557 564 1449803
Strous GJ Dekker J Mucin-type glycoproteins Crit Rev Biochem Mol Biol 1992 27 57 92 1727693
Hareuveni M Tsarfaty I Zaretsky J Kotkes P Horev J Zrihan S Weiss M Green S Lathe R Keydar I A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen. cDNA cloning, expression of the transfected gene and over-expression in breast cancer tissue Eur J Biochem 1990 189 475 486 2112460 10.1111/j.1432-1033.1990.tb15512.x
Shankar V Pichan P Eddy RLJ Tonk V Nowak N Sait SN Shows TB Schultz RE Gotway G Elkins RC Gilmore MS Sachdev GP Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus Am J Respir Cell Mol Biol 1997 16 232 241 9070607
Ho SB Shekels LL Toribara NW Kim YS Lyftogt C Cherwitz DL Niehans GA Mucin gene expression in normal, preneoplastic, and neoplastic human gastric epithelium Cancer Res 1995 55 2681 2690 7780985
Reis CA David L Nielsen PA Clausen H Mirgorodskaya K Roepstorff P Sobrinho-Simoes M Immunohistochemical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody Int J Cancer 1997 74 112 121 9036879 10.1002/(SICI)1097-0215(19970220)74:1<112::AID-IJC19>3.0.CO;2-H
Reis CA David L Correa P Carneiro F de Bolos C Garcia E Mandel U Clausen H Sobrinho-Simoes M Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression Cancer Res 1999 59 1003 1007 10070955
Reis CA David L Carvalho F Mandel U de Bolos C Mirgorodskaya E Clausen H Sobrinho-Simoes M Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas J Histochem Cytochem 2000 48 377 388 10681391
Perrais M Pigny P Buisine MP Porchet N Aubert JP Van Seuningen-Lempire I Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter J Biol Chem 2001 276 15386 15396 11278696 10.1074/jbc.M010534200
Lesuffleur T Zweibaum A Real FX Mucins in normal and neoplastic human gastrointestinal tissues Crit Rev Oncol Hematol 1994 17 153 180 7865137
Aksoy N Corfield AP Sheehan JK Preliminary study pointing out a significant alteration in the biochemical composition of MUC2 in colorectal mucinous carcinoma Clin Biochem 2000 33 167 173 10913513 10.1016/S0009-9120(00)00058-8
Williams SJ McGuckin MA Gotley DC Eyre HJ Sutherland GR Antalis TM Two novel mucin genes down-regulated in colorectal cancer identified by differential display Cancer Res 1999 59 4083 4089 10463611
Zrihan-Licht S Vos HL Baruch A Elroy-Stein O Sagiv D Keydar I Hilkens J Wreschner DH Characterization and molecular cloning of a novel MUC1 protein, devoid of tandem repeats, expressed in human breast cancer tissue Eur J Biochem 1994 224 787 795 7925397 10.1111/j.1432-1033.1994.00787.x
Chu JS Chang KJ Mucin expression in mucinous carcinoma and other invasive carcinomas of the breast Cancer Lett 1999 142 121 127 10424790 10.1016/S0304-3835(99)00161-5
Mommers EC Leonhart AM von Mensdorff-Pouilly S Schol DJ Hilgers J Meijer CJ Baak JP van Diest PJ Aberrant expression of MUC1 mucin in ductal hyperplasia and ductal carcinoma In situ of the breast Int J Cancer 1999 84 466 469 10502721 10.1002/(SICI)1097-0215(19991022)84:5<466::AID-IJC3>3.0.CO;2-#
Guillem P Billeret V Buisine MP Flejou JF Lecomte-Houcke M Degand P Aubert JP Triboulet JP Porchet N Mucin gene expression and cell differentiation in human normal, premalignant and malignant esophagus Int J Cancer 2000 88 856 861 11093805 10.1002/1097-0215(20001215)88:6<856::AID-IJC3>3.0.CO;2-D
Yonezawa S Horinouchi M Osako M Kubo M Takao S Arimura Y Nagata K Tanaka S Sakoda K Aikou T Sato E Gene expression of gastric type mucin (MUC5AC) in pancreatic tumors: its relationship with the biological behavior of the tumor Pathol Int 1999 49 45 54 10227724 10.1046/j.1440-1827.1999.00823.x
Masaki Y Oka M Ogura Y Ueno T Nishihara K Tangoku A Takahashi M Yamamoto M Irimura T Sialylated MUC1 mucin expression in normal pancreas, benign pancreatic lesions, and pancreatic ductal adenocarcinoma Hepatogastroenterology 1999 46 2240 2245 10521973
Terris B Dubois S Buisine MP Sauvanet A Ruszniewski P Aubert JP Porchet N Couvelard A Degott C Flejou JF Mucin gene expression in intraductal papillary-mucinous pancreatic tumours and related lesions J Pathol 2002 197 632 637 12210083 10.1002/path.1146
Ohgami A Tsuda T Osaki T Mitsudomi T Morimoto Y Higashi T Yasumoto K MUC1 mucin mRNA expression in stage I lung adenocarcinoma and its association with early recurrence Ann Thorac Surg 1999 67 810 814 10215233 10.1016/S0003-4975(99)00041-7
Sharma PM Sarkar MG Virmani AK Gazdar AF Sachdev GP Evidence of mucin secretion in human lung adenocarcinoma cell lines NCIH650 and NCIH2077 and effect of select secretagogues on mucin secretion Biosci Rep 1999 19 473 483 10763814 10.1023/A:1020224625088
Yu CJ Yang PC Shew JY Hong TM Yang SC Lee YC Lee LN Luh KT Wu CW Mucin mRNA expression in lung adenocarcinoma cell lines and tissues Oncology 1996 53 118 126 8604237
Hough CD Sherman-Baust CA Pizer ES Montz FJ Im DD Rosenshein NB Cho KR Riggins GJ Morin PJ Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer Cancer Res 2000 60 6281 6287 11103784
Feng H Ghazizadeh M Konishi H Araki T Expression of MUC1 and MUC2 mucin gene products in human ovarian carcinomas Jpn J Clin Oncol 2002 32 525 529 12578901 10.1093/jjco/hyf111
Gipson IK Ho SB Spurr-Michaud SJ Tisdale AS Zhan Q Torlakovic E Pudney J Anderson DJ Toribara NW Hill JA Mucin genes expressed by human female reproductive tract epithelia Biol Reprod 1997 56 999 1011 9096884
Audie JP Tetaert D Pigny P Buisine MP Janin A Aubert JP Porchet N Boersma A Mucin gene expression in the human endocervix Hum Reprod 1995 10 98 102 7745080
D'Cruz OJ Wild RA Medders DE Padhye NV Sachdev GP Antigenic similarities between respiratory and reproductive tract mucins: heterogeneity of mucin expression by human endocervix and endometrium Fertil Steril 1993 60 1011 1019 7694876
D'Cruz OJ Dunn TS Pichan P Hass GGJ Sachdev GP Antigenic cross-reactivity of human tracheal mucin with human sperm and trophoblasts correlates with the expression of mucin 8 gene messenger ribonucleic acid in reproductive tract tissues Fertil Steril 1996 66 316 326 8690123
Gendler SJ Lancaster CA Taylor-Papadimitriou J Duhig T Peat N Burchell J Pemberton L Lalani EN Wilson D Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin J Biol Chem 1990 265 15286 15293 1697589
Gum JR Byrd JC Hicks JW Toribara NW Lamport DT Kim YS Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism J Biol Chem 1989 264 6480 6487 2703501
Gipson IK Spurr-Michaud S Moccia R Zhan Q Toribara N Ho SB Gargiulo AR Hill JA MUC4 and MUC5B transcripts are the prevalent mucin messenger ribonucleic acids of the human endocervix Biol Reprod 1999 60 58 64 9858486
Smith RA Seif MW Rogers AW Li TC Dockery P Cooke ID Aplin JD The endometrial cycle: the expression of a secretory component correlated with the luteinizing hormone peak Hum Reprod 1989 4 236 242 2469695
Hoadley ME Seif MW Aplin JD Menstrual-cycle-dependent expression of keratan sulphate in human endometrium Biochem J 1990 266 757 763 1691631
Dockery P Li TC Rogers AW Cooke ID Lenton EA The ultrastructure of the glandular epithelium in the timed endometrial biopsy Hum Reprod 1988 3 826 834 3182973
Aplin JD Glycans as biochemical markers of human endometrial secretory differentiation J Reprod Fertil 1991 92 525 541 1886107
Hey NA Graham RA Seif MW Aplin JD The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase J Clin Endocrinol Metab 1994 78 337 342 8106621 10.1210/jc.78.2.337
Perez-Vilar J Sheehan JK Randell SH Making More MUCS Am J Respir Cell Mol Biol 2003 28 267 270 12594051 10.1165/rcmb.F262
Dohrman A Miyata S Gallup M Li JD Chapelin C Coste A Escudier E Nadel J Basbaum C Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria Biochim Biophys Acta 1998 1406 251 259 9630659
Surveyor GA Gendler SJ Pemberton L Das SK Chakraborty I Julian J Pimental RA Wegner CC Dey SK Carson DD Expression and steroid hormonal control of Muc-1 in the mouse uterus Endocrinology 1995 136 3639 3647 7628404 10.1210/en.136.8.3639
Aplin JD Hey NA Li TC MUC1 as a cell surface and secretory component of endometrial epithelium: reduced levels in recurrent miscarriage Am J Reprod Immunol 1996 35 261 266 8962658
Ueda M Miura Y Kunihiro O Ishikawa T Ichikawa Y Endo I Sekido H Togo S Shimada H MUC1 overexpression is the most reliable marker of invasive carcinoma in intraductal papillary-mucinous tumor (IPMT) Hepatogastroenterology 2005 52 398 403 15816444
Taylor-Papadimitriou J Burchell JM Plunkett T Graham R Correa I Miles D Smith M MUC1 and the immunobiology of cancer J Mammary Gland Biol Neoplasia 2002 7 209 221 12463741 10.1023/A:1020360121451
Wang JY Chang CT Hsieh JS Lee LW Huang TJ Chai CY Lin SR Role of MUC1 and MUC5AC expressions as prognostic indicators in gastric carcinomas J Surg Oncol 2003 83 253 260 12884239 10.1002/jso.10222
Jang KT Chae SW Sohn JH Park HR Shin HS Coexpression of MUC1 with p53 or MUC2 correlates with lymph node metastasis in colorectal carcinomas J Korean Med Sci 2002 17 29 33 11850585
Nguyen PL Niehans GA Cherwitz DL Kim YS Ho SB Membrane-bound (MUC1) and secretory (MUC2, MUC3, and MUC4) mucin gene expression in human lung cancer Tumour Biol 1996 17 176 192 8638091
Layton GT Devine PL Warren JA Birrell G Xing PX Ward BG McKenzie IF Monoclonal antibodies reactive with the breast carcinoma-associated mucin core protein repeat sequence peptide also recognise the ovarian carcinoma-associated sebaceous gland antigen Tumour Biol 1990 11 274 286 1697426
Yin BW Lloyd KO Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16 J Biol Chem 2001 276 27371 27375 11369781 10.1074/jbc.M103554200
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-6-331615938710.1186/1471-2121-6-33Research ArticleAssociation of the Hermansky-Pudlak syndrome type-3 protein with clathrin Helip-Wooley Amanda [email protected] Wendy [email protected] Heidi [email protected] Mieke [email protected] Raymond E [email protected] William A [email protected] Marjan [email protected] Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda MD, USA2 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands3 Department of Dermatology, University of Cincinnati College of Medicine, OH, USA2005 13 9 2005 6 33 33 24 5 2005 13 9 2005 Copyright © 2005 Helip-Wooley et al; licensee BioMed Central Ltd.2005Helip-Wooley 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
Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172–176.
Results
Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20°C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution.
Conclusion
HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking.
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Background
Hermansky-Pudlak syndrome (HPS [MIM: 203300]) is an autosomal recessive disorder of vesicle biogenesis resulting in the dysfunction of lysosome-related organelles such as melanosomes and platelet dense bodies [1-4]. Affected patients have oculocutaneous albinism presenting as congenital nystagmus, reduced visual acuity, and varying degrees of hypopigmentation of the skin, hair, and irides [5-7]. In addition, a platelet storage pool deficiency, manifesting as absence of platelet dense bodies, causes loss of the secondary aggregation response [2,8,9]. Clinically, this results in easy bruising and epistaxis in childhood, prolonged bleeding during dental extractions and surgeries, and excessive menstrual and postpartum bleeding [9]. Some HPS patients also develop granulomatous colitis or a fatal pulmonary fibrosis [9,10].
To date, seven genes have been identified as causes of human HPS subtypes (HPS-1 through HPS-7), and other genes identified in mouse models of HPS may also cause HPS in humans [4,11]. Of the human subtypes, only HPS-2 ([MIM: 603401]) results from mutations in a gene (AP3B1) with a known function. AP3B1 codes for the β3A subunit of adaptor complex-3 (AP-3), a coat protein that is involved in sorting transmembrane proteins to lysosomes and lysosome-related organelles [12-15]. This recognized function of AP-3 supports the paradigm that all types of HPS result from abnormal vesicle formation and/or trafficking.
Each of the gene products of HPS1 ([MIM: 604982; [16,17]]), HPS3 ([MIM: 606118; [18,19]]), HPS4 ([MIM: 606682; [20,21]]), HPS5 ([MIM: 607521; [22,23]]), HPS6 ([MIM: 607522; [22]]), and HPS7 ([MIM: 607145; [24]]) is unique, although some HPS proteins interact with each other in Biogenesis of Lysosome-related Organelles Complexes or BLOCs [22,24-26]. The fact that these proteins have no homology to any known proteins, to each other, or to known functional domains makes them challenging candidates to investigate using in vitro methods.
In an attempt to understand the function of HPS gene products, we focused on HPS3, a unique protein with a predicted clathrin-binding domain. HPS-3 patients, with mutations in the HPS3 gene, have absent platelet dense bodies, mild to moderate hypopigmentation of skin and hair, iris transillumination, and patchy hypopigmentation of the fundus [19]. The mouse ortholog of human HPS-3, cocoa, demonstrates similar features [27,28]. The HPS3 gene was identified by homozygosity mapping using HPS patients from a central Puerto Rican genetic isolate [18]. HPS3 is located on chromosome 3q24 and has 17 exons and a 3,015-bp open reading frame that codes for a 1004-amino acid protein. The central Puerto Rican founder mutation in HPS3 is a 3.9-kb deletion encompassing exon 1 and its surrounding introns [18]. The central Puerto Rican HPS population is distinct from the HPS isolate in northwest Puerto Rico, in which a founder mutation in the HPS1 gene results in a severe form of HPS [16]. Several non-Puerto Rican HPS3 mutations, as well as an Ashkenazi Jewish founder mutation in HPS3, have now been identified [19].
The human HPS3 protein contains a predicted clathrin-binding motif (LLDFE) at residues 172–176 that conforms to the consensus sequence L(L, I)(D, E, N)(L, F)(D, E). This consensus was determined by amino acid sequence alignment of the clathrin-binding regions in the beta subunits of adaptor proteins 1, 2, and 3 (β1, β2, β3A, and β3B), arrestin 3, and amphiphysin I and II [29]. This 'clathrin box' is sufficient for binding to the amino terminal domain of the clathrin heavy chain [29]. The structures of peptide complexes containing the clathrin-terminal domain and the clathrin-binding motifs of β-arrestin 2 and β3A have been determined by crystallography [30]. Both of these peptides bind, via their clathrin box motifs, to the same site in the clathrin heavy chain's terminal domain, with nearly identical bound conformations.
Clathrin is the main component of protein coats that assist in the formation of vesicles budding from the trans-Golgi network (TGN), plasma membrane, and endosomes. Several clathrin-associated proteins regulate the assembly of clathrin triskelions, composed of three heavy chains and three light chains, into the polyhedral cages that provide structure to intracellular vesicles. Some of these proteins and protein complexes are also involved in sorting cargo into vesicles and directing their transport within the cell [31,32].
HPS-2 disease results from deficiency of the clathrin binding protein β3A [12-15], supporting a possible role for other HPS proteins in clathrin binding. Hence, we investigated the clathrin binding function of the HPS3 protein in melanocytes and fibroblasts.
Results
Several avenues of investigation were pursued to substantiate the predicted clathrin-binding activity of HPS3. These studies employed the techniques of immunoprecipitation, immunofluorescence, live cell imaging, and immunoelectron microscopy.
Immunoprecipitation
Whole cell lysates prepared from normal melanocytes and from HPS3 null melanocytes, i.e., cells from a patient homozygous for the 3.9-kb HPS3 founder deletion [18], exhibited approximately equal amounts of clathrin (Figure 1a, left panel). Each whole cell lysate was then immunoprecipitated with polyclonal HPS3 peptide antibodies. Clathrin, detected by antibodies to its heavy chain, co-immunoprecipitated with HPS3 in the normal lysates but not in the HPS3 null lysates (Figure 1a, right panel). These results were obtained using either of two different monoclonal anti-clathrin heavy chain antibodies.
Figure 1 Association of HPS3 with clathrin. (a) Immunoprecipitation of clathrin with HPS3 antibodies. Western blots of normal (NL) and HPS3-null (HPS3) melanocyte lysates treated with clathrin heavy chain antibodies. Equal amounts of clathrin were detected in both lysates [left panel]. The lysates were immunoprecipitated with HPS3 antibodies, electrophoresed, and immunoblotted using clathrin heavy chain antibodies. HPS3 antibodies immunoprecipitated clathrin only in the normal and not in the HPS3-null melanocyte lysates [right panel]. (b) Amino acid sequences of the predicted clathrin-binding domain (residues 172–176 of human HPS3, shown in blue) in the human, mouse and rat HPS3 proteins, and the surrounding amino acid sequences. (c) Confocal immunofluorescence microscopy of representative normal melanocytes electroporated with GFP-HPS3 [A] and GFP-HPS3-delCBD [B] (in green) and co-stained with antibodies to clathrin (in red). GFP-HPS3 partially co-localized with clathrin on small vesicles (arrowheads) [A], but GFP-HPS3-delCBD did not [B]. (Bar = 20 μm).
Immunofluorescence
At residues 172–176, the HPS3 protein has a predicted clathrin-binding motif (LLDFE) that is conserved in mouse and rat (Figure 1b). Using site-directed mutagenesis, a GFP-HPS3-delCBD construct was created in which the clathrin-binding motif was converted to non-conserved amino acids (AAAPG). The interaction of clathrin-binding proteins with clathrin is a transient phenomena [31,33,34]. To maximize the likelihood of observing the association of GFP-HPS3 with clathrin by immunofluorescence, a 20°C temperature block was employed. Incubation at 20°C for 2 h was used to block trafficking out of the trans-Golgi [35,36] and followed by transfer to 37°C for 5 min to release the temperature block and resume normal trafficking. Normal melanocytes expressing wild-type GFP-HPS3 or GFP-HPS3-delCBD, so treated, were then fixed and stained with clathrin heavy chain antibodies. Cells expressing GFP-HPS3 demonstrated partial co-localization with clathrin on one to several small vesicles in the perinuclear area (representative cell, Figure 1c[A]). In a representative experiment, 9 of 11 GFP-HPS3 expressing melanocytes showed this co-localization. In contrast, 0 of 10 cells exhibited co-localization of GFP-HPS3-delCBD with clathrin (Figure 1c[B]; p < 0.001 by Chi-square analysis).
Live cell imaging
Trafficking of acidic vesicles, stained with Lysotracker Red, was followed in live HPS3 null fibroblasts expressing either GFP-HPS3 (Figure 2a[A]) or GFP-HPS3-delCBD (Figure 2a[B]). Following a 20°C temperature block and transfer to 37°C, acidic vesicles and GFP-HPS3 clustered together in the perinuclear area (Figure 2b[A]). GFP-HPS3 transiently interacted with acidic vesicles and, in some cases (arrows, Figure 2b), emerged together with them from the perinuclear area (Figure 2b and [Additional file 1]). In contrast, following a 20°C block and transfer to 37°C, GFP-HPS3-delCBD was predominantly localized in a punctate pattern in the periphery of the cell; no association of GFP-HPS3-delCBD with acidic vesicles was observed (Figure 2a[B]).
Figure 2 Live cell imaging of HPS3 and acidic vesicles. (a) Lysotracker Red stained acidic vesicle trafficking in live HPS3 null fibroblasts expressing GFP-HPS3 [A] or GFP-HPS3-delCBD [B]. Cells were imaged on a 37°C warming stage following a 2 h incubation at 20°C. Lysotracker Red stained acidic vesicles and GFP-HPS3 transiently co-localized in the perinuclear area [A]. Inset shows the location of the time series shown in (b). GFP-HPS3-delCBD did not localize predominantly to the perinuclear area and showed no co-localization with Lysotracker Red [B]. (b) Time series showing co-trafficking of GFP-HPS3 and Lysotracker Red stained vesicles from the perinuclear area in HPS3 null fibroblasts. Note transient association of GFP-HPS3 with an acidic vesicle as it travels peripherally over a period of time (see [additional file 1]).
Immunoelectron microscopy
Normal melanocytes were fixed approximately 9 h after transfection with GFP-HPS3. Expression of the GFP-HPS3 fusion protein was low at this time, thus minimizing aggregation and other artifacts that can occasionally be observed in overexpressing cells. Normal melanocytes transfected with GFP-HPS3-delCBD were fixed later (approximately 24 h after transfection) because of the very low levels of expression obtained with this construct. No aggregates were observed in the GFP-HPS3-delCBD transfected cells.
GFP-HPS3 localized predominantly to small (50–100 nm) vesicles in the Golgi region (57 of 111 (51%) anti-GFP immunogold particles; Table 1) and the vast majority of these particles (53 of 57 (93%)) co-localized with clathrin (Figure 3, arrowheads; Table 1). At high magnification, GFP-HPS3 labeling was demonstrated on well-defined clathrin-containing vesicles (Figure 3[B,D,E]). Some of these small clathrin-containing vesicles were found near larger endosomal structures (Figure 3[B,D]). GFP-HPS3 labeling was generally less abundant on large endosomal structures than on small vesicles (23 of 111 (21%) of anti-GFP immunogold particles found on endosomes) and fewer of these particles co-localized with clathrin (15 of 23 (65%); Figure 3[B,D], Table 1).
Table 1 Quantitation of immunogold label in normal melanocytes transfected with GFP-HPS3 or GFP-HPS3-delCBD1
Gold Particles Clathrin Association
Intracellular compartment GFP-HPS3 GFP-HPS3-delCBD GFP-HPS3 GFP-HPS3-delCBD
Small vesicles in Golgi area 2,3 57 8 53 1
Golgi stacks 1 1 0 0
Other small vesicles 15 16 9 3
Endosomal structures 3 23 17 15 1
Mitochondria 2 2 0 0
Melanosomes 3 2 0 0
Nucleus 2 1 0 0
Cytoplasm 2 8 63 0 3
Total 3 111 110 77 8
1 Anti-GFP immunogold particles were counted in a total of 28 GFP-HPS3 and 25 GFP-HPS3-delCBD expressing cells
2 Difference in number of gold particles per compartment, for GFP-HPS3-delCBD compared with GFP-HPS3, is statistically significant by Chi square analysis (p < 0.001)
3 Difference in number of gold particles found in association with clathrin, for GFP-HPS3-delCBD compared with GFP-HPS3, is statistically significant by Chi square analysis (p < 0.001)
Figure 3 Immunoelectron micrographs demonstrating co-localization of GFP-HPS3 and clathrin. Double-labeling of anti-GFP (10-nm gold) and anti-clathrin (20-nm gold) [A, B], or the reverse labeled anti-clathrin (10-nm gold) and anti-GFP (15-nm gold) [C, D, E] in normal melanocytes electroporated with GFP-HPS3. Co-localization of the two labels was shown on small (50–100 nm) clathrin-containing vesicles (arrowheads) in the Golgi region [A and C]. Co-localization was observed on small clathrin-labeled vesicles (arrowheads) but not on neighboring large endosomal structures [B and D]. High magnification of a small clathrin containing vesicle labeled with GFP-HPS3 [E]. CM = Cell Membrane, G = Golgi area, E = Endosomal structure. Bar = 100 nm
In contrast, GFP-HPS3-delCBD expressing cells demonstrated very few anti-GFP immunogold particles on small vesicles in the Golgi region (8 of 110 (7%)) and these were only rarely found in association with clathrin (1 of 8 (13%); Figure 4, Table 1). The majority of anti-GFP immunogold particles localized to the cytoplasm of GFP-HPS3-delCBD expressing cells (63 of 110 (57%)), compared with only 7% (8 of 111) in GFP-HPS3 expressing cells (Figures 3 and 4, Table 1). These differences were statistically significant by Chi square analysis (p < 0.001; Table 1).
Figure 4 Immunoelectron micrographs demonstrating no co-localization of GFP-HPS3-delCBD and clathrin.
Double-labeling of anti-clathrin (10-nm gold) and anti-GFP (15-nm gold) in normal melanocytes electroporated with GFP-HPS3-delCBD [A-D]. GFP-HPS3-delCBD (arrowheads) was largely cytoplasmic and distributed throughout the entire cell, from the perinuclear/Golgi region [A,B,D] to the tips [C]. No co-localization of the two labels (GFP-HPS3-delCBD and clathrin) was observed. N=Nucleus, G=Golgi, E=Endosomal structure, M=Melanosome. Bar = 500 nm
No appreciable GFP-HPS3 or GFP-HPS3-delCBD labeling was observed on clearly identifiable Golgi stacks (Figure 3[A,C], Figure 4[D], Table 1). As expected, clathrin labeling appeared much more abundant than GFP labeling in GFP-HPS3 or GFP-HPS3-delCBD expressing melanocytes. Similar amounts of GFP labeling per cell were detected in GFP-HPS3 and GFP-HPS3-delCBD expressing cells and no GFP labeling was observed in untransfected cells. Not all clathrin-containing membranes were associated with GFP-HPS3, but the majority of GFP-HPS3 appeared to be associated with a clathrin-containing membrane.
Discussion
HPS3 is unusual among HPS proteins in that it contains a predicted functional domain, i.e., a clathrin-binding motif (LLDFE) (Figure 1b). In this report, we describe evidence for the association of HPS3 with clathrin. We demonstrated that clathrin co-immunoprecipitates with endogenous HPS3 in normal melanocytes; no clathrin was immunoprecipitated in the absence of HPS3, i.e., in HPS3 null cells. The necessity of the clathrin-binding domain in HPS3 is supported by the partial co-localization of GFP-HPS3 with clathrin only when this domain is present, as demonstrated by both fluorescence and immunoelectron microscopy studies. Furthermore, localization and trafficking of GFP-HPS3 with acidic vesicles (labeled with Lysotracker Red) depends upon an intact clathrin-binding domain.
The presence of a conserved clathrin-binding motif in HPS3, combined with the supportive data mentioned above, indicates that HPS3 most likely binds clathrin directly. We cannot, however, rule out the possibility that HPS3 interacts indirectly with clathrin. Recent studies in mouse [37] and human [38] have shown that HPS3 (cocoa mouse) interacts with HPS5 (ruby-eye-2 mouse) and HPS6 (ruby-eye mouse) in the BLOC-2 complex. Hence, HPS3 may function as an essential component of a complex, such as BLOC-2, in which another member binds clathrin. This seems unlikely, however, since HPS5 and HPS6 have no conserved clathrin-binding sequence motifs.
In our ultrastructural studies, GFP-HPS3 was found primarily on small (50 to 100 nm) clathrin containing vesicles in the perinuclear/Golgi region of normal melanocytes. Mutation of the clathrin-binding domain of HPS3 resulted in a largely cytoplasmic distribution of the fusion protein, suggesting that the clathrin-binding domain is necessary for the correct localization of HPS3 and that clathrin recruits HPS3 to small vesicles. Interestingly, no GFP-HPS3 was seen on Golgi stacks or on vesicles budding from the TGN, nor was it localized to large endosomal structures or mature melanosomes. In melanocytes from HPS3 deficient patients, DOPA histochemistry (used to identify extra-melanosomal sites of functional tyrosinase) detected 50 nm DOPA positive vesicles distributed throughout the cell [39]. This was in contrast to the situation in normal melanocytes, in which the small DOPA-positive vesicles were restricted to the Golgi region. A possible explanation is that HPS3 (and perhaps BLOC-2 as a whole) interacts with the small DOPA-positive vesicles via its clathrin-binding domain, escorting them from the Golgi region to premelanosomes for delivery of their contents. In such a scenario, additional specialized accessory factors (perhaps other HPS proteins or BLOCs) would regulate vesicle targeting, budding and fusion events, clathrin coat assembly and disassembly, and interactions with the cytoskeleton.
Future investigations should pursue the function of BLOC-2 with the recognition that one of its components, HPS3, binds clathrin and may, therefore, bind to vesicles. This understanding allows for hypotheses regarding the roles of HPS5 and HPS6 in BLOC-2. Possible roles could include such functions as binding designated cargo, regulating conformational changes of the complex, or tethering proteins or vesicles for interactions with the BLOC-2 complex as a whole.
Conclusion
HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3 and is supported by immunoprecipitation, immunofluorescence, live cell imaging and immunoelectron microscopy data.
Methods
Patients and cells
Normal human primary epidermal melanocytes used for immunoelectron microscopy were obtained from neonatal foreskin and established as described [40,41]. HPS-3 patient (HPS3 null) primary epidermal melanocytes and primary fibroblast cultures were obtained from skin biopsies and cultured as previously described [14]. All other normal human primary epidermal melanocytes were purchased from Cascade Biologics (Portland, OR). The HPS-3 patients were enrolled in a protocol approved by the National Human Genome Research Institute Institutional Review Board to study the clinical and molecular aspects of HPS. Written informed consent was obtained from the patient or the patient's parent.
Immunoprecipitation and western blotting
Normal and HPS3 null melanocytes cell pellets were resuspended in PBS containing 1% NP-40 and protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). The cell lysates were cleared by centrifugation and the resulting supernatants were incubated with HPS3 polyclonal peptide antibody (developed in rabbit against the peptide KMGDLDMHRNEMKSHS) followed by incubation with protein A/G agarose beads (Oncogene Research Products, San Diego, CA). The agarose beads were washed, boiled in SDS-loading buffer and centrifuged. The resulting supernatants were electrophoresed on 4–12% SDS-PAGE gels (Invitrogen, Carlsbad, CA) and electro-blotted onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The membranes were blocked and incubated with mouse monoclonal anti-clathrin antibodies (1:1000) (BD Biosciences Pharmingen (San Diego, CA) or Affinity BioReagents (Golden, CO), followed by incubation with HRP-conjugated anti-mouse IgG secondary antibodies (1:3000; Amersham Biosciences, Piscataway, NJ). Results were visualized with enhanced chemiluminescence (ECL Western Blotting Detection Reagents, Amersham Biosciences, Piscataway, NJ) and exposure to CL-XPosure film (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions.
GFP-HPS3 plasmid constructs
The HPS3 coding sequence was amplified from normal human cDNA [GenBank: NM_032383] with sequence specific primers and subcloned into pEGFP-C1 (Clontech, Palo Alto, CA). Site-directed mutagenesis to replace the clathrin-binding motif (LLDFE) at residues 172–176 with the non-conserved amino acids AAAPG was performed with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's recommendations, using the forward primer 5'-AATGAGGAATTCTCAGCAGCGGCCCCTGGACGTTCTTTAATTATAC-3' and its reverse complement. All constructs were verified by sequencing before use.
Transfections
All transfections were performed by electroporation in an Amaxa nucleofector electroporator (Amaxa GmbH, Germany). Electroporation of melanocytes was performed as described [42]. Fibroblasts were electroporated using Amaxa reagents and 3 μg of plasmid DNA with the U_23 nucleofector program.
Fluorescence microscopy
Approximately 16 h after transfection with either GFP-HPS3 or GFP-HPS3-delCBD normal melanocytes were incubated at 20°C for 2 h to block trafficking out of the trans-Golgi [35,36]. Cells were transferred to 37°C for 5 min to release the temperature block and resume normal trafficking then fixed in 3% paraformaldehyde. Melanocytes allowed to express GFP-HPS3 or GFP-HPS3-delCBD for longer than 24 h demonstrated some GFP aggregates and decreased viability. Slides were blocked in PBS containing 0.1% saponin, 100 μM glycine, 0.1% BSA and 2% donkey serum followed by incubation with mouse monoclonal clathrin heavy chain antibodies (1:200 dilution; BD Biosciences Pharmingen). The cells were washed and incubated with donkey anti-mouse antibodies conjugated to ALEXA-555 (Molecular Probes), washed again, and mounted in VectaShield (Vector Laboratories, Burlingame, CA).
Live cell imaging was performed on HPS3 null fibroblasts expressing either GFP-HPS3 or GFP-HPS3-delCBD approximately 16 h after transfection. Cells were incubated at 20°C for 2 h followed by a 10 min incubation with the acidic vesicle dye Lysotracker Red (10 nM; Molecular Probes). The cells were then placed in fresh, pre-warmed culture media and imaged on a 37°C warming stage. All cells were imaged with a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Microimaging Inc., Germany) using a 488 Argon and a 543 HeNe laser. Images were acquired using either a Plan Apochromat 63X/1.4 oil DIC or a 100× Plan Apochromat 100X/1.4 oil DIC objective.
Immunoelectron microscopy
Normal melanocytes were fixed approximately 9 h or 24 h after transfection in 2% paraformaldehyde with 0.2% glutaraldehyde in PHEM buffer for 2 h. After embedding, cutting, cryoprotection and snap-freezing of the pellet, ultrathin cryosections were labeled with mouse monoclonal antibodies to clathrin (1:100) (BD Biosciences Pharmingen). The mouse monoclonal antibodies were indirectly labeled with 20-nm protein A-gold particles via a rabbit anti-mouse IgG bridging antibody (1:200) (DakoCytomation, Denmark). The second labeling was performed with a rabbit polyclonal anti-GFP antibody (1:1000) [43], followed by 10-nm protein A-gold incubation. To exclude co-labeling artifacts, ultrathin cryosections were labeled with primary antibodies as above, but incubated with 10-nm protein A-gold in the first labeling and 15-nm protein A-gold in the second labeling. The grids were contrasted with uranyl acetate and imaged with a Philips EM 410 electron microscope (Philips, Eindhoven, The Netherlands). Quantitation of anti-GFP immunogold labeling of cellular compartments and its association with clathrin (anti-clathrin immunogold labeling) was performed on randomly selected cells expressing either GFP-HPS3 (28 cells) or GFP-HPS3-delCBD (25 cells) [44].
List of abbreviations
HPS, Hermansky-Pudlak syndrome; BLOC, biogenesis of lysosome-related organelles complexes; GFP, green fluorescent protein; CBD, clathrin-binding domain; TGN, trans-Golgi network; DOPA, dihydroxyphenylalanine; PBS, phosphate buffered saline; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PHEM, PIPES, HEPES, EGTA, MgCl2
Authors' contributions
AHW performed the immunoprecipitations. AHW and MH prepared the plasmid constructs. AHW and HD carried out the cell culture, transfection experiments, immunofluorescence and live cell confocal imaging. WAG recruited the HPS-3 patient and established the fibroblast cultures. RB established the HPS-3 patient melanocyte cultures. Immunoelectron microscopy and quantitation was performed by WW and MM. WAG, MH, WW and AHW prepared the manuscript.
Supplementary Material
Additional file 1
Time series of GFP-HPS3 and acidic vesicle live cell imaging. GFP-HPS3 expressing HPS3 null fibroblast was imaged at 37°C following a 2 h incubation at 20°C. Time series (from inset in Figure 2a[A] and Figure 2b) showing a transient association of GFP-HPS3 with Lysotracker Red stained vesicles as they exit the perinuclear area.
Click here for file
Acknowledgements
We thank Prof. J. Fransen (Nijmegen, The Netherlands) for providing the polyclonal anti-GFP antibody, Prof. J. Klumperman (Utrecht, The Netherlands) for providing the monoclonal anti-clathrin antibodies, Prof. J. Lambert (Gent, Belgium) for providing normal human melanocytes, J. Onderwater and R. Limpens for assisting with electron microscopy and L. Verschragen for preparation of electron micrographs. This research was supported in part by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health.
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Hermansky F Pudlak P Albinism associated with hemorrhagic diathesis and unusual pigment reticular cells in the bone marrow: report of two cases with histochemical studies Blood 1959 14 162 169 13618373
King RA Hearing VJ Creel DJ Oetting WS Scriver CR, Beaudet AL, Valle DL, Sly WS Albinism The Metabolic and Molecular Bases of Inherited Disease 2001 4 8 New York: McGraw-Hill 5587 5627
Huizing M Anikster Y Gahl WA Hermansky-Pudlak syndrome and related disorders of organelle formation Traffic 2000 1 823 835 11208073 10.1034/j.1600-0854.2000.011103.x
Huizing M Boissy RE Gahl WA Hermansky-Pudlak syndrome: Vesicle formation from yeast to man Pigment Cell Res 2002 15 405 419 12453182 10.1034/j.1600-0749.2002.02074.x
Simon JW Adams RJ Calhoun JH Shapiro SS Ingerman CM Ophthalmic manifestations of the Hermansky-Pudlak syndrome (oculocutaneous albinism and hemorrhagic diathesis) Am J Ophthalmol 1982 93 71 77 7065089
Summers CG Knobloch WH Witkop CJ King RA Hermansky-Pudlak syndrome. Ophthalmic findings Ophthalmology 1988 95 545 554 3174014
Iwata F Reed GF Caruso RC Kuehl EM Gahl WA Kaiser-Kupfer MI Correlation of visual acuity and ocular pigmentation with the 16-bp duplication in the HPS-1 gene of Hermansky-Pudlak syndrome, a form of albinism Ophthalmology 2000 107 783 789 10768343 10.1016/S0161-6420(99)00150-5
Witkop CJ Krumwiede M Sedano H White JG Reliability of absent platelet dense bodies as a diagnostic criterion for Hermansky-Pudlak syndrome Am J Hematol 1987 26 305 311 3120578
Gahl WA Brantly M Kaiser-Kupfer MI Iwata F Hazelwood S Shotelersuk V Duffy LF Kuehl EM Troendle J Bernardini I Genetic defects and clinical characteristics of patients with a form of oculocutaneous albinism (Hermansky-Pudlak syndrome) N Engl J Med 1998 338 1258 1264 9562579 10.1056/NEJM199804303381803
Brantly M Avila NA Shotelersuk V Lucero C Huizing M Gahl WA Pulmonary function and high-resolution CT findings in patients with an inherited form of pulmonary fibrosis, Hermansky-Pudlak syndrome, due to mutations in HPS-1 Chest 2000 117 129 336 10631210 10.1378/chest.117.1.129
Swank RT Novak EK McGarry MP Rusiniak ME Feng L Mouse models of Hermansky-Pudlak syndrome: a review Pigment Cell Res 1998 11 60 80 9585243
Simpson F Peden AA Christopoulou L Robinson MS Characterization of the adaptor-related protein complex, AP-3 J Cell Biol 1997 137 835 845 9151686 10.1083/jcb.137.4.835
Dell'Angelica EC Shotelersuk V Aguilar RC Gahl WA Bonifacino JS Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta-3A subunit of the AP-3 adaptor Mol Cell 1999 3 11 21 10024875 10.1016/S1097-2765(00)80170-7
Huizing M Sarangarajan R Strovel E Zhao Y Gahl WA Boissy RE AP-3 mediates tyrosinase but not TRP-1 trafficking in human melanocytes Mol Biol Cell 2001 12 2075 2085 11452004
Clark RH Stinchcombe JC Day A Blott E Booth S Bossi G Hamblin T Davies EG Griffiths GM Adaptor protein 3-dependent microtubule-mediated movement of lytic granules to the immunological synapse Nature Immunol 2003 4 1111 1120 14566336 10.1038/ni1000
Oh J Bailin T Fukai K Feng GH Ho L Mao JI Frenk E Tamura N Spritz RA Positional cloning of a gene for Hermansky-Pudlak syndrome, a disorder of cytoplasmic organelles Nature Genet 1996 14 300 306 8896559 10.1038/ng1196-300
Dell'Angelica EC Aguilar RC Wolins N Hazelwood S Gahl WA Bonifacino JS Molecular characterization of the protein encoded by the Hermansky-Pudlak syndrome type 1 gene J Biol Chem 2000 275 1300 1306 10625677 10.1074/jbc.275.2.1300
Anikster Y Huizing M White J Shevchenko YO Fitzpatrick DL Touchman JW Compton JG Bale SJ Swank RT Gahl WA Toro JR Mutation of a new gene causes a unique form of Hermansky-Pudlak syndrome in a genetic isolate of central Puerto Rico Nature Genet 2001 28 376 380 11455388 10.1038/ng576
Huizing M Anikster Y Fitzpatrick DL Jeong AB D'Souza M Rausche M Toro JR Kaiser-Kupfer MI White JG Gahl WA Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency Am J Hum Genet 2001 69 1022 1032 11590544 10.1086/324168
Suzuki T Li W Zhang Q Karim A Novak EK Sviderskaya EV Hill SP Bennett DC Levin AV Nieuwenhuis HK Fong CT Castellan C Miterski B Swank RT Spritz RA Hermansky-Pudlak syndrome is caused by mutations in HPS4, the human homolog of the mouse light-ear gene Nature Genet 2002 30 321 324 11836498
Anderson PD Huizing M Claassen DA White J Gahl WA Hermansky-Pudlak syndrome type 4 (HPS-4): clinical and molecular characteristics Hum Genet 2003 113 10 17 12664304
Zhang Q Zhao B Li W Oiso N Novak EK Rusiniak ME Gautam R Chintala S O'Brien EP Zhang Y Roe BA Elliott RW Eicher EM Liang P Kratz C Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6 Nature Genet 2003 33 145 153 12548288 10.1038/ng1087
Huizing M Hess R Dorward H Claassen DA Helip-Wooley A Kleta R Kaiser-Kupfer MI White JG Gahl WA Cellular, molecular and clinical characterization of patients with Hermansky-Pudlak syndrome type 5 Traffic 2004 5 711 722 15296495 10.1111/j.1600-0854.2004.00208.x
Li W Zhang Q Oiso N Novak EK Gautam R O'Brien EP Tinsley CL Blake DJ Spritz RA Copeland NG Jenkins NA Amato D Roe BA Starcevic M Dell'Angelica EC Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) Nature Genet 2003 35 84 89 12923531 10.1038/ng1229
Martina JA Moriyama K Bonifacino JS BLOC-3, a protein complex containing the Hermansky-Pudlak syndrome gene products HPS1 and HPS4 J Biol Chem 2003 278 29376 29384 12756248 10.1074/jbc.M301294200
Nazarian R Falcon-Perez JM Dell'Angelica EC Biogenesis of lysosome-related organelles complex 3 (BLOC-3): A complex containing the Hermansky-Pudlak syndrome (HPS) proteins HPS1 and HPS4 Proc Natl Acad Sci USA 2003 100 8770 8775 12847290 10.1073/pnas.1532040100
Novak EK Sweet HO Prochazka M Parentis M Soble R Reddington M Cairo A Swank RT Cocoa: a new mouse model for platelet storage pool deficiency Br J Haematol 1988 69 371 378 3408670
Suzuki T Li W Zhang Q Novak EK Sviderskaya EV Wilson A Bennett DC Roe BA Swank RT Spritz RA The gene mutated in cocoa mice, carrying adefect of organelle biogenesis, is a homologue of the human Hermansky-Pudlak syndrome-3 gene Genomics 2001 78 30 37 11707070 10.1006/geno.2001.6644
Dell'Angelica EC Klumperman J Stoorvogel W Bonifacino JS Association of the AP-3 adaptor complex with clathrin Science 1998 280 431 434 9545220 10.1126/science.280.5362.431
ter Haar E Harrison SC Kirchhausen T Peptide-in-groove interactions link target proteins to the b-propeller of clathrin Proc Natl Acad Sci USA 2000 97 1096 1100 10655490 10.1073/pnas.97.3.1096
Kirchhausen T Clathrin Annu Rev Biochem 2000 69 699 727 10966473 10.1146/annurev.biochem.69.1.699
Dell'Angelica EC Clathrin-binding proteins: got a motif? Join the network! Trends Cell Biol 2001 11 315 318 11489622 10.1016/S0962-8924(01)02043-8
Rappoport J Simon S Benmerah A Understanding living clathrin-coated pits Traffic 2004 5 327 337 15086782 10.1111/j.1398-9219.2004.00187.x
Ehrlich M Boll W Van Oijen A Hariharan R Chandran K Nibert M Kirchhausen T Endocytosis by random initiation and stabilization of clathrin-coated pits Cell 2004 118 591 605 15339664 10.1016/j.cell.2004.08.017
Matlin KS Simons K Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation Cell 1983 34 233 243 6883510 10.1016/0092-8674(83)90154-X
Griffiths G Pfeiffer S Simons K Matlin K Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane J Cell Biol 1985 101 949 964 2863275 10.1083/jcb.101.3.949
Gautam R Chintala S Li W Zhang Q Tan J Novak EK Di Pietro SM Dell'Angelica EC Swank RT The Hermansky-Pudlak syndrome 3 (cocoa) protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2) J Biol Chem 2004 279 12935 12942 14718540 10.1074/jbc.M311311200
Di Pietro SM Falcon-Perez JM Dell'Angelica EC Characterization of BLOC-2, a complex containing the Hermansky-Pudlak syndrome proteins HPS3, HPS5 and HPS6 Traffic 2004 5 276 283 15030569 10.1111/j.1600-0854.2004.0171.x
Boissy R Richmond B Huizing M Helip-Wooley A Zhao Y Koshoffer A Gahl W Melanocyte-specific proteins are aberrantly trafficked in melanocytes of Hermansky-Pudlak syndrome-type 3 Am J Pathol 2005 165 231 240 15632015
Naeyaert JM Eller M Gordon PR Park HV Gilchrest BA Pigment content of cultured human melanocytes does not correlate with tyrosinase message level Br J Dermatol 1991 125 297 303 1720016
Smit NP Kolb RM Lentjes EG Noz KC van der Meulen H Koerten HK Vermeer BJ Pavel S Variations in melanin formation by cultured melanocytes from different skin types Arch Dermatol Res 1998 290 342 349 9705167 10.1007/s004030050315
Westbroek W Lambert J Bahadoran P Roser B Herteleer MC Smit N Mommaas M Ballotti R Naeyaert JM Interactions of human myosin Va isoforms, endogenously expressed in human melanocytes, are tightly regulated by the tail domain J Invest Dermatol 2003 120 465 475 12603861 10.1046/j.1523-1747.2003.12068.x
Cuppen E Wijers N Schepens J Fransen J Wieringa B Hendriks W A FERM domain governs apical confinement of PTP-BL in epithelial cells J Cell Sci 1999 112 3299 3308 10504335
Mayhew T How to count your gold: A tutorial on TEM immunogold label quantification Micro Anal 2005 19 9 12 (EU)
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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-5-471615938910.1186/1471-2148-5-47Research ArticleA comparison of variation between a MHC pseudogene and microsatellite loci of the little greenbul (Andropadus virens) Aguilar Andres [email protected] Thomas B [email protected] Robert K [email protected] Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095 USA2 Center for Tropical Research, Institute of the Environment, 1609 Hershey Hall, University of California, Los Angeles, CA 90095 USA3 Southwest Fisheries Science Center & Department of Ocean Sciences, 110 Shaffer Road, University of California, Santa Cruz, California 95060 USA2005 13 9 2005 5 47 47 15 3 2005 13 9 2005 Copyright © 2005 Aguilar et al; licensee BioMed Central Ltd.2005Aguilar 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 investigated genetic variation of a major histcompatibility complex (MHC) pseudogene (Anvi-DAB1) in the little greenbul (Andropadus virens) from four localities in Cameroon and one in Ivory Coast, West Africa. Previous microsatellite and mitochondrial DNA analyses had revealed little or no genetic differentiation among Cameroon localities but significant differentiation between localities in Cameroon and Ivory Coast.
Results
Levels of genetic variation, heterozygosity, and allelic diversity were high for the MHC pseudogene in Cameroon. Nucleotide diversity of the MHC pseudogene in Cameroon and Ivory Coast was comparable to levels observed in other avian species that have been studied for variation in nuclear genes. An excess of rare variants for the MHC pseudogene was found in the Cameroon population, but this excess was not statistically significant. Pairwise measures of population differentiation revealed high divergence between Cameroon and Ivory Coast for microsatellites and the MHC locus, although for the latter distance measures were much higher than the comparable microsatellite distances.
Conclusion
We provide the first ever comparison of variation in a putative MHC pseudogene to variation in neutral loci in a passerine bird. Our results are consistence with the action of neutral processes on the pseudogene and suggest they can provide an independent perspective on demographic history and population substructure.
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Background
Portrayed as the paradigm of neutral evolution [1], pseudogenes are thought be free of selective forces that constrain functional genes and this single feature should make pseudogenes highly attractive for population genetic studies. Pseudogenes may be more appealing than introns for population genetic studies, as introns may be closely linked to functional gene regions [2] and therefore may often be under the influence of selection [3]. Though pseudogenes have been the focus of molecular evolutionary studies at the species level, there is a paucity of research that utilizes them for analysis of populations [see [4,5]]. The main reason for the lack of pseudogenes in population level studies may be that few have been isolated for non-model taxa.
Levels of population differentiation and variability depend on the type of molecular marker used. Modes of inheritance [6], mutation rate [7], mutation models [8,9], recombination [10], and natural selection [11] are important factors that can affect estimates of genetic variability, and consequently measures of population differentiation [12]. Microsatellites are 2–5 base pair (bp) repetitive elements found throughout eukaryotic genomes and are hypervariable genetic markers that are commonly used in molecular genetic studies of natural populations [7]. The use of nuclear sequences in population genetic studies is becoming more common in evolutionary studies [13-15]. However, nuclear sequences are often not as attractive for population genetic studies as they generally have much lower mutation rates than microsatellite loci and consequently are less variable. Most recent population genetic studies have utilized non-coding nuclear markers such as microsatellites or nuclear length variants such as amplified fragment length polymorphisms [15-17,19].
The little greenbul (Andropadus virens) is a small passerine that inhabits rainforests in Sub-Saharan Africa [20]. Previous research on the little greenbul with di- and tetra-nucleotide microsatellite loci has revealed extensive gene flow among Cameroon localities [21,22] and showed Cameroon and Ivory Coast populations to be genetically distinct [22]. Analysis of mitochondrial DNA control region variation found Cameroon and Ivory Coast populations define two distinct sequence clades [23]. These phylogeographic units correspond to putative rainforest refugia in lower (Cameroon) and upper (Ivory Coast) Guinea [23,24].
We assessed genetic variation in Anvi-DAB1, a putative MHC pseudogene in the little greenbul. This designation was based on the presence of frame-shift mutations within the reading frame of exon 2 (Aguilar et al., in review). Genetic variation in Anvi-DAB1 should be correlated with that of neutral loci. To test this prediction, we compared genetic variation in Anvi-DAB1 to variation in six microsatellite loci in little greenbuls from Cameroon and Ivory Coast.
Results
We sequenced 16 individuals from Ivory Coast and 55 individuals from Cameroon for variation in the Anvi-DAB1 MHC gene. A total of 17 alleles were found (Table 1) and three alleles, Anvi-DAB1*07, Anvi-DAB1*08, and Anvi-DAB1*14, were shared between Cameroon and Ivory Coast. Eleven of 17 alleles were unique to Cameroon and three were unique to Ivory Coast. Cameroon exhibited much more allelic diversity than Ivory Coast for Anvi-DAB1 (Table 1). An allele previously found containing a frame shift mutation (Anvi-DAB1*05) was found at a frequency of 0.23 in Nkwouak and 0.03 in Ndibi. Observed heterozygosity (Ho) for the Cameroon sites varied from 0.30 (Wakwa) to 1.0 (Tibati) for Anvi-DAB1 (Table 1). Two of the Cameroon populations, Ndibi and Wakwa, exhibited significant deviations from H-W equilibrium (p < 0.05) for the Anvi-DAB1 locus. Per site nucleotide diversity (π) for Cameroon and Ivory Coast was 0.007 and 0.004 (Table 1). The number of segregating sites (S) for the 14 and 4 alleles found in Cameroon and Ivory Coast was 13 and 3, respectively (Table 1). Within Cameroon, the Ndibi site possessed the greatest number of alleles (k = 11) and nucleotide diversity was 0.006 or 0.007 at each site (Table 1).
Table 1 Observed (Ho) and expected (He) heterozygosities, allelic richness (A) for the Anvi-DAB1 and 6 microsatellite loci. Mean allelic richness (A) is give for the 6 microsatellite loci. DNA sequence polymorphism statistics are reported for the Anvi-DAB1 gene (nucleotide diversity – Π; Tajima;s D – D; Fu and Li's F* – F).
Microsatellites Anvi-DAB1
Ho He A Ho He A k S Π D F*
Cameroon 11 13 0.007 -0.84 -1.42
Luna 0.51 0.62 6.8 0.38 0.57 5.0 5 5 0.006 -0.03 0.37
Nkwouak 0.70 0.68 6.8 0.65 0.77 5.7 8 9 0.007 -0.36 -1.05
Ndibi 0.62 0.62 6.4 0.71 0.79 7.1 11 9 0.007 -0.82 -1.86
Wakwa 0.60 0.66 7.1 0.30 0.69 5.7 6 6 0.006 -0.36 -0.83
Ivory Coast 0.42 0.55 5.5 0.41 0.71 4.7 6 9 0.007 -0.76 -0.05
Tajima's D and Fu and Li's F* were both negative for the pooled Cameroon (D = -0.885 and F = -1.330; Table 1) and Ivory Coast sample (D = -0.431 and F* = -0.798; Table 1). However, these values were not significantly different from zero (p > 0.1). All of the sites sampled possessed negative values of Tajima's D (Table 1) whereas all but one of the sites had a negative value for Fu and Li's F* (Luna; Table 1). None of the values for Tajima's D or Fu and Li's F* were significantly different from zero.
Pairwise measures of population divergence based on Anvi-DAB1 and microsatellite data were in general agreement (Table 2). For microsatellite loci and Anvi-DAB1, Ivory Coast had the highest degree of differentiation from all other populations (Table 2). All pairwise FST measures for Anvi-DAB1allelic data between Ivory Coast and Cameroon site were statistically greater than zero (Table 2). Likewise, all four pairwise FST measures between Ivory Coast and Cameroon sites for the six microsatellite loci were significantly greater than zero (Table 2). However, FST values between Cameroon and the Ivory Coast were larger for Anvi-DAB1 (allelic: 0.222 +/- 0.06 [s.d.]; sequence: 0.236 +/- 0.04 [s.d.]) than for the six microsatellite loci (0.086 +/- 0.01 [s.d.]). In contrast, the mean FST for all pairwise comparisons within Cameroon is lower for Anvi-DAB1 allelic (0.004 +/- 0.03 [s.d.]) than for microsatellite data (allelic: 0.012 +/- 0.01 [s.d.]) whereas sequence data has the highest levels of FST (0.026 +/- 0.01 [s.d.]). The FST measures from microsatellites were not significantly different from Anvi-DAB1 allelic (p = 0.23; t = -1.24) and Anvi-DAB1 sequence FST (p = 0.08; t = -1.82). However, all pairwise values within Cameroon are low suggesting high rates of gene flow. The results of the linkage disequilibrium (LD) test for recent divergence versus ongoing gene flow are all positive, indicating that gene flow is most likely occurring among the sampled sites within Cameroon (Table 3). Allelic richness was not highly correlated between the two marker types (r2 = 0.11).
Table 2 Measures of pairwise population differentiation for the 6 microsatellite loci (above diagonal) and for Anvi-DAB1 (below diagonal). For the Anvi-DAB1 FST measures, the numbers on top are based on allelic data and the numbers on bottom are based on sequence data (see Methods). Numbers in bold indicate measures that are significantly different from zero (see Methods).
Luna Nkwouak Ndibi Wakwa Ivory Coast
Luna - 0.016 0.021 0.0 0.080
Nkwouak 0.036
0.039 - 0.027 0.008 0.101
Ndibi 0.036
0.037 0.019
0.034 - 0.003 0.084
Wakwa -0.041
0.027 -0.003
0.001 -0.024
0.016 - 0.077
Ivory Coast 0.301
0.285 0.229
0.214 0.168
0.196 0.191
0.248 -
Table 3 Results of the linkage disequilibrium test for ongoing gene flow versus recent divergence among Cameroon sites for the Anvi-DAB1 locus. Reference group for comparison is listed in rows. NA indicated that less than four pairs of sites were compared and means were not taken.
Reference Population Luna Ndibi Nkwouak Wakwa
Luna - 1.167 1.167 1.167
Ndibi 1.167 - 0.762 0.733
Nkwouak 0.867 0.381 - 0.400
Wakwa 1.167 NA NA -
Pairwise values of FST for allelic and sequence Anvi-DAB1 information were highly correlated (r2 = 0.944) and both statistics were correlated with values of FST for microsatellite loci (r2 = 0.889, r2 = 0.876, respectively). However, none of these relationships were significantly based on the Mantel's test likely reflecting the small number of matrix entries (n = 4). The Mantel's test was also preformed omitting the pairwise measures from Lamto, and a non-significant positive correlation was still found (r2 = 0.864; p = 0.167).
Population level relationships based on genetic distance measures varied with distance measure used and with locus type (Figure 1). All neighbor-joining trees showed that Ivory Coast is divergent from the Cameroon sites (Figure 1). However, high bootstrap support distinguishing Ivory Coast from Cameroon is only evident in the tree constructed using DS with Anvi-DAB1 sequence data (Figure 1B). There was not any support within trees or consistency among trees with regard to the relationships among the Cameroon sites (Figure 1).
Figure 1 Unrooted neighbor-joining trees for the five A. virens populations using Nei's standard distance (Ds) for the allelic data from the Anvi-DAB1 locus (A) and 6 microsatellite loci (B). Bootstrap support above 50% is shown (see methods).
Discussion
We have shown that measures of population differentiation for a MHC pseudogene, Anvi-DAB1, are not significantly differently different from those of six unlinked neutral microsatellite loci. A high degree of differentiation for the Anvi-DAB1 pseudogene as measured by FST was found between sites in Cameroon and Ivory Coast, a result that has been previously found in studies that utilized microsatellite loci [22], and mitochondrial DNA [23]. Within sampled Cameroon sites, high levels of gene flow, as evidenced by low pairwise FST measures, was found for the Anvi-DAB1 locus. This again is concordant with results from microsatellite and mitochondrial DNA [21-23].
Evidence for Anvi-DAB1 being a pseudogene is based on the observation that an allele containing a frame-shift mutation (Anvi-DAB1*05) is homozygous in three individuals, nearly equal rates of synomonous and nonsynomonous substitutions, absence from a survey of transcribed genes in the little greenbul, high divergence in sequence type when compared to classical transcribed MHC sequences, and a lack of conserved MHC class II vertebrate amino acid residues (Aguilar et al., in review). Pseudogenes are rarely used in studies of natural populations, yet they may be valuable tool for quantifying genetic variation and differentiation. For example, polymorphism at the psGBA pseudogene in humans was found to be concordant with previous studies of neutral genes [5]. Nucleotide diversity in Anvi-DAB1 was found to be low, and was similar to that found for another avian MHC pseudogene (Came-DAB1: π = 0.03 [38]). This level of polymorphisms is also low compared to functional MHC genes isolated from other birds and vertebrate taxa [25]. However, study of a human MHC class I pseudogene (HLA-H) found elevated levels of genetic variation, and this was attributed to the linkage of HLA-H to functional HLA loci [26]. Therefore, although pseudogenes maybe useful loci in population genetic studies, comparison of their genetic variability to neutral markers is needed to determine if levels of genetic variability may be influenced by selection.
Negative D and F* values suggest an excess of rare mutants in the pooled Cameroon population though these values were not statistically significant. Similarly, all individual sites possessed negative values for Tajima's D and Fu and Li's F* but again these were not statistically significant. An overabundance of rare mutants in a sample can be caused by recent population expansions [27,28], selective sweeps [29,30], or from pooling samples [31,32]. Further sampling of sites within Cameroon and Ivory Coast, the inclusion of other loci, and establishing fine-scale patterns of population structure will elucidate the significance of the excess of rare mutants for the Anvi-DAB1 gene.
Levels of differentiation were high and significant between Ivory Coast and Cameron for the MHC pseudogene (allelic and sequence data) and microsatellite loci suggesting long-term isolation. In contrast, low mean FST for within Cameroon comparisons, for both Anvi-DAB1 and microsatellites, is indicative of a high level of gene flow among the sampled Cameroon sites. The results of the LD test, coupled with the low FST values, indicate that gene flow is still occurring within the samples Cameroon sites. The positive correlation of pairwise measures of population differentiation for Anvi-DAB1 and the six microsatellite loci and the lack of significant differences in levels of within and between population variation supports drift and migration are the primary influences on the observed genetic variation at Anvi-DAB1.
The lack of any significant correlation between allelic richness measures from microsatellites and the MHC gene could be due to the small-observed differences in allelic richness across populations and/or the low number of populations sampled. High gene flow, as well as large effective population sizes, could account for low discrepancy in allelic richness. To determine if drift is an important factor affecting allelic richness at the two marker sets we would need to sample populations with low effective population size, where we would expect a concordant decrease in microsatellites and MHC variation.
Null alleles could account for the deficiency of heterozygotes observed in many samples. Other factors that could contribute to the deviations from Hardy-Weinberg expectations include sampling artifacts, family structure, and non-random mating. Further work that could elucidate the role of null alleles in generating the observed pattern in heterozygosity would include the re-designing of PCR primers and the use of less stringent PCR conditions. However, such modifications could lead to the amplification of non-orthologous closely related loci.
The unrooted neighbor-joining dendrograms showed that Ivory Coast was topologically distinct from Cameroon localities for both Anvi-DAB1 and microsatellite data. The main difference in the neighbor-joining trees was the degree of genetic distance observed for both marker types, as the Anvi-DAB1 dendrogram constructed with Ds showed much lower differentiation between Ivory Coast and Cameron than the dendrogram based on microsatellite loci (Figure 1). This difference is most likely due to both the limited sample from Ivory Coast and the effect of using a single locus. Unrepresentative allele frequencies as well as the biases associated with a single locus might suggest the average distance measures based on the six microsatellite loci more accurately reflect population history.
The observed genetic differences between Cameroon and Ivory Coast little greenbul populations are a result of geographic isolation two million years ago [23]. Reciprocally monophyletic clades representing the Upper and Lower Guinea refugia were found using mitochondrial NADH dehydrogenase subunit 2 sequence data [23]. The corrected sequence divergence between the two clades was 4.7%, and the estimated time of gene divergence was 2 mya. A more rigorous analysis of 10 microsatellite loci revealed elevated FST between Cameroon and Ivory Coast and these two population groups were also recovered using a Bayesian clustering approach [22]. Examination of population differentiation within Cameroon sites has revealed low levels of gene flow among lowland forest sites [21-23]. Similar results, based on Anvi-DAB1, indicate that this locus is reflecting historical population separations and the contemporary effects of gene flow.
Conclusion
Comparable measures of population differentiation and similarity in population level phylogenetic trees indicate that the processes that are operating on Anvi-DAB1 are analogous to those acting on the typed microsatellite loci. These results suggest that pseudogenes may be useful as molecular tools in population level studies. However, several pseudogenes should be used to decrease locus specific effects and comparisons should be made to other nuclear loci that are unlikely to be under selection (such as microsatellites) so that the influences of selection on pseudogenes can be evaluated. Though pseudogenes may not be as readily available for use, they may become more common as researchers continue large scale sequencing projects on non-model organisms (see [38] and others).
Methods
Little greebul blood samples were collected by T. B. Smith in Cameroon and Ivory Coast. A total of 71 individuals were genotyped at Anvi-DAB1, 55 were from Cameroon, and 16 from Ivory Coast. From Cameroon, localities Luna (n = 8), Nkwouak (n = 20), Ndibi (n = 17), and Wakwa (n = 10) were sampled. The lone site from Ivory Coast was Lamto (n = 16) (see [22] for locality detail). DNA was extracted from blood samples by digestion with proteinase-K followed by phenol-choloroform extraction [33] or by use of a commercially available DNA extraction kit (Qiagen Inc.). The microsatellite dataset used here was from Smith et al. [22] and contained scores on six tetranucleotide microsatellite loci.
The nuclear pseudogene used was the Anvi-DAB1 MHC gene isolated from the little greenbul (Aguilar et al. in review). SSCP [34] was used to identify unique alleles. Briefly, both primers were end-labeled with α-32P [33] and these radio-labeled primers were used in a PCR reaction with the following conditions: 10 ng genomic DNA, 1 mM of each primer (Anviex2F.1 [TGC CAT GGA CGC TTA CAC T] and Int2R.1 [CCG AGG GGA CAC GCT CT] [35], 1 mM dNTPs, 1 x PCR buffer (Sigma), 0.5 units of Taq polymerase (Sigma) and 1.0 mM MgCl2 in a 25 μL reaction volume. These primer pairs target a 267 bp portion of exon 2 from the Anvi-DAB1 pseudogene. Reactions were run with the following temperature cycles: an initial 3 min denaturing step at 94°C, 30 sec at 94°C, 30 sec at 58°C, 30 sec at 72°C, and a final 5 min extension at 72°C. Five μL of the PCR reaction were mixed with two μL of stop solution (95% formamide and 0.05% bromophenol blue), heated for 5 min at 95°C then cooled immediately on ice. Two μL of this cocktail were loaded into a 5% non-denaturing polyacrylamide gel containing 5% glycerol (v/v) and run at 20 W for 8–10 hours at room temperature. Gels were transferred to 3 M Whatman paper, dried, and exposed to autoradiographic film for 12–48 hours (depending upon activity of 32P). Unique alleles, as identified from SSCP, were isolated from dried gels and re-amplified [34]. PCR products were separated on 1% agarose gels, isolated, and sequenced using forward and reverse primers. Alleles having the same confirmation were sequenced from multiple individuals to assure identity in sequence. Sequencing was done either on an ABI 377 or a Beckman CEQ2000 following manufacture's protocols. Sequences were then imported into SEQUENCHER (GeneCodes, Inc.) and aligned. Sequences have been deposited in [Genbank: AY437894-AY437899; DQ113429-DQ113439].
Observed and expected heterozygosity for Anvi-DAB1 and the microsatellite loci were calculated using GENETIX [36]. Deviations from Hardy-Weinberg equilibrium were assessed with the exact test implemented in GENEPOP [37]. We also calculated Tajima's D [38] and Fu and Li's F* [39] for each site and pooled samples from Cameroon to assess any deviations from neutral evolution using DNAsp [40]. Both Tajima's D and Fu and Li's F statistics test for deviations from neutrality by examining the frequency spectra of mutations in the sample. Statistical significance from neutrality was assessed for Tajima's D and Fu and Li's F* using 10,000 coalescent simulations in DNAsp [40].
Pairwise population differentiation (FST or θ) was calculated from allelic data [41] with GENETIX [41] for Anvi-DAB1 and for the six microsatellite loci. Significance of pairwise FST measures was assessed with 500 bootstrap replicates in GENETIX. We calculated FST from sequence data using the method of Hudson et al. [42] implemented in DNAsp [40]. The statistical significance of correlations among pairwise measures of FST (for Anvi-DAB1 and microsatellite loci) was assessed with a Mantel's test (5000 permutations) using GENETIX. Allelic richness, a measure of allelic variation that takes into account differences in sample sizes among populations, was estimated with the rarefaction method [43]. The rarefaction estimate was based on sampling 16 genes per population.
We used the approach of Machado et al. [44] to distinguish between ongoing gene flow and recent divergence among the Cameroon populations. This method compares the difference in LD between all shared polymorphisms (DSS) between two populations and the LD from pairs of nucleotide sites that are shared between populations and exclusive to one reference population (DSX). This difference has previously been reported as x. LD, was estimated as D', and x were estimated with the program SITES [45]. Ongoing gene flow is expected to produce positive x values, while the lack of gene flow will produce x values close to zero [44].
Unrooted neighbor-joining dendrograms also were constructed from genotype data using Nei's standard genetic distance (Ds) [46] calculated between each population pair with the program POPULATIONS [47]. Five hundred bootstrap replicates were preformed to assess the support for branching nodes.
Authors' contributions
This work started out of a collaborative effort between the laboratories of TBS and RKW. AA designed the study, carried out the laboratory work and statistical analyses, and drafted the manuscript. TBS collected samples and TBS and RKW participated in the design and drafting of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank the Government of the Republic of Cameroon for permission to conduct the field research. This research was supported by grants from the National Geographic Society, National Environmental Research Council, Royal Society, and the National Science Foundation grants DEB-9726425 and IRCEB9977072 to T.B.S, an Academic Senate grant (UCLA) to A.A. and R.K.W. and an NSF-DDIG to A.A. We thank J. Pollinger for laboratory assistance and S. V. Edwards and an anonymous reviewer for helpful comments on the manuscript.
==== Refs
Li WH Gojobri T Nei M Pseudogenes as a paradigm of neutral evolution Nature 1981 292 237 239 7254315 10.1038/292237a0
Nachman MW Crowell SL Contrasting evolutionary histories of two introns of the Duchenne muscular dystrophy gene, Dmd, in humans Genetics 2000 155 1855 1864 10924480
Leicht BG Muse SV Hanczyc M Clarck AG Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila Genetics 1995 139 299 308 7535717
Prager EM Orrego C Sage RD Genetic variation and phylogeography of central Asian and other House Mice, including a major new mitochondrial lineage in Yemen Genetics 1998 150 835 861 9755213
Martinez-Arias R Calafell F Mateu E Comas D Andrés A Bertranpetit J Sequence variability of a human pseudogene Genome Res 2001 11 1071 1085 11381033 10.1101/gr.GR-1677RR
Avise JC Molecular markers, natural history, and evolution 1994 New York: Chapman and Hall
Ellegren H Microsatellite mutations in the germline: implications for evolutionary inference Trends Genet 2000 16 551 558 11102705 10.1016/S0168-9525(00)02139-9
Kimura M Crow JF The number of alleles that can be maintained in a finite population Genetics 1964 49 725 738 14156929
Ohta T Kimura M A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population Genet Res 1973 22 201 204 4777279
Begun DJ Aquadro CF Levels of naturally occurring DNA polymorphism correlate with recombination rates in Drosophila melanogaster Nature 1992 356 519 520 1560824 10.1038/356519a0
Kreitman M Akashi H Molecular evidence for natural selection Ann Rev Ecol Syst 1995 26 403 422 10.1146/annurev.es.26.110195.002155
Hedrick PW Perspective: Highly variable loci and their interpretation in evolution and conservation Evolution 1999 53 313 318
Palumbi SR Baker CS Contrasting population structure from nuclear intron and mtDNA of humpback whales Mol Biol Evol 1994 11 426 435 7912407
Friesen VL Congdon BC Walsh HE Birt TP Intron variation in marbled murrelets detected using analyses of single-stranded conformational polymorphisms Mol Ecol 1997 6 1047 1058 9394463 10.1046/j.1365-294X.1997.00277.x
Congdon BC Piatt JF Martin K Freisen V Mechanisms of population differentiation in marbled murrelets: Historical versus contemporary processes Evolution 2000 54 974 986 10937270
Karl SA Avise JC PCR-based assays of Mendelian polymorphisms from anonymous single-copy nuclear DNA: Techniques and applications for population genetics Mol Biol Evol 1993 10 342 361 8098128
Holder K Montgomerie R Friesen VL A test of the glacial refugium hypothesis using patterns of mitochondrial and nuclear DNA sequence variation in rock ptarmigam (Lagopus mutus) Evolution 1999 53 1936 1950
Vallianatos M Lougheed SC Boag PT Conservation of the loggerhead shrike (Lanius ludovicianus) in central and eastern North America Cons Genet 2002 3 1 13 10.1023/A:1014232326576
Brumfield RT Beerli P Nickerson DA Edwards SV The utility of single nucleotide polymorphisms in inferences of population history Trends Ecol Evol 2003 18 249 256 10.1016/S0169-5347(03)00018-1
Keith S Urban EK Fry CH The birds of Africa 1992 4 New York: Academic Press
Smith TB Wayne RK Girman DJ Bruford MW A role for ecotones in generating rainforest biodiversity Science 1997 276 1855 1857 10.1126/science.276.5320.1855
Smith TB Calsbeek R Wayne RK Pires DB Bardeleben C Testing alternative mechanisms of evolutionary divergence in an African rain forest passerine bird J Evol Biol 2005 18 257 268 15715832 10.1111/j.1420-9101.2004.00825.x
Maley J Weber W, White LJT, Vedder A The impact of arid phases on the African rainforest through geologic history African rain forest ecology and conservation 2001 New Haven: Yale University Press 68 87
Smith TB Schneider CJ Holder K Refugual isolation versus ecological gradients Genetica 2001 112–113 383 398 10.1023/A:1013312510860
Hess CM Gasper J Hoekstra HE Hill CE Edwards SV Mhc class II pseudogene and genomic signature of a 32-kb cosmid in the house finch (Carpodacus mexicanus) Genome Res 2000 10 613 623 10810083 10.1101/gr.10.5.613
Grimsley C Mather KA Ober C HLA-H: Pseudogene with increased variation due to balancing selection at neighboring loci Mol Biol Evol 1998 15 1581 1588 9866194
Slatkin M Hudson RR Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations Genetics 1991 129 555 562 1743491
Rogers AR Harpending H Population growth makes waves in the distribution of pairwise genetic differences Mol Biol Evol 1992 9 552 569 1316531
Pogson GH Nucleotide polymorphism and natural selection at the pantophysin (Pan I) locus in the Atlantic cod, Gadus morhua (L.) Genetics 2001 157 317 330 11139512
Saez AG Tatarenkov A Barrio E Becerra NH Ayala FJ Patterns of DNA sequence polymorphism at Sod vicinities in Drosophila melanogaster: unraveling the footprint of a recent selective sweep Proc Natl Acad Sci (USA) 2003 100 1793 1798 12578968 10.1073/pnas.242746799
Ptak SE Przeworski M Evidence for population growth in humans is confounded by fine-scale population structure Trend Genetics 2002 18 559 563 10.1016/S0168-9525(02)02781-6
Hammer MF Blackmer F Garrigan D Nachman MW Wilder JA Human population structure and its effects on sampling Y chromosome sequence variation Genetics 2003 164 1495 1509 12930755
Sambrook J Fritsch EF Maniatis T Molecular Cloning: a laboratory manual 1989 New York: Cold Spring Harbor Laboratory Press
Sunnucks P Wilson ACC Beheregary LB Zenger K French J Taylor AC SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology Mol Ecol 2000 9 1699 1710 11091307 10.1046/j.1365-294x.2000.01084.x
Edwards SV Gasper J March M Genomics and polymorphism of Agph-DAB1, an Mhc class II gene in Red-winged Blackbirds (Agelaius phoecineus) Mol Biol Evol 1998 15 236 250 9501491
Belkhir K GENETIX (v4.04): A Windows program for population genetic analysis 1999 Laboratorie Genome, Populations: Interactions UPR 5000 du CNRS, Universite Montpellier II, Montpellier (France)
Raymond M Rousset F GENEPOP (Version 1.2): Population genetics software for exact tests and ecumenicism J Heredity 1995 86 248 249
Tajima F Statistical methods for testing the neutral mutation hypothesis by DNA polymorphism Genetics 1989 123 585 595 2513255
Fu YX Li WH Statistical tests of neutrality of mutations Genetics 1993 133 693 709 8454210
Rosas J Rosas R DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis Bioinformatics 1999 15 174 175 10089204 10.1093/bioinformatics/15.2.174
Weir BS Cockerham CC Estimating F statistics for the analysis of population structure Evolution 1984 38 1358 1370
Hudson RR Slatkin M Maddison WP Estimation of levels of gene flow from DNA sequence data Genetics 1992 132 582 589
Kalinowski ST HP-RARE: a computer program for performing rarefaction on measures of allelic richness Mol Ecol 2005 5 187 189 10.1111/j.1471-8286.2004.00845.x
Machado CA Kliman RM Markert JA Hey J Inferring the history of speciation from multilocus DNA sequence data: the case of Drosophila pseudoobscura and close relatives Mol Biol Evol 2002 19 472 288 11919289
Hey J Wakeley J A coalescent estimator of the population recombination rate Genetics 1997 145 833 846 9055092
Nei M Molecular Evolutionary Genetics 1987 New York: Columbia University Press
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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-5-471615938910.1186/1471-2148-5-47Research ArticleA comparison of variation between a MHC pseudogene and microsatellite loci of the little greenbul (Andropadus virens) Aguilar Andres [email protected] Thomas B [email protected] Robert K [email protected] Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095 USA2 Center for Tropical Research, Institute of the Environment, 1609 Hershey Hall, University of California, Los Angeles, CA 90095 USA3 Southwest Fisheries Science Center & Department of Ocean Sciences, 110 Shaffer Road, University of California, Santa Cruz, California 95060 USA2005 13 9 2005 5 47 47 15 3 2005 13 9 2005 Copyright © 2005 Aguilar et al; licensee BioMed Central Ltd.2005Aguilar 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 investigated genetic variation of a major histcompatibility complex (MHC) pseudogene (Anvi-DAB1) in the little greenbul (Andropadus virens) from four localities in Cameroon and one in Ivory Coast, West Africa. Previous microsatellite and mitochondrial DNA analyses had revealed little or no genetic differentiation among Cameroon localities but significant differentiation between localities in Cameroon and Ivory Coast.
Results
Levels of genetic variation, heterozygosity, and allelic diversity were high for the MHC pseudogene in Cameroon. Nucleotide diversity of the MHC pseudogene in Cameroon and Ivory Coast was comparable to levels observed in other avian species that have been studied for variation in nuclear genes. An excess of rare variants for the MHC pseudogene was found in the Cameroon population, but this excess was not statistically significant. Pairwise measures of population differentiation revealed high divergence between Cameroon and Ivory Coast for microsatellites and the MHC locus, although for the latter distance measures were much higher than the comparable microsatellite distances.
Conclusion
We provide the first ever comparison of variation in a putative MHC pseudogene to variation in neutral loci in a passerine bird. Our results are consistence with the action of neutral processes on the pseudogene and suggest they can provide an independent perspective on demographic history and population substructure.
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Background
Portrayed as the paradigm of neutral evolution [1], pseudogenes are thought be free of selective forces that constrain functional genes and this single feature should make pseudogenes highly attractive for population genetic studies. Pseudogenes may be more appealing than introns for population genetic studies, as introns may be closely linked to functional gene regions [2] and therefore may often be under the influence of selection [3]. Though pseudogenes have been the focus of molecular evolutionary studies at the species level, there is a paucity of research that utilizes them for analysis of populations [see [4,5]]. The main reason for the lack of pseudogenes in population level studies may be that few have been isolated for non-model taxa.
Levels of population differentiation and variability depend on the type of molecular marker used. Modes of inheritance [6], mutation rate [7], mutation models [8,9], recombination [10], and natural selection [11] are important factors that can affect estimates of genetic variability, and consequently measures of population differentiation [12]. Microsatellites are 2–5 base pair (bp) repetitive elements found throughout eukaryotic genomes and are hypervariable genetic markers that are commonly used in molecular genetic studies of natural populations [7]. The use of nuclear sequences in population genetic studies is becoming more common in evolutionary studies [13-15]. However, nuclear sequences are often not as attractive for population genetic studies as they generally have much lower mutation rates than microsatellite loci and consequently are less variable. Most recent population genetic studies have utilized non-coding nuclear markers such as microsatellites or nuclear length variants such as amplified fragment length polymorphisms [15-17,19].
The little greenbul (Andropadus virens) is a small passerine that inhabits rainforests in Sub-Saharan Africa [20]. Previous research on the little greenbul with di- and tetra-nucleotide microsatellite loci has revealed extensive gene flow among Cameroon localities [21,22] and showed Cameroon and Ivory Coast populations to be genetically distinct [22]. Analysis of mitochondrial DNA control region variation found Cameroon and Ivory Coast populations define two distinct sequence clades [23]. These phylogeographic units correspond to putative rainforest refugia in lower (Cameroon) and upper (Ivory Coast) Guinea [23,24].
We assessed genetic variation in Anvi-DAB1, a putative MHC pseudogene in the little greenbul. This designation was based on the presence of frame-shift mutations within the reading frame of exon 2 (Aguilar et al., in review). Genetic variation in Anvi-DAB1 should be correlated with that of neutral loci. To test this prediction, we compared genetic variation in Anvi-DAB1 to variation in six microsatellite loci in little greenbuls from Cameroon and Ivory Coast.
Results
We sequenced 16 individuals from Ivory Coast and 55 individuals from Cameroon for variation in the Anvi-DAB1 MHC gene. A total of 17 alleles were found (Table 1) and three alleles, Anvi-DAB1*07, Anvi-DAB1*08, and Anvi-DAB1*14, were shared between Cameroon and Ivory Coast. Eleven of 17 alleles were unique to Cameroon and three were unique to Ivory Coast. Cameroon exhibited much more allelic diversity than Ivory Coast for Anvi-DAB1 (Table 1). An allele previously found containing a frame shift mutation (Anvi-DAB1*05) was found at a frequency of 0.23 in Nkwouak and 0.03 in Ndibi. Observed heterozygosity (Ho) for the Cameroon sites varied from 0.30 (Wakwa) to 1.0 (Tibati) for Anvi-DAB1 (Table 1). Two of the Cameroon populations, Ndibi and Wakwa, exhibited significant deviations from H-W equilibrium (p < 0.05) for the Anvi-DAB1 locus. Per site nucleotide diversity (π) for Cameroon and Ivory Coast was 0.007 and 0.004 (Table 1). The number of segregating sites (S) for the 14 and 4 alleles found in Cameroon and Ivory Coast was 13 and 3, respectively (Table 1). Within Cameroon, the Ndibi site possessed the greatest number of alleles (k = 11) and nucleotide diversity was 0.006 or 0.007 at each site (Table 1).
Table 1 Observed (Ho) and expected (He) heterozygosities, allelic richness (A) for the Anvi-DAB1 and 6 microsatellite loci. Mean allelic richness (A) is give for the 6 microsatellite loci. DNA sequence polymorphism statistics are reported for the Anvi-DAB1 gene (nucleotide diversity – Π; Tajima;s D – D; Fu and Li's F* – F).
Microsatellites Anvi-DAB1
Ho He A Ho He A k S Π D F*
Cameroon 11 13 0.007 -0.84 -1.42
Luna 0.51 0.62 6.8 0.38 0.57 5.0 5 5 0.006 -0.03 0.37
Nkwouak 0.70 0.68 6.8 0.65 0.77 5.7 8 9 0.007 -0.36 -1.05
Ndibi 0.62 0.62 6.4 0.71 0.79 7.1 11 9 0.007 -0.82 -1.86
Wakwa 0.60 0.66 7.1 0.30 0.69 5.7 6 6 0.006 -0.36 -0.83
Ivory Coast 0.42 0.55 5.5 0.41 0.71 4.7 6 9 0.007 -0.76 -0.05
Tajima's D and Fu and Li's F* were both negative for the pooled Cameroon (D = -0.885 and F = -1.330; Table 1) and Ivory Coast sample (D = -0.431 and F* = -0.798; Table 1). However, these values were not significantly different from zero (p > 0.1). All of the sites sampled possessed negative values of Tajima's D (Table 1) whereas all but one of the sites had a negative value for Fu and Li's F* (Luna; Table 1). None of the values for Tajima's D or Fu and Li's F* were significantly different from zero.
Pairwise measures of population divergence based on Anvi-DAB1 and microsatellite data were in general agreement (Table 2). For microsatellite loci and Anvi-DAB1, Ivory Coast had the highest degree of differentiation from all other populations (Table 2). All pairwise FST measures for Anvi-DAB1allelic data between Ivory Coast and Cameroon site were statistically greater than zero (Table 2). Likewise, all four pairwise FST measures between Ivory Coast and Cameroon sites for the six microsatellite loci were significantly greater than zero (Table 2). However, FST values between Cameroon and the Ivory Coast were larger for Anvi-DAB1 (allelic: 0.222 +/- 0.06 [s.d.]; sequence: 0.236 +/- 0.04 [s.d.]) than for the six microsatellite loci (0.086 +/- 0.01 [s.d.]). In contrast, the mean FST for all pairwise comparisons within Cameroon is lower for Anvi-DAB1 allelic (0.004 +/- 0.03 [s.d.]) than for microsatellite data (allelic: 0.012 +/- 0.01 [s.d.]) whereas sequence data has the highest levels of FST (0.026 +/- 0.01 [s.d.]). The FST measures from microsatellites were not significantly different from Anvi-DAB1 allelic (p = 0.23; t = -1.24) and Anvi-DAB1 sequence FST (p = 0.08; t = -1.82). However, all pairwise values within Cameroon are low suggesting high rates of gene flow. The results of the linkage disequilibrium (LD) test for recent divergence versus ongoing gene flow are all positive, indicating that gene flow is most likely occurring among the sampled sites within Cameroon (Table 3). Allelic richness was not highly correlated between the two marker types (r2 = 0.11).
Table 2 Measures of pairwise population differentiation for the 6 microsatellite loci (above diagonal) and for Anvi-DAB1 (below diagonal). For the Anvi-DAB1 FST measures, the numbers on top are based on allelic data and the numbers on bottom are based on sequence data (see Methods). Numbers in bold indicate measures that are significantly different from zero (see Methods).
Luna Nkwouak Ndibi Wakwa Ivory Coast
Luna - 0.016 0.021 0.0 0.080
Nkwouak 0.036
0.039 - 0.027 0.008 0.101
Ndibi 0.036
0.037 0.019
0.034 - 0.003 0.084
Wakwa -0.041
0.027 -0.003
0.001 -0.024
0.016 - 0.077
Ivory Coast 0.301
0.285 0.229
0.214 0.168
0.196 0.191
0.248 -
Table 3 Results of the linkage disequilibrium test for ongoing gene flow versus recent divergence among Cameroon sites for the Anvi-DAB1 locus. Reference group for comparison is listed in rows. NA indicated that less than four pairs of sites were compared and means were not taken.
Reference Population Luna Ndibi Nkwouak Wakwa
Luna - 1.167 1.167 1.167
Ndibi 1.167 - 0.762 0.733
Nkwouak 0.867 0.381 - 0.400
Wakwa 1.167 NA NA -
Pairwise values of FST for allelic and sequence Anvi-DAB1 information were highly correlated (r2 = 0.944) and both statistics were correlated with values of FST for microsatellite loci (r2 = 0.889, r2 = 0.876, respectively). However, none of these relationships were significantly based on the Mantel's test likely reflecting the small number of matrix entries (n = 4). The Mantel's test was also preformed omitting the pairwise measures from Lamto, and a non-significant positive correlation was still found (r2 = 0.864; p = 0.167).
Population level relationships based on genetic distance measures varied with distance measure used and with locus type (Figure 1). All neighbor-joining trees showed that Ivory Coast is divergent from the Cameroon sites (Figure 1). However, high bootstrap support distinguishing Ivory Coast from Cameroon is only evident in the tree constructed using DS with Anvi-DAB1 sequence data (Figure 1B). There was not any support within trees or consistency among trees with regard to the relationships among the Cameroon sites (Figure 1).
Figure 1 Unrooted neighbor-joining trees for the five A. virens populations using Nei's standard distance (Ds) for the allelic data from the Anvi-DAB1 locus (A) and 6 microsatellite loci (B). Bootstrap support above 50% is shown (see methods).
Discussion
We have shown that measures of population differentiation for a MHC pseudogene, Anvi-DAB1, are not significantly differently different from those of six unlinked neutral microsatellite loci. A high degree of differentiation for the Anvi-DAB1 pseudogene as measured by FST was found between sites in Cameroon and Ivory Coast, a result that has been previously found in studies that utilized microsatellite loci [22], and mitochondrial DNA [23]. Within sampled Cameroon sites, high levels of gene flow, as evidenced by low pairwise FST measures, was found for the Anvi-DAB1 locus. This again is concordant with results from microsatellite and mitochondrial DNA [21-23].
Evidence for Anvi-DAB1 being a pseudogene is based on the observation that an allele containing a frame-shift mutation (Anvi-DAB1*05) is homozygous in three individuals, nearly equal rates of synomonous and nonsynomonous substitutions, absence from a survey of transcribed genes in the little greenbul, high divergence in sequence type when compared to classical transcribed MHC sequences, and a lack of conserved MHC class II vertebrate amino acid residues (Aguilar et al., in review). Pseudogenes are rarely used in studies of natural populations, yet they may be valuable tool for quantifying genetic variation and differentiation. For example, polymorphism at the psGBA pseudogene in humans was found to be concordant with previous studies of neutral genes [5]. Nucleotide diversity in Anvi-DAB1 was found to be low, and was similar to that found for another avian MHC pseudogene (Came-DAB1: π = 0.03 [38]). This level of polymorphisms is also low compared to functional MHC genes isolated from other birds and vertebrate taxa [25]. However, study of a human MHC class I pseudogene (HLA-H) found elevated levels of genetic variation, and this was attributed to the linkage of HLA-H to functional HLA loci [26]. Therefore, although pseudogenes maybe useful loci in population genetic studies, comparison of their genetic variability to neutral markers is needed to determine if levels of genetic variability may be influenced by selection.
Negative D and F* values suggest an excess of rare mutants in the pooled Cameroon population though these values were not statistically significant. Similarly, all individual sites possessed negative values for Tajima's D and Fu and Li's F* but again these were not statistically significant. An overabundance of rare mutants in a sample can be caused by recent population expansions [27,28], selective sweeps [29,30], or from pooling samples [31,32]. Further sampling of sites within Cameroon and Ivory Coast, the inclusion of other loci, and establishing fine-scale patterns of population structure will elucidate the significance of the excess of rare mutants for the Anvi-DAB1 gene.
Levels of differentiation were high and significant between Ivory Coast and Cameron for the MHC pseudogene (allelic and sequence data) and microsatellite loci suggesting long-term isolation. In contrast, low mean FST for within Cameroon comparisons, for both Anvi-DAB1 and microsatellites, is indicative of a high level of gene flow among the sampled Cameroon sites. The results of the LD test, coupled with the low FST values, indicate that gene flow is still occurring within the samples Cameroon sites. The positive correlation of pairwise measures of population differentiation for Anvi-DAB1 and the six microsatellite loci and the lack of significant differences in levels of within and between population variation supports drift and migration are the primary influences on the observed genetic variation at Anvi-DAB1.
The lack of any significant correlation between allelic richness measures from microsatellites and the MHC gene could be due to the small-observed differences in allelic richness across populations and/or the low number of populations sampled. High gene flow, as well as large effective population sizes, could account for low discrepancy in allelic richness. To determine if drift is an important factor affecting allelic richness at the two marker sets we would need to sample populations with low effective population size, where we would expect a concordant decrease in microsatellites and MHC variation.
Null alleles could account for the deficiency of heterozygotes observed in many samples. Other factors that could contribute to the deviations from Hardy-Weinberg expectations include sampling artifacts, family structure, and non-random mating. Further work that could elucidate the role of null alleles in generating the observed pattern in heterozygosity would include the re-designing of PCR primers and the use of less stringent PCR conditions. However, such modifications could lead to the amplification of non-orthologous closely related loci.
The unrooted neighbor-joining dendrograms showed that Ivory Coast was topologically distinct from Cameroon localities for both Anvi-DAB1 and microsatellite data. The main difference in the neighbor-joining trees was the degree of genetic distance observed for both marker types, as the Anvi-DAB1 dendrogram constructed with Ds showed much lower differentiation between Ivory Coast and Cameron than the dendrogram based on microsatellite loci (Figure 1). This difference is most likely due to both the limited sample from Ivory Coast and the effect of using a single locus. Unrepresentative allele frequencies as well as the biases associated with a single locus might suggest the average distance measures based on the six microsatellite loci more accurately reflect population history.
The observed genetic differences between Cameroon and Ivory Coast little greenbul populations are a result of geographic isolation two million years ago [23]. Reciprocally monophyletic clades representing the Upper and Lower Guinea refugia were found using mitochondrial NADH dehydrogenase subunit 2 sequence data [23]. The corrected sequence divergence between the two clades was 4.7%, and the estimated time of gene divergence was 2 mya. A more rigorous analysis of 10 microsatellite loci revealed elevated FST between Cameroon and Ivory Coast and these two population groups were also recovered using a Bayesian clustering approach [22]. Examination of population differentiation within Cameroon sites has revealed low levels of gene flow among lowland forest sites [21-23]. Similar results, based on Anvi-DAB1, indicate that this locus is reflecting historical population separations and the contemporary effects of gene flow.
Conclusion
Comparable measures of population differentiation and similarity in population level phylogenetic trees indicate that the processes that are operating on Anvi-DAB1 are analogous to those acting on the typed microsatellite loci. These results suggest that pseudogenes may be useful as molecular tools in population level studies. However, several pseudogenes should be used to decrease locus specific effects and comparisons should be made to other nuclear loci that are unlikely to be under selection (such as microsatellites) so that the influences of selection on pseudogenes can be evaluated. Though pseudogenes may not be as readily available for use, they may become more common as researchers continue large scale sequencing projects on non-model organisms (see [38] and others).
Methods
Little greebul blood samples were collected by T. B. Smith in Cameroon and Ivory Coast. A total of 71 individuals were genotyped at Anvi-DAB1, 55 were from Cameroon, and 16 from Ivory Coast. From Cameroon, localities Luna (n = 8), Nkwouak (n = 20), Ndibi (n = 17), and Wakwa (n = 10) were sampled. The lone site from Ivory Coast was Lamto (n = 16) (see [22] for locality detail). DNA was extracted from blood samples by digestion with proteinase-K followed by phenol-choloroform extraction [33] or by use of a commercially available DNA extraction kit (Qiagen Inc.). The microsatellite dataset used here was from Smith et al. [22] and contained scores on six tetranucleotide microsatellite loci.
The nuclear pseudogene used was the Anvi-DAB1 MHC gene isolated from the little greenbul (Aguilar et al. in review). SSCP [34] was used to identify unique alleles. Briefly, both primers were end-labeled with α-32P [33] and these radio-labeled primers were used in a PCR reaction with the following conditions: 10 ng genomic DNA, 1 mM of each primer (Anviex2F.1 [TGC CAT GGA CGC TTA CAC T] and Int2R.1 [CCG AGG GGA CAC GCT CT] [35], 1 mM dNTPs, 1 x PCR buffer (Sigma), 0.5 units of Taq polymerase (Sigma) and 1.0 mM MgCl2 in a 25 μL reaction volume. These primer pairs target a 267 bp portion of exon 2 from the Anvi-DAB1 pseudogene. Reactions were run with the following temperature cycles: an initial 3 min denaturing step at 94°C, 30 sec at 94°C, 30 sec at 58°C, 30 sec at 72°C, and a final 5 min extension at 72°C. Five μL of the PCR reaction were mixed with two μL of stop solution (95% formamide and 0.05% bromophenol blue), heated for 5 min at 95°C then cooled immediately on ice. Two μL of this cocktail were loaded into a 5% non-denaturing polyacrylamide gel containing 5% glycerol (v/v) and run at 20 W for 8–10 hours at room temperature. Gels were transferred to 3 M Whatman paper, dried, and exposed to autoradiographic film for 12–48 hours (depending upon activity of 32P). Unique alleles, as identified from SSCP, were isolated from dried gels and re-amplified [34]. PCR products were separated on 1% agarose gels, isolated, and sequenced using forward and reverse primers. Alleles having the same confirmation were sequenced from multiple individuals to assure identity in sequence. Sequencing was done either on an ABI 377 or a Beckman CEQ2000 following manufacture's protocols. Sequences were then imported into SEQUENCHER (GeneCodes, Inc.) and aligned. Sequences have been deposited in [Genbank: AY437894-AY437899; DQ113429-DQ113439].
Observed and expected heterozygosity for Anvi-DAB1 and the microsatellite loci were calculated using GENETIX [36]. Deviations from Hardy-Weinberg equilibrium were assessed with the exact test implemented in GENEPOP [37]. We also calculated Tajima's D [38] and Fu and Li's F* [39] for each site and pooled samples from Cameroon to assess any deviations from neutral evolution using DNAsp [40]. Both Tajima's D and Fu and Li's F statistics test for deviations from neutrality by examining the frequency spectra of mutations in the sample. Statistical significance from neutrality was assessed for Tajima's D and Fu and Li's F* using 10,000 coalescent simulations in DNAsp [40].
Pairwise population differentiation (FST or θ) was calculated from allelic data [41] with GENETIX [41] for Anvi-DAB1 and for the six microsatellite loci. Significance of pairwise FST measures was assessed with 500 bootstrap replicates in GENETIX. We calculated FST from sequence data using the method of Hudson et al. [42] implemented in DNAsp [40]. The statistical significance of correlations among pairwise measures of FST (for Anvi-DAB1 and microsatellite loci) was assessed with a Mantel's test (5000 permutations) using GENETIX. Allelic richness, a measure of allelic variation that takes into account differences in sample sizes among populations, was estimated with the rarefaction method [43]. The rarefaction estimate was based on sampling 16 genes per population.
We used the approach of Machado et al. [44] to distinguish between ongoing gene flow and recent divergence among the Cameroon populations. This method compares the difference in LD between all shared polymorphisms (DSS) between two populations and the LD from pairs of nucleotide sites that are shared between populations and exclusive to one reference population (DSX). This difference has previously been reported as x. LD, was estimated as D', and x were estimated with the program SITES [45]. Ongoing gene flow is expected to produce positive x values, while the lack of gene flow will produce x values close to zero [44].
Unrooted neighbor-joining dendrograms also were constructed from genotype data using Nei's standard genetic distance (Ds) [46] calculated between each population pair with the program POPULATIONS [47]. Five hundred bootstrap replicates were preformed to assess the support for branching nodes.
Authors' contributions
This work started out of a collaborative effort between the laboratories of TBS and RKW. AA designed the study, carried out the laboratory work and statistical analyses, and drafted the manuscript. TBS collected samples and TBS and RKW participated in the design and drafting of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank the Government of the Republic of Cameroon for permission to conduct the field research. This research was supported by grants from the National Geographic Society, National Environmental Research Council, Royal Society, and the National Science Foundation grants DEB-9726425 and IRCEB9977072 to T.B.S, an Academic Senate grant (UCLA) to A.A. and R.K.W. and an NSF-DDIG to A.A. We thank J. Pollinger for laboratory assistance and S. V. Edwards and an anonymous reviewer for helpful comments on the manuscript.
==== Refs
Li WH Gojobri T Nei M Pseudogenes as a paradigm of neutral evolution Nature 1981 292 237 239 7254315 10.1038/292237a0
Nachman MW Crowell SL Contrasting evolutionary histories of two introns of the Duchenne muscular dystrophy gene, Dmd, in humans Genetics 2000 155 1855 1864 10924480
Leicht BG Muse SV Hanczyc M Clarck AG Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila Genetics 1995 139 299 308 7535717
Prager EM Orrego C Sage RD Genetic variation and phylogeography of central Asian and other House Mice, including a major new mitochondrial lineage in Yemen Genetics 1998 150 835 861 9755213
Martinez-Arias R Calafell F Mateu E Comas D Andrés A Bertranpetit J Sequence variability of a human pseudogene Genome Res 2001 11 1071 1085 11381033 10.1101/gr.GR-1677RR
Avise JC Molecular markers, natural history, and evolution 1994 New York: Chapman and Hall
Ellegren H Microsatellite mutations in the germline: implications for evolutionary inference Trends Genet 2000 16 551 558 11102705 10.1016/S0168-9525(00)02139-9
Kimura M Crow JF The number of alleles that can be maintained in a finite population Genetics 1964 49 725 738 14156929
Ohta T Kimura M A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population Genet Res 1973 22 201 204 4777279
Begun DJ Aquadro CF Levels of naturally occurring DNA polymorphism correlate with recombination rates in Drosophila melanogaster Nature 1992 356 519 520 1560824 10.1038/356519a0
Kreitman M Akashi H Molecular evidence for natural selection Ann Rev Ecol Syst 1995 26 403 422 10.1146/annurev.es.26.110195.002155
Hedrick PW Perspective: Highly variable loci and their interpretation in evolution and conservation Evolution 1999 53 313 318
Palumbi SR Baker CS Contrasting population structure from nuclear intron and mtDNA of humpback whales Mol Biol Evol 1994 11 426 435 7912407
Friesen VL Congdon BC Walsh HE Birt TP Intron variation in marbled murrelets detected using analyses of single-stranded conformational polymorphisms Mol Ecol 1997 6 1047 1058 9394463 10.1046/j.1365-294X.1997.00277.x
Congdon BC Piatt JF Martin K Freisen V Mechanisms of population differentiation in marbled murrelets: Historical versus contemporary processes Evolution 2000 54 974 986 10937270
Karl SA Avise JC PCR-based assays of Mendelian polymorphisms from anonymous single-copy nuclear DNA: Techniques and applications for population genetics Mol Biol Evol 1993 10 342 361 8098128
Holder K Montgomerie R Friesen VL A test of the glacial refugium hypothesis using patterns of mitochondrial and nuclear DNA sequence variation in rock ptarmigam (Lagopus mutus) Evolution 1999 53 1936 1950
Vallianatos M Lougheed SC Boag PT Conservation of the loggerhead shrike (Lanius ludovicianus) in central and eastern North America Cons Genet 2002 3 1 13 10.1023/A:1014232326576
Brumfield RT Beerli P Nickerson DA Edwards SV The utility of single nucleotide polymorphisms in inferences of population history Trends Ecol Evol 2003 18 249 256 10.1016/S0169-5347(03)00018-1
Keith S Urban EK Fry CH The birds of Africa 1992 4 New York: Academic Press
Smith TB Wayne RK Girman DJ Bruford MW A role for ecotones in generating rainforest biodiversity Science 1997 276 1855 1857 10.1126/science.276.5320.1855
Smith TB Calsbeek R Wayne RK Pires DB Bardeleben C Testing alternative mechanisms of evolutionary divergence in an African rain forest passerine bird J Evol Biol 2005 18 257 268 15715832 10.1111/j.1420-9101.2004.00825.x
Maley J Weber W, White LJT, Vedder A The impact of arid phases on the African rainforest through geologic history African rain forest ecology and conservation 2001 New Haven: Yale University Press 68 87
Smith TB Schneider CJ Holder K Refugual isolation versus ecological gradients Genetica 2001 112–113 383 398 10.1023/A:1013312510860
Hess CM Gasper J Hoekstra HE Hill CE Edwards SV Mhc class II pseudogene and genomic signature of a 32-kb cosmid in the house finch (Carpodacus mexicanus) Genome Res 2000 10 613 623 10810083 10.1101/gr.10.5.613
Grimsley C Mather KA Ober C HLA-H: Pseudogene with increased variation due to balancing selection at neighboring loci Mol Biol Evol 1998 15 1581 1588 9866194
Slatkin M Hudson RR Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations Genetics 1991 129 555 562 1743491
Rogers AR Harpending H Population growth makes waves in the distribution of pairwise genetic differences Mol Biol Evol 1992 9 552 569 1316531
Pogson GH Nucleotide polymorphism and natural selection at the pantophysin (Pan I) locus in the Atlantic cod, Gadus morhua (L.) Genetics 2001 157 317 330 11139512
Saez AG Tatarenkov A Barrio E Becerra NH Ayala FJ Patterns of DNA sequence polymorphism at Sod vicinities in Drosophila melanogaster: unraveling the footprint of a recent selective sweep Proc Natl Acad Sci (USA) 2003 100 1793 1798 12578968 10.1073/pnas.242746799
Ptak SE Przeworski M Evidence for population growth in humans is confounded by fine-scale population structure Trend Genetics 2002 18 559 563 10.1016/S0168-9525(02)02781-6
Hammer MF Blackmer F Garrigan D Nachman MW Wilder JA Human population structure and its effects on sampling Y chromosome sequence variation Genetics 2003 164 1495 1509 12930755
Sambrook J Fritsch EF Maniatis T Molecular Cloning: a laboratory manual 1989 New York: Cold Spring Harbor Laboratory Press
Sunnucks P Wilson ACC Beheregary LB Zenger K French J Taylor AC SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology Mol Ecol 2000 9 1699 1710 11091307 10.1046/j.1365-294x.2000.01084.x
Edwards SV Gasper J March M Genomics and polymorphism of Agph-DAB1, an Mhc class II gene in Red-winged Blackbirds (Agelaius phoecineus) Mol Biol Evol 1998 15 236 250 9501491
Belkhir K GENETIX (v4.04): A Windows program for population genetic analysis 1999 Laboratorie Genome, Populations: Interactions UPR 5000 du CNRS, Universite Montpellier II, Montpellier (France)
Raymond M Rousset F GENEPOP (Version 1.2): Population genetics software for exact tests and ecumenicism J Heredity 1995 86 248 249
Tajima F Statistical methods for testing the neutral mutation hypothesis by DNA polymorphism Genetics 1989 123 585 595 2513255
Fu YX Li WH Statistical tests of neutrality of mutations Genetics 1993 133 693 709 8454210
Rosas J Rosas R DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis Bioinformatics 1999 15 174 175 10089204 10.1093/bioinformatics/15.2.174
Weir BS Cockerham CC Estimating F statistics for the analysis of population structure Evolution 1984 38 1358 1370
Hudson RR Slatkin M Maddison WP Estimation of levels of gene flow from DNA sequence data Genetics 1992 132 582 589
Kalinowski ST HP-RARE: a computer program for performing rarefaction on measures of allelic richness Mol Ecol 2005 5 187 189 10.1111/j.1471-8286.2004.00845.x
Machado CA Kliman RM Markert JA Hey J Inferring the history of speciation from multilocus DNA sequence data: the case of Drosophila pseudoobscura and close relatives Mol Biol Evol 2002 19 472 288 11919289
Hey J Wakeley J A coalescent estimator of the population recombination rate Genetics 1997 145 833 846 9055092
Nei M Molecular Evolutionary Genetics 1987 New York: Columbia University Press
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-6-391618803610.1186/1471-2296-6-39Research ArticleA qualitative study of the impact of the implementation of advanced access in primary healthcare on the working lives of general practice staff Ahluwalia Sanjiv [email protected] Maxine [email protected] Watling Medical Centre, 108 Watling Avenue, HA8 0NR, London, UK2 Faculty of Health and Human Sciences, University of Hertfordshire, College Lane, Hatfield, AL10 9AB, UK2005 27 9 2005 6 39 39 13 5 2005 27 9 2005 Copyright © 2005 Ahluwalia and Offredy; licensee BioMed Central Ltd.2005Ahluwalia and Offredy; 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 North American model of 'advanced access' has been emulated by the National Primary Care Collaborative in the UK as a way of improving patients' access in primary care. The aim of this study was to explore the impact of the implementation of advanced access on the working lives of general practice staff.
Methods
A qualitative study design, using semi-structured interviews, was conducted with 18 general practice staff: 6 GPs, 6 practice managers and 6 receptionists. Two neighbouring boroughs in southeast England were used as the study sites. NUD*IST computer software assisted in data management to identify concepts, categories and themes of the data. A framework approach was used to analyse the data.
Results
Whilst practice managers and receptionists saw advanced access as having a positive effect on their working lives, the responses of general practitioners (GPs) were more ambivalent. Receptionists reported improvements in their working lives with a change in their role from gatekeepers for appointments to providing access to appointments, fewer confrontations with patients, and greater job satisfaction. Practice managers perceived reductions in work stress from fewer patient complaints, better use of time, and greater flexibility for contingency planning. GPs recognised benefits in terms of improved consultations, but had concerns about the impact on workload and continuity of care.
Conclusion
AA has improved working conditions for receptionists, converting their perceived role from gatekeeper to access facilitator, and for practice managers as patients were more satisfied. GP responses were more ambivalent, as they experienced both positive and negative effects.
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Background
Patients' access to general practitioner (GP) appointments has been a key concern of the current British Labour government. Two national surveys [1,2] published in 1998 and 2000 highlighted the difficulty in accessing GPs, citing inconvenient surgery hours and long waiting. The government's response to this was to promise that "by the year 2004, patients will be able to see a primary care professional within 24 hours and a GP within 48 hours" [2]. Managing patients' requests for appointment has become increasingly important in general practice, as it is a national and local imperative, and features in the new GP contract in the United Kingdom (UK).
The National Primary Care Development Team (NPCDT) was set up to deliver the government's modernisation agenda in primary care by using the National Primary Care Collaborative (the Collaborative) to implement change. A key role of the Collaborative is to work with general practices and primary care trusts (PCTs) to help them modernise their services to better meet the needs of their patients. A priority for the Collaborative is improving access to primary care using the Advanced Access (AA) model, developed in the United States (US) [3-5]. Murray and Tantau's [5] solution for addressing the problem is borrowed from queuing theory and lean thinking, which are used in engineering and manufacturing, respectively. The underpinning principle is "doing today's work today". The solution is based on five principles, namely:
• understand the access demand on the practice
• clear the backlog of appointments
• review the appointment system
• develop contingency plans
• widen the mode of patient consultation.
The literature on AA focuses on: Improving access [5,6]; standards of access to quality in primary care [7,8]; drivers and barriers to implementing AA [9-11]; continuity of care versus quick access [12,13] and evaluation of AA [14]. Few of these articles provide empirical data. Those that provide empirical data demonstrate that AA improves access for patients to see GPs [14], AA adversely impacts on the ability of patients to see a GP of their own choice [13], and describes the barriers and drivers for implementing AA [11].
A wealth of literature suggests that the implementation and sustainability of innovations is dependent upon the perceptions of the users (patients and staff) of that innovation [15-17]. Moreover, the link between quality of working life and staff recruitment, retention, morale with ensuing effects on patient care is well recognised, and reflected in recent UK government policy [2,18]. There is, however, a real paucity of research into the impact of AA on the quality of working life amongst general practitioner staff. One questionnaire study explored the problems associated with working with the Collaborative and Advanced Access but to date there are no data on the effect of AA on other practice staff. Mays and Pope [19] suggest that qualitative methods are especially useful for understanding the perspectives of staff affected by health service reform. This research is therefore timely in its concentration on understanding the perceptions of receptionists, practice managers and GPs in relation to AA and its impact on their working lives.
AIM
The purpose of this study was to explore staff's perspectives of the effect of introducing AA in their general practice on their workload and job satisfaction.
Methods
Settings
The study site was two boroughs situated in the south east of England with a resident population of approximately 207,000 and 306,000 people [20]. Both boroughs have ethnically and socio-economically diverse populations. The major causes of death in the boroughs are circulatory disorders and cancer. The six practices (table 1) in which the research was conducted were in different parts of the borough and represented different levels of deprivation and ethnic mix. Prior to the implementation of AA, the waiting time for a routine appointment in these practices was up to 10 days. After implementation the waiting time for an appointment to see a healthcare professional was less than 48 hours.
Table 1 Characteristics of the study's general practices
Practice No. of patients Number of GPs Full-time equivalent (fte) GPs Patients per fte No. of PMs No. of practice nurses Nurse roles Number of receptionists
A 11,700 6 6 1950 2 3 Chronic disease management 8 (part-time)
B 7,200 4 4 1800 1 2 Chronic disease, minor illness, telephone triage 3 (part-time)
C 6,800 2 2 3400 1 2 Chronic disease management 4 (part-time)
D 12000 7 5.5 2180 1 4 Chronic disease management 12 (part-time)
E 15000 9 7 2140 1 4 Chronic disease management, minor illness, telephone triage 14 (part-time)
F 10500 5 4.5 2330 2 3 Chronic disease management, minor illness, telephone triage 10 (part-time)
The practices varied in size from 7000 to 15000 patients. The numbers of doctors in the practices varied from 2 to 9. The number of patients per full time equivalent GP varied from 1800 to 3400. 5 practices were training practices. 4 practices had previously participated in research. For comparison, the average list size by weighted time equivalent for England was 1956 in 2003 [21] and median number of partners was between two and three [21].
Ethical issues
Ethical approval was granted from the relevant research ethics committee covering the boroughs' two PCTs. Written consent was obtained from all participants prior to inclusion in the study.
Participants
The selection of the six practices was purposive, reflecting the need for these practices to have implemented AA to meet the government's 48-hour access target. From all the practices invited to attend, one practice refused on the grounds that they had not implemented AA. None of the individual healthcare professionals approached for this study refused to participate. The researchers (SA & MO) attended practice meetings to inform the practice staff about the project and to seek their cooperation. GPs and practice managers with lead involvement in the implementation of AA in their practices were chosen for interview to ensure that they could share their experiences of advantages and disadvantages of previous appointment systems and AA. The receptionists on duty on the day of visit were asked to participate in the study. A total of 18 staff participated: 6 GPs, 6 practice managers and 6 receptionists. Information leaflets about the study were given to each participant.
Interviews
Semi-structured interviews were conducted with all participants, at a time and place convenient to them. The interviews were conducted informally and in a conversational style, which encouraged expansion of ideas. Each interview was tape-recorded and transcribed verbatim. The issues covered in the interviews are indicated in figure 1.
Figure 1 Interview schedule.
Analysis
Participants were given a number to protect their identity. Tape recordings generated by the 18 participants were transcribed verbatim using framework analysis [22] to identify concepts, categories and themes in the data. This analytic process provides systematic and visible stages of data analysis, broadly divided in to five stages shown in figure 2. It was developed in the context of applied policy research to meet specific information needs and provide outcomes or recommendations, often within a short timescale [23]. NUD*IST [24] software was used to manage the transcripts.
Figure 2 Data analysis stages.
Several approaches were used to ensure the quality of the research (figure 3). Validity was ensured by respondent validation, triangulation of data with literature and field notes. Generating an audit trail, keeping systematic field notes, and asking external researchers to code the data for a subset of the transcripts were ways of ensuring reliability. Where there were discrepancies these were discussed with the researchers. These methods of ensuring quality of research are consistent with established practice [25,26].
Figure 3 Approaches used to ensure the quality of the research.
Results
The effects of AA are presented according to its impact on the working lives of receptionists (figure 4), practice managers (figure 5) and GPs (figure 6).
Figure 4 Themes for receptionists.
Figure 5 Themes for practice managers.
Figure 6 Themes for GPs.
Receptionists
From gatekeeper to access facilitator
Receptionists commented on changes in their levels of stress that took place with the introduction of AA. A significant problem for receptionists (R) under the previous appointment booking system was the stress and heightened tension encountered with patients, particularly when there was a shortage of appointments. This led to confrontations between receptionists and patients. Receptionists thus felt that patients perceived them as barriers or gatekeepers to being able to access GPs.
"It was very uncomfortable for receptionists to tell them about the waiting time because the patients saw it as our fault. We tended to get the blame; we took the brunt of it because when patients could not get to see the doctor of their choice and they did not want to wait 3–4 weeks because their problem is immediate, their aggravation and frustration would be taken out on us" (R2).
The way in which receptionists saw the change in their jobs with the introduction of AA was also enlightening. With the removal of the pressure of being seen as barriers to appointment, receptionists were able to adopt a role offering patients choice and access. They viewed their job as facilitating patients in being able to make appropriate appointments for their needs. The excerpt below underlines the point:
"With this system you can offer them (patients) an appointment on the day; it may not necessarily be the doctor of their choice though. You always finish the conversation with offering the patient an appointment. It is then their choice whether they take it or not... So I go off the phone feeling that I have offered them everything – three appointments – if it doesn't fit into their busy schedule then that is their choice. I am not denying them a health care professional" (R4).
Stress reduction through reduced confrontation with patients
Receptionists reported that the implementation of AA had reduced the level of stress of dealing with the public. With the implementation of AA long waits for an appointment to see a GP had disappeared. As a result, receptionists did not feel the need to have to triage patients, thereby being perceived as barriers or gatekeepers by patients. As a result, confrontations with patients had abated and receptionists were less stressed.
"For us receptionists, it has made our lives easier because patients phone up in the morning and they say can I have an appointment, and we say yes that's fine. The turnaround is much quicker; we are not on the phone for so long. We don't have to triage patients, which really is unacceptable. So it's made life much easier for us" (R5).
Improved sense of control over working day
Prior to the implementation of AA in their practices, receptionists would spend the majority of their time answering telephones, and would try and fit the rest of the job at quieter times. With the introduction of AA, receptionists found that demand for appointments was greatest early in the morning, which meant that they had a lot more time during the rest of the day, to complete their remaining duties, giving them a greater sense of control over their working day.
"But now you get the bulk of it (telephone calls) in the morning and then you get a set time of it in the afternoon, so it weighs itself out a little bit better.... Yes we can decide when to do scripts,(prescriptions) the rota and spend time showing the receptionists how to do things and sort their training out". (R4)
Greater job satisfaction
The shift in receptionists' views of their job from having to act as a barrier to GP appointments (prior to the introduction of AA) to becoming facilitators of choice for patients in provision of appointments (with the introduction of AA) and greater control over their working day increased their sense of job satisfaction as indicated by a receptionist:.
"It is so much better when you can offer the patient an appointment on the day; you come off the phone feeling satisfied rather than the hassle you used to have bargaining with them about how ill they are and why can't they come tomorrow" (R2).
Improved working relationships
Other benefits of implementing AA included improved working relationships as a consequence of feeling less stressed. Less confrontation at the reception desk with patients meant that receptionists felt they had a greater capacity to cope with the demands placed upon them by other members of the practice team. This led to better relationships between team members.
"I think the receptionists are far more relaxed and are relating to everyone else in a much more relaxed manner instead of being uptight because of patients. When someone (doctor or nurse) comes from a room the receptionists do not respond in such an uptight manner. It has definitely eased everything" (R3)
"Some of us are even smiling more, even with patients!" (R6)
Practice managers
Better use of time
Prior to the introduction of AA, practice managers perceived high stress levels arising from the appointments system. The practice managers perceived several reasons for this stress. There was a high bureaucratic workload and time spent having to deal with complaints from patients as a result of poor access to GPs, pressure from having to cancel booked surgeries or find locums at short notice, and having to support stressed receptionists. The practice managers in this study reported that the introduction of AA improved their bureaucratic workload and use of time. They perceived a reduction in the numbers of complaints from patients. This had the effect of reducing paperwork and overall workload. It also meant that practice managers perceived patients were happier with the new appointments system.
"The advantages of the system to myself, is I don't get the complaints, it is a lot easier to organise, holidays with doctors, everyone is having a bit of a better life" (PM1).
Managing emergencies better
Coping with sudden illness or other unforeseen circumstances was easier and less stressful with AA than with the previous appointment booking system as exemplified below.
"As there are only a few pre-bookable appointments each session, it is much easier to rearrange appointments. Before this system (i.e. AA) came in, it was simply hell trying to cancel appointments because you know that you would be getting a lot of abuse from the patients" (PM4)
Planning service provision
Reductions in workload, stress from having to manage appointment crises at short notice, and reduced pressure from having to support staff meant that for the practice managers in the study were able to spend more time planning for the future.
"The advantage of the system is that I can plan in advance. I know exactly how many appointments we are going to have to offer each day and therefore I can plan if we need extra cover particularly for annual leave and training days." (PM5).
Improved job satisfaction
The overall impact of implementing AA meant practice managers perceived improvements for staff and patients. This in turn created a sense of job satisfaction for practice managers.
General Practitioners
GPs in the study described the impact of implementing AA by its effect on the consultation between doctor and patient, fear of loss of autonomy, its impact on continuity of care, and anxieties about the future of general practice.
1 Impact on the doctor patient consultation
The implementation of AA had reduced the time it took for a patient to be seen by a GP of their choice. As a result the GPs in this study perceived themselves as dealing with fewer complaints about long waits for an appointment or dealing with long problem lists (as patients were now better able to get an appointment quickly). They felt they were seeing patients nearer the time of their appointment and this improved the quality of the consultation.
2 Increased workload for GPs
GPs perceived consultation rates as being higher with the additional burden of seeing more patients and therefore longer surgeries, as indicated below:
"..we find that because people are actually ringing in on the day when we run out of appointments, we feel we actually need to increase the number of appointments to accommodate them We often do that, and it just ends up with us seeing a lot more patients than we used to."(GP1)
Higher perceived consultation rates occurred for several reasons. These included fewer missed appointments with AA, improving access exposing unmet need for GP care, minor illness presenting earlier and mismatches between demand for appointments and supply of appointments.
2a Difficulties with doctor flexibility
GPs understood the need for the appointments system to be closely related to the profile of demand for appointments from patients. However, this required doctors to be flexible with their time so that they did surgeries at times of high demand for appointments. However, such flexibility from doctors was limited. This was because of non-clinical commitments such as training, management, family and childcare issues.
"We (GPs) all try to be flexible; however, if there are partners who have got children and have other commitments, it is difficult for them to swap around (their sessions)" (GP2)
This meant that there was not enough doctor time at busy times to meet the demand for appointments requiring doctors to work longer sessions during these busy times.
2b Earlier presentation of minor illness
Higher consultation rates also appeared to be partly related to earlier presentations of minor and self-limiting conditions. With the previous appointments systems long waits to get an appointment meant that patients with self limiting conditions would have got better by the due date of their appointment. With AA, this delay had disappeared, and therefore patients were being seen much sooner. This was seen as a source of frustration by GPs.
"We are going back to, 'I will just check before the weekend', or they will come in with a common cold and I will say "what would you like me to do for you? You have come with a cold". You feel they really need to see the Nurse Practitioner, not a GP."(GP2).
3 Impact on continuity of care
The GPs in the study described the impact of AA on continuity of care. They described how AA affects continuity of care because patients were able to see a doctor or other healthcare professional other than their registered doctor; and there were difficulties gaining an appointment with a doctor of the patient's choice.
The idea of a single GP providing continuous care appeared to be changing. As a consequence, GPs found that they needed to re-assess patients every time, which they regarded as stressful and labour-intensive.
"She (the patient) doesn't really care whom she sees, and she doesn't perceive it as being important who she sees. But then there's tremendous work for doctors, picking up each other's work. The doctors are used to seeing the same patients coming back to them so they know where they are, and they know where they stand. Often you are starting a fresh all the time with patients we don't always know" (GP2).
Other GPs replaced the idea of continuous care by a single GP with continuous care provided by a team of practitioners.
"If there is somebody with an acute illness that may be related to their chronic illness, does it matter that they see somebody else? It only matters if you don't keep good clinical records and if you don't communicate with your partners." (GP1).
A combination of increased perceived workload and the erosion of continuity of care meant that GPs feared a loss of autonomy over their working lives.
4 Anxiety about the future of general practice
The perceived increase in minor illness and workload meant that practices had to consider alternative ways of providing healthcare. Various options were considered by the practices studied. These included the use of telephone triage, self-help and education material for patients, nurse telephone advice and triage, pharmacist and nurse consultations for minor illness, and providing consultations by alternative means such as emails.
"We have a very successful nurse advice line which the doctors do use to some extent to follow up a patient. However, we really do need more time for that. So we are looking to develop the advice line in the afternoon and that the nurse is available to do triage in the afternoons."(PM2)
These alternatives to GP-led healthcare suggested that in the long term GPs would stop being the frontline providers of healthcare. Such a role would be taken over by nurses and other professional groups. GPs expressed a fear that the holistic nature of providing care in the future would be replaced by a system where doctors became specialists caring for chronic disease.
"The nature of general practice will change because the patient will not have continuity of care. They will not come to you for their minor bits and pieces (as with the previous appointments system), they will only come for the major things, and GPs will become consultants in general practices. I think that is probably the way it is going to go. It will change the exclusive character of British general practice, which I think will be sad." (GP3).
With the implementation of AA, GPs perceived doctors popular with patients being busier than GPs less popular. GPs were seeing a greater variety of patients. However, because the demand for appointments was greater than the supply of appointments, many patients with long term relationships were being seen by other GPs and healthcare professionals. In some cases it has encouraged better use of the whole team whilst maintaining flexibility for doctors to be able to see whoever they felt was appropriate.
"It has meant for me that some of my hangers-on are prepared to see other people, so we are using the whole team and they are beginning to build relationships with the other members of the team, which is really positive for me. I don't feel bereaved over the loss of these patients." (GP4).
Discussion
Summary of main findings
This paper has elucidated some of the advantages and disadvantages to general practice staff arising from the implementation of AA in their practices. Whilst practice managers and receptionists see AA as having a positive effect on their working lives, GPs had a more ambivalent reaction.
For GPs in this study, the perception of stress from patient contact has reduced because patients have complained less about poor access; they now attend the consultation with fewer, more trivial complaints. Other benefits included better time management and better use of the whole team, particularly for doctors perceived as being popular with patients. Concerns included a perception of increased workload, related to fewer non-attendances by patients, earlier presentation of minor illness and difficulties in getting doctors to work more closely to the profile of demand from patients causing a mismatch between demand for appointments and supply. Other concerns were the effect of AA on continuity of care, and the impact of AA on the future of GP work. These concerns seemed to reduce job satisfaction and engendered a fear of loss of autonomy.
Implications of this study and relationship to other work
Higher perceived workload
This study suggested that GPs perceive their workload as having increased with the advent of AA. This is consistent with the British Medical Association's survey of GP workload in 2001 [27], and Pickin et al's [14] study on AA in UK general practice. It is unclear from this study whether AA has fuelled the increase in workload and demand for appointments, or whether AA has permitted the unmasking of previously unmet healthcare related needs. It is also unclear what the consequences are from a resource perspective with increasing workloads.
AA has had the perceived impact of increasing GP workload, increasing patient demand for appointments, and changing the way care is provided by GPs so that the greater variability in case-mix along with fragmentation of care become sources of increased stress and reduced job satisfaction. Gosden et al [28] have shown that major work-related stressors for GPs include increasing patient demand, and increasing workloads. Similarly, less control over how care is provided is a significant factor in reducing job satisfaction.
Continuity of care
Some GPs, in this study, defined continuity of care as being provided by individual GPs. These doctors felt that AA had eroded the personal relationship between doctor and patient. By contrast, other GPs defined continuity of care as being provided by groups of healthcare professionals. These GPs did not see AA as adversely affecting continuity of care. Windridge et al [13], in their qualitative patient study, highlighted that AA adversely impacted on the ability of patients to see a GP of their choice. Yet, doctors and patients regard continuity of care as being important [29]. This contrasts with government policy to increase interprofessional working, and offer patients greater choice in accessing healthcare [30-33].
Impact on practice managers
The beneficial effects of AA in reducing work-related stress for practice managers are particularly welcome in view of the previously identified causes of psychological morbidity in this group [34].
Strengths and limitations of the study
The study is the first of its kind to provide qualitative insights into the working lives of GPs, practice managers and receptionists following the introduction of AA in their practices. It highlights that the effects on GPs remain unclear and there is need for further research in this area. The practices selected for this study were diverse and not dissimilar to the profile of English general practices overall. The interviewers were independent and not perceived as having a vested interest. Outside researchers validated the analytical framework. However, its limitation is that the six practices used in the study may not be representative of the practices that have implemented AA because they could be seen as early adopters of change, even though they included ethnically and socio-economically diverse populations. It is possible that these practices were influenced by the same PCT policies and PCT access facilitator, thereby reducing the potential generalisability of such a study. The focus on the views of doctors who had a lead involvement in introducing AA may have introduced perceptions more favourable than otherwise.
Conclusion
AA has improved the working conditions for receptionists, converting their perceived role from gatekeeper to facilitator, and practice managers were also more satisfied with their jobs. GP responses were more ambivalent, as they experienced both positive and negative effects.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SA and MO designed the study, carried out the analyses, and contributed equally to the writing of this article.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We are indebted to the participating practices' staff whose commitment made this work possible. NOCTEN is thanked for their financial support. Thanks are due to Professor Chris Salisbury and Dr Mark Harris for their helpful and constructive comments. We are grateful to Dr Elizabeth Murray for her guidance and advice in writing this article.
==== Refs
Airey C Errens B National Surveys of NHS Patients: General Practice 1998 1999 London , NHS Executive 10350446
DoH The NHS Plan
Kendrick T Kerry S How many surgery appointments should be offered to avoid undesirable numbers of 'extras'? Br J Gen Pract 1999 49 273 276 10736903
Murray M Patient care: access British Medical Journal 2000 320 1594 1596 10845975
Murray M Tantau C Same-day appointments: exploding the access paradigm Fam Pract Manag 2000 7 45 50 11183460
Freeman GK Horder JP Howie JG Hungin AP Hill AP Shah NC Wilson A Evolving general practice consultation in Britain: issues of length and context Bmj 2002 324 880 882 11950738 10.1136/bmj.324.7342.880
Murfin D Standards of access and quality in primary care J R Soc Med 2001 94 Suppl 39 43 45 11383430
Campbell S Steiner A Robison J Webb D Raven A Roland M Is the quality of care in general medical practice improving? Results of a longitudinal observational study Br J Gen Pract 2003 53 298 304 12879830
Longridge L Divert the demand Primary Care Management 2001 11 28 30
Lamb A Why advanced access is a retrograde step. Br J Gen Pract 2002 52 1035
Solberg LI Hroscikoski MC Sperl-Hillen JM O'Connor PJ Crabtree BF Key issues in transforming health care organizations for quality: the case of advanced access Jt Comm J Qual Saf 2004 30 15 24 14738032
Stoddart H Evans M Peters TJ Salisbury C The provision of 'same-day' care in general practice: an observational study Fam Pract 2003 20 41 47 12509369 10.1093/fampra/20.1.41
Windridge K Tarrant C Freeman GK Baker R Boulton M Low J Problems with a 'target' approach to access in primary care: a qualitative study Br J Gen Pract 2004 54 364 366 15113520
Pickin M O'Cathain A Sampson FC Dixon S Evaluation of advanced access in the national primary care collaborative Br J Gen Pract 2004 54 334 340 15113514
Kitson A Harvey G McCormack B Enabling the implementation of evidence-based practice: a conceptual framework. Qual Health Care 1998 7 149 158 10185141
Meyers PW Sivakumar K Nakata C Implementation of industrial process innovations: factors, effects, and marketing implications. Journal of Product Innovation Management 1999 16 295–311 10.1016/S0737-6782(98)00044-7
Gustafson DH Sainfort F Eichler M Adams L Bisognano M Steudel H Developing and testing a model to predict outcomes oforganizational change Health Services Research 2003 38 751 776 12785571 10.1111/1475-6773.00143
DoH Improving working lives in the NHS
Pope C Mays N Qualitative Research: Reaching the parts other methods cannot reach: an introduction to qualitative methods in health and health services research BMJ 1995 311 42 45 7613329
ONS Census 2001: report for England and Wales 2001 London , Office for National Statistics
RCGP Profile of UK practices 2004 London , Royal College of General Practitioners
Spradley J The ethnographic interview 1979 New York , Holt, Rhinehart and Winston
Ritchie JSL Bryman , Burgess Qualitative data analysis for applied policy research Analysing Qualitative Data 1994 London , Routledge 173 194
Qualitative Solutions and Research NUD*IST Non-numerical Unstructured Data Indexing Searching and Theorising (QSR NUD*IST 4.0) 1997 London , Sage
Mays N Pope C Qualitative research in health care: Assessing quality in qualitative research BMJ 2000 320 50 52 10617534 10.1136/bmj.320.7226.50
Silverman D Doing qualitative research- A practical handbook 2000 London , Sage
BMA National Survey of GP opinion 2001 London , British Medical Association.
Gosden T Williams J Petchey R Leese B Sibbald B Salaried contracts in UK general practice: a study of job satisfaction and stress J Health Serv Res Policy 2002 7 26 33 11822258 10.1258/1355819021927647
Freeman G Hjortdahl P What future for continuity of care in general practice? Bmj 1997 314 1870 1873 9224130
DoH The New NHS: Modern and Dependable 1997 London , Department of Health
DoH Health D A First Class Service: Quality in the New NHS 1998 London , HMSO
DoH Health D Working together- Learning together 2001 London , HMSO
DoH Health D Building on the Best- Choice, Responsiveness, and Equity in the NHS 2003 London , HMSO
Sheikh A Hurwitz B Psychological morbidity in general practice managers: a descriptive and explanatory study Br J Gen Pract 2000 50 203 206 10750229
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-311617908710.1186/1471-230X-5-31Research ArticleComplement activation capacity in plasma before and during high-dose prednisolone treatment and tapering in exacerbations of Crohn's disease and ulcerative colitis Zimmermann-Nielsen Erik [email protected]ønbæk Henning [email protected] Jens Frederik [email protected] Gunnar [email protected] Ole [email protected] Department of Surgery K, Hospital of Funen, Svendborg, Valdemarsgade 53, Denmark 5700 Svendborg2 Department of Medicine V, Aarhus University Hospital, Nørrebrogade 44, Denmark 8000 Aarhus C3 Department of Surgical Gastroenterology, Haukeland University Hospital, Norway Bergen4 Department of Surgical Gastroenterology A, Aalborg Hospital, Hobrovej 18-22, Postbox 365, Denmark 9100 Aalborg2005 22 9 2005 5 31 31 7 3 2005 22 9 2005 Copyright © 2005 Zimmermann-Nielsen 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
Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by intestinal inflammation mainly caused by a disturbance in the balance between cytokines and increased complement (C) activation. Our aim was to evaluate possible associations between C activation capacity and prednisolone treatment.
Methods
Plasma from patients with exacerbations of UC (n = 18) or CD (n = 18) were collected before and during high dose prednisolone treatment (1 mg/kg body weight) and tapering. Friedman's two way analysis of variance, Mann-Whitney U test and Wilcoxon signed-rank sum test were used
Results
Before treatment, plasma from CD patients showed significant elevations in all C-mediated analyses compared to the values obtained from 38 healthy controls (p < 0.02), and in mannan binding lectin (MBL)-concentration and MBL-C4-activation capacity (AC) values compared to UC patients (p < 0.02). Before treatment, plasma from UC patients showed significant elevations only in the classical pathway-mediated C3-AC compared to values obtained from healthy controls (p < 0.01). After treatment was initiated, significant reductions, which persisted during follow-up, were observed in the classical pathway-mediated C3-AC and MBL-C4-AC in plasma from CD patients (p < 0.05).
Conclusion
Our findings indicate that C activation capacity is up-regulated significantly in plasma from CD patients. The decreases observed after prednisolone treatment reflect a general down-regulation in immune activation.
==== Body
Background
The complement (C) system consists of more than 20 proteins and a number of cell associated regulator molecules and receptors [1]. The C system is activated through either of three pathways, initiated by e.g. microorganisms, immune complexes (IC), and tissue injuries.
The classical pathway (CP) is initiated by IgM and IgG-molecules, bound to antigens, which trigger the activation of proenzymes leading to cleavage of C2 and C4, and eventually to cleavage of C3.
Cleavage of C3 is a key reaction in the C sequence as the classical and alternative pathways (AP) converge here, and from this point on potent anaphylactic and chemotactic C split-products are generated. Alternative pathway activation is initiated by CP-generated C3b or by a continuous low 'tick-over' activation of C3, generating C3b which binds randomly to available cell surfaces. Mannan-binding lectin (MBL) is the only known collectin known to activate the C system and binds multivalently to terminal mannose, N-acetylglucosamine, glucose and fucose on yeast cells and Gram-negative bacteria, thereby promoting phagocytosis of the microorganisms without the involvement of antibodies.
The MBL-pathway converges with the CP at the level of C4 which may be activated to generate C4b, which binds randomly to available surfaces. The activity of C, and especially C3b/iC3b and C4b/iC4b, is strictly regulated by proteins present ubiquitously in fluid-phases and expressed on almost all cell surfaces. Further insights into C continue to emerge, e.g. that MBL utilizes two specific serine proteases (MASP-1, MASP-2) to initiate the activation of C, and the function of these proteases may influence the pathogenesis of more diseases.
A role of the C system in the pathogenesis of ulcerative colitis (UC) and Crohn's disease (CD), or in maintaining inflammation, has been established by several observations: Mucosal cells show a down-regulation in their expression of C regulators within the affected areas of UC and CD [2]. A concomitant occurrence of IC and C activation has been demonstrated in plasma from UC and CD patients during clinical exacerbations [3-5]. Deposits of several C components in colonic mucosal are found to correlate significantly to the degree of inflammation in UC and CD [6,7]. A putative auto-antigen is demonstrable in colonic mucosal and in the extraintestinal tissues affected by UC [7,8], this auto-antigen may trigger C activation. The C split-product C5a may participate in the formation of the granulomas observed in colonic tissue affected by CD [9]. Thus, both AP and CP-mediated C activation have been suggested to be involved in UC and CD [10].
Glucocorticoids suppress inflammatory processes, e.g. by down-regulation of the transcription/translation of proinflammatory cytokines such as tumor necrosis factor alpha and interleukin-6 [11], presumeable by an inhibition of nuclear factor kappa B [12]. The effects of glucocorticoids on C activation have been reported inconsistently. This is partly explained by the dependency on the type and dosage of glucocorticoid administered, and the biphasic response in C activation with time after administration [13]. Glucocorticoids may enhance the synthesis of C1-inhibitor and interfere with the function of C3 convertases, thereby exerting an anti-complementary effect [14-16]. Glucocorticoids may inhibit C-mediated chemotaxis [17].
Measurement of C activation has been impeded by the complexity, lability and reactivity of the system. The methods currently in use for routine analyses show rather low sensitivity, and do normally not differentiate between the pathways, and the serum concentration of MBL varies considerably amongst individuals influenced by mutations and polymorphism in the promoter region [18]. We have standardized two functional assays which measure the C activation capacity of the classical, alternative and MBL pathways in vitro as function of incubation time. Our aim was to evaluate C in plasma before and during high-dose prednisolone treatment and tapering to analyze for a possible association between C activation capacity and treatment.
Methods
Patients with clinical exacerbation of UC (n = 18) or CD (n = 18), necessitating hospitalization and requiring high-dose prednisolone to induce remission, were included consecutively at the Medical Department V, Aarhus University Hospital in the period December 2000 to July 2001. Patients were excluded by the presence of obvious bacterial infection or acute abdomen.
All patients had a well-established diagnosis of UC or CD according to clinical, biochemical, endoscopy, histopathological criteria, and small bowel follow through.
The group of patients with UC consisted of 6 women and 12 men, median 39 years of age (range 21–78). There were 12 women and 6 men among the patients with CD, median 29 years of age (range 18–78).
The disease activity was evaluated clinically by the Harvey-Bradshaw Index in both UC and CD patients [19]. The UC patients had a median score of 7 (range 5–19), and CD patients revealed a median score of 7 (range 3–24). Seventeen UC patients had extensive colitis (pancolitis) and 1 left sided colitis. Patients with CD had either colon affection, small bowel affection, or a combination.
At inclusion, UC patients were treated with combinations of steroids (rectal enema)(n = 6), 5-aminosalicylic acid (n = 12), antibiotics (n = 1), whereas 6 patients received no immunomodulatory medication at all. The CD patients were treated with combinations of steroids (rectal enema)(n = 1), 5-aminosalicylic acid (n = 7), azathioprine (n = 1), antibiotics (n = 1), whereas 4 patients received no immunomodulatory medication at all.
All treatments except for rectal steroid enema (except for 1 patient) and antibiotics were continued. The treatment with prednisolon was started instantly, and administrated intravenously for the first 3–7 days (1 mg/kg body-weight), followed by oral administration by day 7 where the dose were tapered to 40–60 mg/day, and further tapering typically 5 mg/week until 0.
Blood samples were collected from all patients before high-dose prednisolone treatment was initiated, after 7 days of high-dose prednisolone treatment, after 6 weeks when the prednisolone dosage was approximately 0.5 mg/kg, and after 12 weeks when prednisolone was stopped for most patients (except in 6 UC- and 4 CD patients). The patients still treated with prednisolon after 12 weeks were all well characterized by clinical and/or biochemical relapse during the last tapering period.
Plasma samples from healthy blood donors (n = 38) from the Department of Clinical Immunology, Aalborg Hospital were included as controls. In addition, plasma samples from some of these donors were heat inactivated at 56°C for 30 min and used as negative internal controls.
The blood samples were collected in citrate tubes by venepuncture without stasis, placed on ice for a maximum of 10 min, and centrifuged at 4°C and 2,000 g for 20 min. Duplicate blood samples were collected in glass tubes and allowed to clot at room temperature before centrifuged. Plasma/serum was withdrawn and stored in aliquots of 0.5 ml at minus 80°C.
Plasma/serum samples were analyzed for alternative- and classical C pathway mediated factor C3 activation capacity (C3-AC), mannan-binding lectin (MBL), MBL-C4-AC, leucocyte count, C-Reactive Protein (CRP) and orosomucoid.
The AC-derived measurements were performed at the Surgical Gastroenterological Research Unit whereas the MBL-assay was performed as a routine method at the Department of Clinical Immunology, Aalborg Hospital, as described in details elsewhere [20-22].
C3-AC-assay
The enzyme linked immunosorbent assay (ELISA) measures plasma derived C3b/iC3b deposition on IC during in vitro C activation. The activation capacity of the C system is defined as the amount of C3b/iC3b generated and bound to the solid-phase IC as a function of incubation time. The assay differentiates between activation mediated by AP and CP. Briefly, the plasma was diluted 1/5 in MgEGTA-buffer (10 mM Mg2+ and 10 mM EGTA) when measuring the AP and 1/200 in CaMg-buffer (0.30 mM Ca2+ and 1.0 mM Mg2+) when measuring the CP. The AP is activated at low plasma dilutions only and the CP activation requires Ca2+. Upon C activation, the generated C3b molecules bind covalently to the IC and are eventually degraded to iC3b. Bound C3b/iC3b fragments were detected by the addition of biotinylated rabbit anti-C3c-antibodies (Dako, Glostrup, Denmark), avidin alkaline phosphatase, and para-nitrophenylphosphate as enzyme substrate. In standard dose response curves the absorbance values for plasma dilutions 1/5 (AP) and 1/200 (CP) were designated 100%. Plasma from 1 healthy donor was heat inactivated at 56°C for 30 min, and used as negative control. Test samples were analyzed in duplicate and the mean value was converted to 'per cent of the standard' [20].
A second ELISA measured the plasma concentration of MBL. Briefly, microplates were coated with anti-MBL-antibodies, plasma was diluted 1/100 in dilution-buffer (6.7 g NaCl, 4.6 g NaH2PO4·2H2O, 1.1 g KH2PO4, 5 mM EDTA, 200 μl mouse IgG). Bound MBL were detected by the addition of biotinylated anti-MBL-antibodies, avidin-peroxidase conjugate, and 1,2-phenylene diamine dihydrochloride as enzyme substrate. A plasma standard diluted 1/25-1/3,200 was included in all plates. Plasma from 3 healthy donors with high, moderate and low concentration of MBL were included as controls. Test samples were analyzed in duplicate and the mean value was used in the calculations [21].
The third ELISA measured the plasma derived C4b/iC4b of the MBL-pathway deposited on mannan during in vitro C activation. Briefly, microtiter plates were coated with mannan, and plasma was diluted 1/10 in diluent-buffer (10 mM tris hydroxy aminomethan, 10 mM CaCl2, 1 M NaCl, 15 mM NaN3, pH 7.8). The assay does not measure activation of the AP at the plasma dilution (1/10) used and the CP was not initiated due to the high NaCl-concentration in the diluent buffer (1 M). Bound C4b/iC4b fragments were detected by the addition of biotinylated rabbit anti-C4c-antibodies (Dako, Glostrup, Denmark), avidin alkaline phosphatase, and para-nitrophenylphosphate as enzyme substrate. A plasma standard diluted 1/5-1/100 was included. Test samples were analyzed in duplicate and the mean value was converted to 'per cent of the standard'. Plasma from 2 healthy donors with high and low MBL-C4-AC were included as controls. Plasma from 1 healthy donor was heat inactivated at 56°C for 30 min, and used as negative control [22].
The measurements of leucocyte count, CRP, and orosomucoid were performed by routine methods at the Department of Clinical Chemistry, Aarhus University Hospital.
Ethics
This investigation has been approved by the regional Ethical Committees of Northern Jutland and Aarhus Counties, and wasin accordance with the standards of the Declaration of Helsinki II.
Statistics
Non-parametric descriptive (median – range) and comparative statistics were used with a probality value of < 0.05 considered statistically significant. Data were analyzed by non-parametric methods. The calculations and analyses were performed with Prism 3.0 (GraphPad Software Inc., Microsoft Corp., USA). First, overall comparison over time (before treatment, 1 week, 6 weeks and 12 weeks after treatment) of a variable in the same group of patients (e.g. CD patients) was done by Friedman's two-way analysis. Secondly, paired comparison in a group of patients of a variable between two different time points (matched pair) were by Wilcoxon signed-rank test. Furthermore, the Mann-Whitney U test was used comparing one variable between two different groups at a specific time point (e.g. plasma value of MBL before prednisolone treatment in CD patients versus UC patients). Correlation between two variables were calculated using Spearman's rank correlation coefficient rho. Finally, to allow for multiple comparisons the Bonferroni correction was used, the corrected p-values are stated.
Results
All C data showed a significant variation over time (before and 1, 6 and 12 weeks after initiation of high dose prednisolone treatment) in both UC and CD patients (in both groups: p < 0.0001, Friedman's test).
Table 1 shows the C-activation
Before treatment
Plasma from CD patients revealed significant elevations in the activating capacity of all C pathways compared to the values obtained from healthy controls (p < 0.02, Mann-Whitney test). In plasma from UC patients, only the median classical pathway-mediated C3-AC was significantly elevated compared to the values obtained from healthy controls (p < 0.01, Mann-Whitney test). Plasma from CD patients revealed significant elevations in MBL-concentration and MBL-C4-AC values compared to the values obtained from UC patients (p < 0.02, Mann-Whitney test). No significant difference was observed in the alternative or classical pathway-mediated C3-AC between the patient groups.
After treatment
No significant difference was observed neither in the alternative nor in the classical pathway-mediated C3-AC between the patient groups. The MBL-concentration and MBL-C4-AC values obtained were significantly higher at week 6 and 12 in plasma from CD patients compared to UC patients (p < 0.04, Mann-Whitney test).
In CD patients
The classical pathway-mediated C3-AC and MBL-C4-AC remained significantly reduced over time in plasma from CD patients (p < 0.05, Wilcoxon test). In UC patients: No significant difference over time was observed.
Other parameters
The medians of leucocyte count, CRP, and orosomucoid which were obtained from blood/plasma of CD and UC patients are stated in Table 2. Before treatment, plasma from CD patients revealed significant elevations in CRP and orosomucoid (p < 0.04, Mann-Whitney test). After treatment, the medians of all parameters were comparable.
The degrees of correlation were calculated for all C-values against CRP, orosomucoid, leucocyte count, and the Harvey-Bradshaw index, respectively. No signification correlation was observed
Discussion
Our findings indicate that C activation capacity is up-regulated in plasma from CD patients compared to both healthy controls and UC patients, and that the decreases observed after treatment with prednisolone reflect a general down-regulation in immune activation as indicated by the concomitant trends in CRP and orosomucoid. The initial increase in leucocyte count may reflect a mobilization of premature leucocytes from the bone marrow. The demonstrated association between C activation capacity and treatment with prednisolone seems to support the finding that glucocorticoids may interfere with C activation capacity [13-16]. However, the concentrations of C components in plasma depend on a complex balance, e.g. between synthesis and degradation rates, and degree of binding to other proteins.
The C system has been suggested to be involved in the pathogeneses of UC and CD, although differently. Thus, a putative auto-antigen, with a potential to initiate the classical pathway, is demonstrable in tissues affected by UC [7,8], whereas C activation may occur through the alternative pathway in patients with CD [10]. The lectin-mediated C activation has not been considered in earlier studies of this topic.
More clinical features differ between UC and CD. Thus, UC is characterized by inflammatory infiltrations within the mucosa of the colon and rectum, whereas the infiltrations in CD may affect all layers of the intestinal wall. The clinical exacerbations, frequently necessitating hospitalization and glucocorticoid treatment, may vary widely between UC and CD, and by the duration of disease. These variations in clinical features may not be detectable with the Harvey-Bradshaw index.
Our results show more differences in plasma derived C activation capacity between patients with UC and CD, which is in concordance with the overall immune activation as indicated by CRP, orosomucoid and leucocyte count, and by the fact that CRP may activate C by initiating the classical pathway [23]. The up-regulation in C activation capacity is more pronounced in plasma from CD-patients compared to UC, in accordance with in vitro studies [24,25].
The demonstrated reduction of MBL-concentration and MBL-C4-AC in plasma from UC patients before treatment, compared to values obtained from either healthy controls or CD patients, is unexpected. Thus, the MBL gene variants, correlating with reduced MBL concentrations, occur less frequently in UC patients compared to CD patients and healthy controls [26]. However, our finding may be secondary to the use of immunomodulators at study entry, which was administered more frequently to the UC patients, or to differences in clinical features between UC and CD.
The significantly increased MBL-concentration and MBL-C4-AC values in plasma from CD patients, compared to values obtained from healthy controls, are opposite to our results in plasma from CD patients complicated by fistulizing ano-rectal disease [27] despite their comparability in disease activity as indicated by the Harvey-Bradshaw values. The discrepancy in MBL-concentration and MBL-C4-AC values of these two groups of CD-patients may be due to the circumstance that the patients with fistulizing disease were highly selected as chronic out-patients with a low degree of inflammation as indicated by CRP. In the present study only one of the CD patients had fistulizing ano-rectal disease.
The demonstrated association between C activation capacity and treatment with prednisolone seems to reflect a treatment-induced down-regulation in inflammation, as estimated by CRP and orosomucoid.
Conclusion
We found that C activation capacity is up-regulated in plasma from CD patients compared to UC patients and healthy controls. The plasma MBL-concentration and MBL-mediated C activation capacity were reduced in UC patients compared to healthy controls. The changes in C activation capacity during prednisolone treatment seems to reflect a general down regulation in immune activation especially in CD patients.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
EZ-N participated to the conception and design of the study, carried out the immunoassays, participated to analysis and interpretation of data, and drafted the manuscript. HG participated to the conception and design of the study, collection of data, and helped to draft the manuscript. JFD participated to the design of the study, and was involved in revising the article critically. GB participated to the conception and design of the study, carried out the immunoassays, participated to analysis and interpretation of data, and drafted the manuscript. OTU participated to the conception and design of the study, to analysis and interpretation of data, and was involved in revising the article. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We are indepted to Mrs. Annette Andreasen, the Surgical Gastroenterological Research Unit, Aalborg Hospital and Mrs. Bente Knøsgaard, the Department of Clinical Immunology, Aalborg Hospital for skilful technical assistance.
The work was supported by grants from Nordjyllands Amts Forskningsråd, Nordjyllands Lægekredsforenings Forskningsfond, Speciallæge Heinrich Kopp's legat, and Carla Cornelia Storck Møllers legat.
Figures and Tables
Table 1 Complement activation in plasma from patients with Crohn's disease (CD), patients with ulcerative colitis (UC) and healthy subjects (HS) before and after 1, 6 and 12 weeks high-dose treatment with prednisolone.
before treatment after 1 week after 6 weeks after 12 weeks
Number of patients Number of patients Number of patients Number of patients
CD 18 18 18 18
UC 18 18 18 16
HS 38 -- -- --
C3-AC, alternative % of standard % of standard % of standard % of standard
CD 61.5 (53–82) 51.5 (49–71) 55.5 (49–70) 59 (49–75)
UC 58 (49–78) 49 (49–70) 49 (49–78) 59 (49–70)
HS 49 (49–67) -- -- --
C3-AC, classical % of standard % of standard % of standard % of standard
CD 109 (81–172) 97.5 (39–124) 94.5 (70–141) 99.5 (53–143)
UC 97 (39–148) 88 (58–129) 91 (59–115) 94 (39–160)
HS 82.5 (68–108) -- -- --
MBL-concentration ng/ml ng/ml ng/ml ng/ml
CD 3251 (14 – 6085) 3071 (10 – 4636) 2911 (10 – 5178) 3257 (10 – 4718)
UC 1082 (6 – 3926) 1171 (3 – 4845) 1141 (3 – 4590) 1292 (3 – 4147)
HS 1219 (2 – 3577) -- -- --
MBL-C4-AC % of standard % of standard % of standard % of standard
CD 118 (21–187) 63 (25–192) 62.5 (21–163) 87 (17–192)
UC 54 (12–201) 54 (11–192) 45 (9–163) 55 (12–192)
HS 66.5 (9–162) -- -- --
Median and range (in parenthesis) of alternative and classical pathway-mediated C3-activation capacity(C3-AC), mannan-binding lectin (MBL) concentration, MBL-C4-AC.
Table 2 Acute phase reactants (median values) in plasma/blood from patients with Crohn's disease (CD), patients with ulcerative colitis (UC) before and after 1,6 and 12 weeks high-dose treatement with prednisolone.
before treatment after 1 week after 6 weeks after 12 weeks
C Reactive Protein
(nmol/L)
CD 372 47 47 62.5
UC 47 47 47 33.5
Orosomucoid
(μmol/L)
CD 39 33 28 25
UC 25 25 20 24
Leucocyte count
(109/L)
CD 10.9 13.5 9.7 8.1
UC 8.3 13.6 8.6 7.9
==== Refs
Walport MJ Review articles Advances in immunology: Complement N Engl J Med 2001 344 1058 1066 1140–1144 11287977 10.1056/NEJM200104053441406
Scheinin T Bohling T Halme L Kontiainen S Bjorge L Meri S Decreased expression of protectin (CD59) in gut epithelium in ulcerative colitis and Crohn's disease Hum Pathol 1999 30 1427 1430 10667419 10.1016/S0046-8177(99)90163-6
Nielsen H Binder V Daugharty H Svehag S-E Circulating immune complexes in ulcerative colitis. I. Correlation to disease activity Clin Exp Immunol 1978 31 72 80 346271
Nielsen H Petersen PH Svehag S-E Circulating immune complexes in ulcerative colitis. II. Correlation with serum protein concentrations and complement conversion products Clin Exp Immunol 1978 31 81 91 639352
Petersen NE Elmgreen J Teisner B Svehag SE Activation of the classical pathway complement in chronic inflammation. Elevated levels of circulating C3d and C4d split products in rheumatoid arthritis and Crohn's disease Acta Med Scand 1988 223 557 560 3389208
Ueki T Mizuno M Uesu T Kiso T Nasu J Inaba T Kihara Y Matsuoka Y Okada H Fujita T Tsuji T Distribution of activated complement, C3b, and its degraded fragments, iC3b/C3dg, in the colonic mucosa of ulcerative colitis (UC) Clin Exp Immunol 1996 104 286 292 8625522 10.1046/j.1365-2249.1996.17721.x
Halstensen TS Das KM Brandtzaeg P Epithelial deposits of immunoglobulin G1 and activated complement colocalise with the Mr 40kD putative autoantigen in ulcerative colitis Gut 1993 34 650 657 8504966
Takahashi F Das KM Isolation and characterization of a colonic autoantigen specifically recognized by colon tissue-bound immunoglobulin G from idiopathic ulcerative colitis J Clin Invest 1985 76 311 318 4019782
Cavaillon JM Fitting C Haeffner-Cavaillon N Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages Eur J Immunol 1990 20 253 257 1690130
Halstensen TS Mollnes TE Garred P Fausa O Brandtzaeg Surface epithelium related activation of complement differs in Crohn's disease and ulcerative colitis Gut 1992 33 902 908 1379568
Meduri GU Tolly EA Choussos GP Stentz F Prolonged methyl prednisolone treatment suppresses systemic inflammation in patients with unresolving acute respiratory distress syndrome Am J Respir Crit Care Med 2002 165 983 991 11934726
Auphan N DiDonato JA Rosette C Helmberg A Karin M Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis Science 1995 270 286 290 7569976
Atkinson JP Frank MM Effect of cortisone therapy on serum complement components J Immunol 1973 111 1061 1066 4728675
Jansen NJ van Oeveren W van Vliet M Stoutenbeek CP Eysman L Wildevuur CR The role of different types of corticosteroids on the inflammatory mediators in cardiopulmonary bypass Eur J Cardiothorac Surg 1991 5 211 217 1711873 10.1016/1010-7940(91)90032-F
Roeise O Garred P Mollnes TE Stadaas JO Aasen AO Methylprednisolon in high doses gives different effects on the early and the late part of complement Eur Surg Res 1990 22 41 49 2199200
Brandslund I Peters ND Ejstrup L Steroids reduce complement activation in rheumatoid arthritis Int J Tissue React 1985 7 161 165 3875589
Rhodes JM Bartholomew TC Jewell DP Inhibition of leucocyte motility by drugs used in ulcerative colitis Gut 1981 22 642 647 6116649
Madsen HO Garred P Thiel S Kurzhals J Lamm LU Ryder LP Svejgaard J Interplay between promoter and structural gene variants control basal levels of mannan-binding protein J Immunol 1995 155 3013 3020 7673719
Harvey RF Bradshaw JM A simple index of Crohn's disease activity Lancet 1980 1 514 6102236 10.1016/S0140-6736(80)92767-1
Zimmermann-Nielsen E Svehag S-E Thorlacius-Ussing O Baatrup G ELISA for incorporation of plasma derived complement split-products C3b/iC3b into solid-phase immune complexes J Immunol Methods 2001 249 43 51 11226462 10.1016/S0022-1759(00)00281-7
Super M Thiel S Lu J Turner MW Association of low levels of mannan-binding protein with a common defect in opsonization Lancet 1989 ii 1236 1239 10.1016/S0140-6736(89)91849-7
Zimmermann-Nielsen E Baatrup G Thorlacius-Ussing O Agnholt J Svehag S-E Complement activation mediated by mannan-binding lectin in plasma from healthy individuals and patients with SLE, Crohn's disease and colorectal cancer. Suppressed activation by SLE plasma Scand J Immunol 2002 55 105 110 11841698 10.1046/j.1365-3083.2002.01035.x
Claus DR Siegel J Petras K Osmand AP Gewurz H Interaction of C-reactive protein with the first component of human complement J Immunol 1977 119 187 192 17637
Reinecker HC Steffen M Witthoeft T Pflueger I Schreiber S Enhanced secretion of tumor necrosis factor alpha, IL-6, and IL-1β by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease Clin Exp Immunol 1993 94 174 181 8403503
Breese EJ Michie CA Nicholls SW Murch SH Williams CB Domizio P Walker-Smith JA MacDonald TT Tumor necrosis factor α-producing cells in the intestinal mucosa of children with inflammatory bowel disease Gastroenterology 1994 106 1455 1466 8194690
Rector A Lemey P Laffut W Keyaerts E Struyf F Wollants E Vermeire S Rutgeerts P Van Raust M Mannan-binding lectin (MBL) gene polymorphisms in ulcerative colitis and Crohn's disease Genes Immun 2001 2 323 328 11607788 10.1038/sj.gene.6363784
Zimmermann-Nielsen E Agnholt J Thorlacius-Ussing O Dahlerup JF Baatrup G Complement activation in plasma before and after infliximab treatment in Crohn's Disease Scand J Gastroent 2003 38 1050 1054 14621279 10.1080/00365520310005767
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-461617428910.1186/1471-2156-6-46Research ArticleMAOA haplotypes associated with thrombocyte-MAO activity Jansson Mårten [email protected] Shane [email protected] Patrick F [email protected] Paul [email protected] Björn [email protected] Lars [email protected] Martin [email protected] Nancy L [email protected] Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden2 Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden3 Departments of Genetics, Psychiatry & Epidemiology, University of North Carolina at Chapel Hill, NC, USA4 Department of Neuroscience, Uppsala University, Uppsala, Sweden5 Department of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden6 Department of Psychology, University of Southern California, Los Angeles, USA2005 20 9 2005 6 46 46 17 1 2005 20 9 2005 Copyright © 2005 Jansson et al; licensee BioMed Central Ltd.2005Jansson et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The aim was to ascertain whether thrombocyte MAO (trbc-MAO) activity and depressed state are genetically associated with the MAO locus on chromosome X (Xp11.3 – 11.4). We performed novel sequencing of the MAO locus and validated genetic variants found in public databases prior to constructing haplotypes of the MAO locus in a Swedish sample (N = 573 individuals).
Results
Our results reveal a profound SNP desert in the MAOB gene. Both the MAOA and MAOB genes segregate as two distinct LD blocks. We found a significant association between two MAOA gene haplotypes and reduced trbc-MAO activity, but no association with depressed state.
Conclusion
The MAO locus seems to have an effect on trbc-MAO activity in the study population. The findings suggest incomplete X-chromosome inactivation at this locus. It is plausible that a gene-dosage effect can provide some insight into the greater prevalence of depressed state in females than males.
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Background
Monoamine oxidase A (MAOA) and B (MAOB) are enzymes that deaminate monoamines such as serotonin, dopamine and noradrenaline. The genes encoding MAOA and B are located on the X chromosome in a tail-to-tail orientation and separated by approximately 20 kilobases (kb) [1,2]. Although MAOA and MAOB span 65 kb and 116 kb, respectively, both genes display a high degree of homology and most certainly have a common ancestry [3]. The frequencies of confirmed polymorphisms in the two genes vary widely among different ethnic groups [4-6]. Only two common haplotype variants of the MAOA locus were found among individuals of northern European ancestry [5].
Both enzymes are localized in the outer mitochondrial membrane [7]. They are also present in glial cells [8], although MAOA is less expressed than MAOB [9]. The enzymes differ in their expression patterns not only peripherally in the body but also in the central nervous system (CNS) [10]. MAOB is the only form that is expressed in human blood cells. MAOA is primarily expressed in catecholaminergic neurons in the human brain [10,11], whereas MAOB is expressed in serotonergic [10] and histaminergic neurons [8]. The two MAO-enzymes also differ on substrate preferences; MAOA preferentially metabolizes serotonin and norepinephrine while MAOB has a much higher affinity for phenylethylamine [12,13] and benzylamine [14].
Thrombocyte-MAO activity (Trbc-MAO) has been associated with cerebrospinal fluid (CSF) levels of serotonin metabolites in humans [15] and is higher in women than men [16-18]. This difference has been speculated to be an effect of sex steroids altering the enzyme's activity or a matter of escaped X-inactivation [19]. The proportion of variance in trbc-MAO activity explained by genetic factors (its heritability) in a Swedish population is 77% [20]. Trbc-MAO activity is weakly associated with a C/T polymorphism in intron 13 of the MAOB gene in a Swedish population [21] and is also influenced by smoking and specific medications; smokers can have a 30–40% lower trbc-MAO activity than non-smokers [22]. Trbc-MAO activity is also associated with several psychiatric syndromes [23], personality traits and mood disorders e.g. [24-28].
In the present study we address issues concerning genetic variation in MAOA and MAOB genes, activity levels of trbc-MAO, and associations with depressed state. Genetic variation was analyzed by sequencing the regulatory region of both MAOA and MAOB, and validating SNPs reported in public databases. We used multiple SNPs covering the MAO gene locus to generate haplotypes on a population level. Finally, we investigated associations between depressed state and trbc-MAO activity and genetic variants in the MAO locus in a large elderly Swedish population.
Results
Trbc-MAO activity and depressed state
We found a clearly significant difference between males (mean; 10.7) and females (mean; 12.1) (t = 4.69; p ≤ 0,0001), as well as between smokers and non-smokers in mean trbc-MAO activity (t = 5,86; p =< 0,0001). Smokers showed a 23% lower trbc-MAO activity compared to non-smokers. Females with a depressed state showed a significantly higher mean trbc-MAO activity than unaffected females (t = 2,02; p = 0,04).
Genetic variants and haplotype construction
Approximately 4.5 kb of both the MAOA and MAOB gene promotor, including the first exons, totaling 9 kb, were sequenced from a total of 148 X chromosomes. Power to discover SNPs with frequencies greater than 1% and 3% for this sample size was 77% and 100%, respectively. No variants were found in the MAOB gene. In contrast, one previously reported variation was confirmed (rs3788863) for the MAOA gene, lying within the first intron, as well as two additional variants further down stream with a minor allele frequency greater than 1%. Both the recorded and most distal variants showed complete LD with each other, therefore only the recorded variant was chosen for further analysis.
The genotyping error rate was calculated at 0,4% through males scoring as heterozygotes and from MZ twins where both twins in a pair were genotyped. These errors could not be scored differently from the sequence and therefore most likely reside in the handling of samples, e.g. contamination or labelling error.
In addition to resequencing the upstream regions, we genotyped reported SNPs in the remainder of the gene clusters by Pyrosequencing. Six of the previously reported SNPs could not be confirmed as polymorphic (rs1014876, rs3027464, rs6324, rs1040398 and two SNPs reported by Balciuniene et al.) The remaining nine polymorphic variants; one in the Norrie gene (rs766117), four SNPs in MAOB (rs1181252, rs2283729, rs3027452 and rs1799836) and four SNPs in MAOA (rs1801291, rs979605, rs6323, rs388863) were sequenced in the total sample.
The LD map (Figure 2) displays a clear structure of the MAO locus with strong LD across the MAOA gene in a distinct block spanning approximately 65 kb. The MAOB gene also displays a similar block-like structure, although the pattern of LD is not as robust as for MAOA. This is perhaps due to the inconsistencies in allele frequencies across MAOB. Interestingly, weak LD is observed at the tail ends between the two MAO loci.
Figure 2 LD map. Pair-wise LD map with one individual from each female pair (N = 356). D' is shown below the diagonal and Δ2 above the diagonal. Color code D': Red: ≥0,8 Orange: 0,5–0,8 Yellow: 0,3–0,5 White: <0,3. Color code Δ2: Red: ≥0,30 Orange: 0,1–0,30 Yellow: 0,05–0,1 White: <0,05.
Furthermore, because there was no LD between the Norrie gene variant, located >66 kb upstream of MAOB, and any other variant in the MAO region, we decided not use this variant further in the haplotype assessment. Modest deviations from Hardy-Weinberg equilibrium were noted in rs766117 in the Norrie gene (p = 0,022) and rs979605 in intron 10 of the MAOA gene (p = 0,028). This could reflect the underlying LD structure [29], as demographic influences would act over larger regions [30]. However to clarify this, a denser set of SNPs would need to be genotyped.
In the male population we could identify five distinct haplotypes in the MAOB gene and four in the MAOA gene with frequencies ≥1% (Figure 1.). When analyzing the MAO locus as one large block using eight SNPs, we found ten distinct locus haplotypes with a frequencies ≥1% (data not shown). In the female population, "PHASE" assembled identical higher frequency haplotypes as were identified in the male sample, with minor discrepancies in lower frequency haplotypes due to unknown phase (Figure 1).
Figure 1 Genetic structure of the MAO locus. Haplotype and common allele frequencies in the total sample. dbSNP rs numbers for all genotyped SNPs are presented with major allele frequencies. Haplotypes frequencies illustrated for MAOA and B separately as well as the genes combined (See Text). NDP was not used in the haplotype frequency estimations.
Associations with SNPs
In the total sample, no single variant of any of the individual SNPs was associated with trbc-MAO activity. However, in females the C/C and C/T genotypes of rs979605 in the MAOA gene were associated with a significant decrease in trbc-MAO activity, (-2,9; CI 95%: -5,2 – -0,6) and (-2,4; CI 95%: -4,7 – -0,1) respectively.
Analyzed by gender, depressed state was associated with the A-allele of MAOB SNP rs1181252 in males (OR = 4,5; CI 95%: 1,0 – 21,7) and both GG and GA of rs766117 (OR = 2,2; CI 95%: 1,1 – 4,3) in females. It should be noted that the "A" allele of rs1181252 only had a population frequency of 6%.
Associations with haplotypes
There was no association between any of the MAOB haplotypes and trbc-MAO activity. Two MAOA haplotypes, A1 and A3, both sharing identical alleles at the three first haplotype positions (CCA-) (Figure 1), were associated with a significant decrease in trbc-MAO activity (Table 1). Analyses of the entire MAO locus and trbc-MAO activity did not reveal any significant findings (data not shown). We could not find any significant associations between depressed state and any specific haplotype in men or women (Table 2).
Table 1 Associations between MAO haplotypes and trbc-MAO activity, reported as unit change in mean trbc-MAO activity per allele and controlling for gender and smoking status.
Total sample N = 340 Males N = 156 Females N = 184
per allele Hemizygous per allele
Haplotypes Estimates (Unit change in mean trbc-MAO activity per allele)
B1 -0,38 (-1,3 – 0,5) 0 (ref) -0,3 (-1,1 – 0,5)
B2 -0,63 (-1,8 – 0,5) -0,08 (-1,6 – 1,5) -0,4 (-1,5 – 0,7)
B3 -0,18 (-1,6 – 1,3) -0,8 (-2,9 – 1,2) -0,2 (-1,5 – 1,0)
B4 -1,3 (-3,5 – 0,8) 0,7 (-2,8 – 4,3) -1,0 (-2,8 – 0,8)
B5 -1,7 (-5,1 – 1,6) NA -0,7 (-3,8 – 2,4)
Male gender -2,1 (-3,3 – -0,9)*
Non-smokers 2,3 (1,1 – 3,5)* 1,5 (0,2 – 2,8) 3,5 (2,0 – 5,0)
A1 -1,1 (-1,9 – -0,3)* -1,8 (-3,2 – -0,5)* -1,0 (-1,7 – -0,3)*
A2 0,1 (-0,9 – 1,2) 0 (ref) 0,6 (-0,4 – 1,5)
A3 -3,1 (-6,1 – -0,14)* -2,3 (-5,8 – 1,2) -4,1 (-7,4 – -0,7)*
A4 -0,02 (-3,8 – 3,7) NA -0,5 (-3,7 – 2,7)
Male gender -2,3 (-3,5 – -1,1)*
Non-smokers 2,4 (1,1 – 3,6)* 1,4 (0,03 – 2,8) 3,4 (1,9 – 4,8)
Table 2 MAO haplotypes and depressive state, reported as odds ratios per allele. Without genetic information in the model male gender was significant [OR: 0,5 (0,3 – 0,8)] for depressive state. *Homozygote compared to heterozygote.
MAO haplotypes and depressive state
Total sample N = 573 Males N = 239 Females N = 334
per allele Hemizygous per allele Homozygotes*
Odds Ratio with 95% CI
B1 1,2 (0,6 – 2,5) 1,0 (ref) 1,4 (0,6 – 2,9) 1,3 p = 0,57
B2 1,5 (0,7 – 3,3) 1,7 (0,8 – 3,6) 1,5 (0,6 – 3,3) 1,2 p = 0,80
B3 1,3 (0,5 – 3,0) 0,7 (0,3 – 1,7) 1,7 (0,7 – 4,2) 1,9 p = 0,51
B4 2,0 (0,8 – 5,2) 3,7 (0,7 – 18,7) 2,0 (0,7 – 5,3) 1,7 p = 0,59
B5 0,5 (0,1 – 2,8) 2,4 (0,4 – 14,5) 0,3 (0,04 – 2,5) NA
Male gender 0,7 (0,3 – 1,5)
A1 3,0 (0,8 – 12,2) 1,0 (ref) 2,2 (0,6 – 8,4) 5,5 p = 0,08
A2 2,5 (0,6 – 10,6) 0,9 (0,4 – 1,8) 1,7 (0,4 – 7,1) 1,3 p = 0,80
A3 2,8 (0,7 – 11,4) 1,2 (0,3 – 4,4) 1,7 (0,4 – 6,9) NA
A4 3,2 (0,6 – 18,6) NA 2,8 (0,4 – 17,3) NA
Male gender 1,4 (0,3 – 6,0)
When the model was analyzed without genetic information, males have a significantly lower risk for being affected with depressed state compared to women (OR = 0,5). This gender effect may be explained by the genetic information (even though no associations were found with any of the haplotypes), because the risk for depressed state due to the male gender is differs in the analyses of the MAOA locus (OR = 1,4; non-significant) and the MAOB locus, where the estimate is similar to the model without genetic information.
Interestingly, in females all MAOB homozygote haplotypes displayed greater odds ratios with depressed state than that for heterozygotes (Table 2), indicating an additive effect. The same was true for MAOA (Table 2).
Discussion
Monoamine oxidase A and B constitute two important molecules in the human body in general and in the central nervous system (CNS) in particular. Numerous studies suggest a contribution of these two mitochondrial enzymes to complex human behaviors [26-28,31-33]. In the present study we searched the MAO locus for novel genetic variants and evaluated the genetic and haplotype structure in a Swedish population. We also assessed associations between trbc-MAO activity and depressed state, and their respective associations with the genetic structure of the MAO locus. The key findings of this study are first: the profound lack of variation at functional regions of the two MAO genes and a pattern of two distinct genetic LD blocks, one for each gene. Second: we replicated the gender differences in trbc-MAO activity and demonstrated an association between trbc-MAO activity and depressed state in women. Third: two MAOA haplotype variants were associated with decreased trbc-MAO activity although we could not replicate a previously reported genetic association between the MAOB gene and trbc-MAO activity. Fourth: we could not find any significant associations between the genetic variants and depressed state. On the other hand, there was an interesting, although not significant dose-response effect of haplotypes displayed in women, with greater odds ratios in homozygotes than heterozygotes.
Considering the size and importance of the MAO locus, relatively few polymorphic sites have been verified. We observed two new variants through sequence screening a partial region of MAOA intron 1, but in MAOB neither the previously reported nor any novel variants were found in the areas sequenced. It is surprising that so few SNPs were discovered given our power to detect variants with very low frequencies. SNP deserts have been previously noted on the q arm of the X chromosome [34]. Gilad and colleagues [4] have described similar features across MAOA, where extensive LD and low nucleotide diversity suggest recent action by population structure forces and perhaps a recent positive selection sweep [35]. Although we could not evaluate the influence of such forces, evidence of strong LD and the lack of decay across MAOA in our Swedish sample complement these previous findings. Linkage disequilibrium decays rapidly between the two MAO genes (separated by approximately 20 kb). Perhaps selection is in action much more locally than would be expected in each MAO gene, both separated by regions of higher recombination than that within each gene.
Previous studies have indicated that the MAOA gene may harbour relatively few haplotypes within a block structure [5]. We observed similar results here with two haplotypes encompassing 95% of the haplotypic variation. We found similar results for the MAOB gene, with a distinct block structure in which three haplotypes explain 93% of the variation. So few haplotypes over such long distances have been observed previously (McCarthy et al, manuscript) and are proposed signatures of selection and population substructure on the X chromosome [36,37].
A previous Swedish association between the MAOB gene and trbc-MAO activity [21] could not be replicated nor distinctly refuted, as we found a small non-significant effect of the same allele in males. However, none of the haplotype blocks carrying this allele could strengthen or support this effect, suggesting that this allele is not in high LD with a larger region of the MAOB gene.
Two MAOA haplotypes (A1 and A3) showed a significant association with reduced trbc-MAO activity. Both haplotypes shared the initial sequence variants [CCA], but varied at the fourth allele [T/C]. Given that only MAOB is expressed in platelets there is no clear explanation for this finding. Given the minor kinetic differences between platelet and brain MAO-B [38] and the correlation of MAOB and MAOA levels in regions of the brain [39], this association may reflect MAOA activity in the brain. On the other hand, it is possible that the MAOA locus holds cis-acting regulatory elements affecting MAOB expression. Another possible explanation could be that one or several single-base variants affected by methylation cause changes in the expression pattern [40].
Our study is based on a relatively large population-based sample of normally aging adults, although it is not without its limitations. We have controlled for smoking, but were unable to do so for intake of certain medications. The study sample was included in a larger study where associations between depressed state and the serotonin receptor 2A and the serotonin transporter were evaluated [41]. The influence of these genes has not been corrected for in the analysis.
Conclusion
Good et al [19] demonstrated that trbc-MAO activity is related to the number of X chromosomes. We replicated a significant difference in trbc-MAO activity between males and females reported by others e.g. [17]. The findings suggest incomplete X chromosome inactivation at this locus and are consistent with other findings of genes escaping inactivation on the X chromosome [42,43]. It has been hypothesized that this dosage imbalance between males and females might be crucial for gender characteristics [19,44]. Recently it was demonstrated that a number of genes, including MAOA, escape X-inactivation [45]. Furthermore, the X-inactivation pattern, which shows a substantial heritability [46], increases in the elderly. Although we could not find a significant association between variants of MAOB or MAOA and depressed state in this population, we found an interesting dose-response effect in women, with a higher risk for depressed state with homozygosity. Whether levels of trbc-MAO activity are correlated with the number of X chromosomes and whether this might be linked to the higher prevalence of depressive symptoms in females deserves further investigation. Nevertheless it is plausible that a partially doubled gene activity on the X chromosome can explain difference in prevalence of depressive state in men and women.
Methods
Participants
The participants were taken from a longitudinal twin study of aging, the Swedish Adoption/Twin Study of Aging (SATSA) with up to five occasions of measurement [47]. SATSA is a sub-sample of the population based Swedish Twin Registry [48]. All participants are Caucasian and born in Sweden. For the present analyses we selected all individuals who participated in an in-person testing session during which questionnaires were administered and a blood sample was drawn. The mean age of the sample was 61,3 years at the time of testing. Twenty two percent of the participants were current smokers; 35% of the males and 15% of the females.
Zygosity was initially based on self-reports of similarity and confirmed by serological analyses and comparisons of up to 10 DNA markers.
For preliminary screening of the promoter, the first exon and intron regions for novel variants, 94 Swedish male blood donors were randomly selected from a larger sample set collected to study MAOB regulation. All were between the ages of 20 to 40 years and non-smokers.
This study was reviewed and approved by the Ethics Committee of the Karolinska Institute, the Swedish Data Inspection Board, and the IRBs at the University of Southern California and the Pennsylvania State University. All subjects provided informed consent.
DNA and trbc-MAO activity
DNA samples were available from 573 twins. Trbc-MAO activity measures were available from 565 twins. The trbc-MAO activity is expressed as nmoles of 2-phenylethylamine oxidized per minute and per 1010 platelets. Trbc-MAO activity measures have previously been described in detail [20].
Depressed state
Depressive symptoms were measured with the Center for Epidemiologic Studies Depression Scale (CES-D), a 20-item self-report instrument developed for use in the community and well established for use with older adults [49,50]. The scale has been shown to have minimal overlap with physical illness [51] and assesses current symptoms during the past week. Respondents scoring 16 or higher on the CES-D scale are considered to have a clinically relevant depressed state. In this study population of 574 participants, 144 were classified as having a depressed state, 17.9% of the males and 30.2% of females.
Genotyping & sequencing
Approximately 4.5 kb of each gene was initially sequenced in search of novel SNPs in both MAOA and MAOB, first in 94 Swedish males and later 45 twins with CES-D scores (36 males and 9 females). Power to detect minor allele frequencies (q) between 1 and 5% was determined as by Glatt et al. [52], 1-(1-q)N where N is the number of chromosomes. Amplification and nested sequencing primers were designed with the CPrimers programme from Genbank entry GI:8671203 containing the promoter, coding exon 1 and flanking intronic sequence of MAOA (~5.0 kb, nucleotides 46490–51454) and Genbank entry GI:2440066 spanning the same characterized sequences of MAOB (~4 kb nucleotides 35033–39021).
Direct sequencing reactions were performed using DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences) and separated using a Megabace 1000. Reads were base called with Phred [53], assembled using Phrap and viewed using Consed Version 13 [54]. All SNPs were documented and cross validated with dbSNP at NCBI.
Twelve SNPs identified from public databases (dbSNP at NCBI) and two novel SNPs previously reported (introns 3 and 10 of MAOB) a Swedish sample [5] were sequenced in 95 participants (142 chromosomes) by Pyrosequencing to confirm their presence in this population. For Pyrosequencing, either the forward or the reverse primer in each primer pair was biotinylated. Sequencing primers with a length of 14 and 18 bases were placed within one base of the SNP. The PCR reaction was performed in a 50 μl reaction volume, containing 5 ng of genomic DNA, 10 pmoles of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2 and 1.5 U of Taq. Thermal cycling was performed in a PTC-225 DNA machine (MJ Research Inc., Cambridge, MA, USA) at 95°C for 5 min followed by 50 cycles of 95°C for 30 s, 45 s of annealing at an optimized temperature, followed by 72°C for 30 s and a final extension of 5 min at 72°C. The biotinylated PCR product was immobilized onto streptavidin-coated sepharose beads and DNA strands were separated by denaturation with 0.2 M NaOH. The pyrosequencing reaction was performed on a PSQ96™ Instrument from Pyrosequencing AB (Uppsala, Sweden) as described by [55,56]. Detailed primer and assay information are available upon request.
Statistical analysis
Male haplotypes could be extrapolated directly since the MAO locus is located on the X chromosome and males are thereby hemizygous. Female bi-allelic haplotypes were estimated using an EM algorithm (Sham 1998) and the pair-wise LD measures D' [57] and Δ2 [58]. We used "PHARE" (by David G Cox, available at ) to create input files for "PHASE" [59,60] to construct female haplotypes.
We used linear regression to estimate the association between trbc-MAO activity and genotypic information using a generalized estimating equation (GEE) approach and alternating logistic regression (ALR) [61] to estimate the association between depressed state and genotypic information. We first modeled the association between single SNPs and each of the two outcomes and then modeled the association between haplotype constructs and the two outcomes. All estimates were adjusted for current smoking status. We estimated both dominance and co-dominance models. Explanatory variables in the dominance models were binary whereas in the co-dominance models they were coded as the number of reference alleles (i.e., 0, 1, or 2 for females and 0 or 1 for males). The parameter estimates for the co-dominance models represent the change in the outcome (trbc-MAO activity or odds of being in a depressed state) per affected allele. Due to the continuous nature of the trbc-MAO measure, only one individual from each complete twin pair and single participating individuals were analyzed (N = 340). Among females we also estimated the effect of being homozygote compared to heterozygote. If the co-dominance model is a good fit to the data then these estimates should be similar to the "per allele" estimates from the co-dominance model. All statistical analyses were performed in SAS 8.01 using GENMOD procedure (SAS Institute Inc. Cary, NC).
Authors' contributions
MJ: Design of the study, performed data analysis and interpretation of data. Carried out the molecular genetic studies (genotyping) and drafted the manuscript.
SM: Participated in the design of the study. Carried out the molecular genetic studies (sequencing), sequence alignment and critically revised the manuscript.
PFS: Participated in the design of the study and critically revised the manuscript for important intellectual content.
PD: Planed and performed the statistical analysis.
BA: Participated in the design of the study and critically revised the manuscript.
LO: Substantially revised the manuscript for important intellectual content.
MS: Participated in the design of the study and critically revised the manuscript.
NLP: Participated in the design of the study and substantially revised the manuscript for important intellectual content.
All authors read and approved the final manuscript.
Acknowledgements
SATSA is supported by grants AG 04563, AG 10175, the Swedish Council for Social Research, and the MacArthur Foundation Research Network on Successful Aging. The work herein is also supported by Kapten Artur Erikssons fund, Organons stiftelse för stöd till forskning inom gynekologi-obstertik och psykiatri, VR 10909 and 4145, the Söderström Königska Stiftelsen and funds from Karolinska Institutet and the Karolinska Hospital. Sequencing of the genetic loci was supported by the Genome Program of the Swedish Foundation for Strategic research.
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Kochersperger LM Parker EL Siciliano M Darlington GJ Denney RM Assignment of genes for human monoamine oxidases A and B to the X chromosome J Neurosci Res 1986 16 601 616 3540317 10.1002/jnr.490160403
Grimsby J Chen K Wang LJ Lan NC Shih JC Human monoamine oxidase A and B genes exhibit identical exon-intron organization Proc Natl Acad Sci U S A 1991 88 3637 3641 2023912
Chen ZY Denney RM Breakefield XO Norrie disease and MAO genes: nearest neighbors Hum Mol Genet 1995 4 Spec No 1729 1737 8541872
Gilad Y Rosenberg S Przeworski M Lancet D Skorecki K Evidence for positive selection and population structure at the human MAO-A gene Proc Natl Acad Sci U S A 2002 99 862 867 11805333 10.1073/pnas.022614799
Balciuniene J Syvanen AC McLeod HL Pettersson U Jazin EE The geographic distribution of monoamine oxidase haplotypes supports a bottleneck during the dispersion of modern humans from Africa J Mol Evol 2001 52 157 163 11231895
Tivol EA Shalish C Schuback DE Hsu YP Breakefield XO Mutational analysis of the human MAOA gene Am J Med Genet 1996 67 92 97 8678123 10.1002/(SICI)1096-8628(19960216)67:1<92::AID-AJMG16>3.0.CO;2-K
Greenawalt JW Schnaitman C An appraisal of the use of monoamine oxidase as an enzyme marker for the outer membrane of rat liver mitochondria J Cell Biol 1970 46 173 179 5460462 10.1083/jcb.46.1.173
Westlund KN Denney RM Rose RM Abell CW Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brainstem Neuroscience 1988 25 439 456 3399053 10.1016/0306-4522(88)90250-3
Saura J Bleuel Z Ulrich J Mendelowitsch A Chen K Shih JC Malherbe P Da Prada M Richards JG Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry Neuroscience 1996 70 755 774 9045087 10.1016/S0306-4522(96)83013-2
Thorpe LW Westlund KN Kochersperger LM Abell CW Denney RM Immunocytochemical localization of monoamine oxidases A and B in human peripheral tissues and brain J Histochem Cytochem 1987 35 23 32 3025289
Arai R Kimura H Nagatsu I Maeda T Preferential localization of monoamine oxidase type A activity in neurons of the locus coeruleus and type B activity in neurons of the dorsal raphe nucleus of the rat: a detailed enzyme histochemical study Brain Res 1997 745 352 356 9037433 10.1016/S0006-8993(96)01239-5
Fowler CJ Oreland L Substrate- and stereoselective inhibitor of human brain monoamine oxidase by 4-dimethylamino-alpha, 2-dimethylphenethylamine (FLA 336) J Pharm Pharmacol 1981 33 403 406 6115022
Arai Y Kinemuchi H Hamamichi N Satoh N Tadano T Kisara K Inhibition of rat brain monoamine oxidase by some analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium ion Neurosci Lett 1986 66 43 48 3487053 10.1016/0304-3940(86)90163-1
White HL Glassman AT Multiple binding sites of human brain and liver monoamine oxidase: substrate specificities, selective inhibitions, and attempts to separate enzyme forms J Neurochem 1977 29 987 997 599351
Oreland L Wiberg A Asberg M Traskman L Sjostrand L Thoren P Bertilsson L Tybring G Platelet MAO activity and monoamine metabolites in cerebrospinal fluid in depressed and suicidal patients and in healthy controls Psychiatry Res 1981 4 21 29 6164071 10.1016/0165-1781(81)90004-4
Bridge TP Soldo BJ Phelps BH Wise CD Francak MJ Wyatt RJ Platelet monoamine oxidase activity: demographic characteristics contribute to enzyme activity variability J Gerontol 1985 40 23 28 3965558
Harro M Eensoo D Kiive E Merenakk L Alep J Oreland L Harro J Platelet monoamine oxidase in healthy 9- and 15-years old children: the effect of gender, smoking and puberty Prog Neuropsychopharmacol Biol Psychiatry 2001 25 1497 1511 11642650 10.1016/S0278-5846(01)00212-3
Sandler M Reveley MA Glover V Human platelet monoamine oxidase activity in health and disease: a review J Clin Pathol 1981 34 292 302 6453137
Good CD Lawrence K Thomas NS Price CJ Ashburner J Friston KJ Frackowiak RS Oreland L Skuse DH Dosage-sensitive X-linked locus influences the development of amygdala and orbitofrontal cortex, and fear recognition in humans Brain 2003 126 2431 2446 12958079 10.1093/brain/awg242
Pedersen NL Oreland L Reynolds C McClearn GE Importance of genetic effects for monoamine oxidase activity in thrombocytes in twins reared apart and twins reared together Psychiatry Res 1993 46 239 251 8493293 10.1016/0165-1781(93)90092-U
Garpenstrand H Ekblom J Forslund K Rylander G Oreland L Platelet monoamine oxidase activity is related to MAOB intron 13 genotype J Neural Transm 2000 107 523 530 11072748 10.1007/s007020070075
Berlin I Anthenelli RM Monoamine oxidases and tobacco smoking Int J Neuropsychopharmacol 2001 4 33 42 11343627 10.1017/S1461145701002188
Oreland L Platelet monoamine oxidase, personality and alcoholism: the rise, fall and resurrection Neurotoxicology 2004 25 79 89 14697883 10.1016/S0161-813X(03)00115-3
Stalenheim EG von Knorring L Oreland L Platelet monoamine oxidase activity as a biological marker in a Swedish forensic psychiatric population Psychiatry Res 1997 69 79 87 9109175 10.1016/S0165-1781(96)03056-9
Verkes RJ Van der Mast RC Kerkhof AJ Fekkes D Hengeveld MW Tuyl JP Van Kempen GM Platelet serotonin, monoamine oxidase activity, and [3H]paroxetine binding related to impulsive suicide attempts and borderline personality disorder Biol Psychiatry 1998 43 740 746 9606528 10.1016/S0006-3223(97)00317-X
Kirk KM Whitfield JB Pang D Heath AC Martin NG Genetic covariation of neuroticism with monoamine oxidase activity and smoking Am J Med Genet 2001 105 700 706 11803517 10.1002/ajmg.1555
Oreland L Damberg M Hallman J Garpenstrand H Smoking only explains part of the associations between platelet monoamine oxidase activity and personality J Neural Transm 2002 109 963 975 12111482 10.1007/s007020200079
Shih JC Chen K Ridd MJ Monoamine oxidase: from genes to behavior Annu Rev Neurosci 1999 22 197 217 10202537 10.1146/annurev.neuro.22.1.197
Nielsen DM Ehm MG Weir BS Detecting marker-disease association by testing for Hardy-Weinberg disequilibrium at a marker locus Am J Hum Genet 1998 63 1531 1540 9867708 10.1086/302114
Weir BS Hill WG Cardon LR Allelic association patterns for a dense SNP map Genet Epidemiol 2004 27 442 450 15543640 10.1002/gepi.20038
Buchsbaum MS Coursey RD Murphy DL The biochemical high-risk paradigm: behavioral and familial correlates of low platelet monoamine oxidase activity Science 1976 194 339 341 968488
von Knorring AL Bohman M von Knorring L Oreland L Platelet MAO activity as a biological marker in subgroups of alcoholism Acta Psychiatr Scand 1985 72 51 58 4036659
Eensoo D Paaver M Pulver A Harro M Harro J Low platelet MAO activity associated with high dysfunctional impulsivity and antisocial behavior: evidence from drunk drivers Psychopharmacology (Berl) 2003
Miller RD Taillon-Miller P Kwok PY Regions of low single-nucleotide polymorphism incidence in human and orangutan xq: deserts and recent coalescences Genomics 2001 71 78 88 11161800 10.1006/geno.2000.6417
Przeworski M The signature of positive selection at randomly chosen loci Genetics 2002 160 1179 1189 11901132
Nachman MW Crowell SL Estimate of the mutation rate per nucleotide in humans Genetics 2000 156 297 304 10978293
Przeworski M Hudson RR Di Rienzo A Adjusting the focus on human variation Trends Genet 2000 16 296 302 10858659 10.1016/S0168-9525(00)02030-8
Fowler CJ Ekstedt B Egashira T Kinemuchi H Oreland L The interaction between human platelet monoamine oxidase, its monoamine substrates and oxygen Biochem Pharmacol 1979 28 3063 3068 518704 10.1016/0006-2952(79)90614-2
Fowler CJ Wiberg A Oreland L Marcusson J Winblad B The effect of age on the activity and molecular properties of human brain monoamine oxidase J Neural Transm 1980 49 1 20 7441234 10.1007/BF01249185
Van Laere AS Nguyen M Braunschweig M Nezer C Collette C Moreau L Archibald AL Haley CS Buys N Tally M Andersson G Georges M Andersson L A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig Nature 2003 425 832 836 14574411 10.1038/nature02064
Jansson M Gatz M Berg S Johansson B Malmberg B McClearn GE Schalling M Pedersen NL Association between depressed mood in the elderly and a 5-HTR2A gene variant Am J Med Genet 2003 120B 79 84 10.1002/ajmg.b.20016
Carrel L Cottle AA Goglin KC Willard HF A first-generation X-inactivation profile of the human X chromosome Proc Natl Acad Sci U S A 1999 96 14440 14444 10588724 10.1073/pnas.96.25.14440
Carrel L Willard HF Heterogeneous gene expression from the inactive X chromosome: an X-linked gene that escapes X inactivation in some human cell lines but is inactivated in others Proc Natl Acad Sci U S A 1999 96 7364 7369 10377420 10.1073/pnas.96.13.7364
Disteche CM Escapees on the X chromosome Proc Natl Acad Sci U S A 1999 96 14180 14182 10588671 10.1073/pnas.96.25.14180
Carrel L Willard HF X-inactivation profile reveals extensive variability in X-linked gene expression in females Nature 2005 434 400 404 15772666 10.1038/nature03479
Kristiansen M Knudsen GP Bathum L Naumova AK Sorensen TI Brix TH Svendsen AJ Christensen K Kyvik KO Orstavik KH Twin study of genetic and aging effects on X chromosome inactivation Eur J Hum Genet 2005 13 599 606 15756296 10.1038/sj.ejhg.5201398
Finkel D Pedersen NL Processing Speed and Longitudinal Trajectories of Change for Cognitive Abilities: The Swedish Adoption / Twin Study of Aging. Aging, Neuropsychology and Cognition 2004 In press
Lichtenstein P De Faire U Floderus B Svartengren M Svedberg P Pedersen NL The Swedish Twin Registry: a unique resource for clinical, epidemiological and genetic studies J Intern Med 2002 252 184 205 12270000 10.1046/j.1365-2796.2002.01032.x
Radloff LS The CES-D scale: a self report depression scale for research in the general population. Appl Psychol Meas 1977 1 385 401
Blazer DG Depression in late life: review and commentary J Gerontol A Biol Sci Med Sci 2003 58 249 265 12634292
Berkman LF Berkman CS Kasl S Freeman DHJ Leo L Ostfeld AM Cornoni-Huntley J Brody JA Depressive symptoms in relation to physical health and functioning in the elderly Am J Epidemiol 1986 124 372 388 3740038
Glatt CE DeYoung JA Delgado S Service SK Giacomini KM Edwards RH Risch N Freimer NB Screening a large reference sample to identify very low frequency sequence variants: comparisons between two genes Nat Genet 2001 27 435 438 11279528 10.1038/86948
Ewing B Hillier L Wendl MC Green P Base-calling of automated sequencer traces using phred. I. Accuracy assessment Genome Res 1998 8 175 185 9521921
Gordon D Abajian C Green P Consed: a graphical tool for sequence finishing Genome Res 1998 8 195 202 9521923
Nordfors L Jansson M Sandberg G Lavebratt C Sengul S Schalling M Arner P Large-scale genotyping of single nucleotide polymorphisms by Pyrosequencingtrade mark and validation against the 5'nuclease (Taqman((R))) assay Hum Mutat 2002 19 395 401 11933193 10.1002/humu.10062
Ronaghi M Uhlen M Nyren P A sequencing method based on real-time pyrophosphate Science 1998 281 363, 365 9705713 10.1126/science.281.5375.363
Lewontin RC The Interaction of Selection and Linkage. I. General considerations; heterotic models Genetics 1964 49 49 67 17248194
Hill WG Robertson A The effects of inbreeding at loci with heterozygote advantage Genetics 1968 60 615 628 5728744
Stephens M Donnelly P A comparison of bayesian methods for haplotype reconstruction from population genotype data Am J Hum Genet 2003 73 1162 1169 14574645 10.1086/379378
Stephens M Smith NJ Donnelly P A new statistical method for haplotype reconstruction from population data Am J Hum Genet 2001 68 978 989 11254454 10.1086/319501
Carey VJ Zeger SL Modelling multivariate binary data with alternating logistic regressions. Biometrica 1993 517 526
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BMC GenomicsBMC Genomics1471-2164BioMed Central 1471-2164-6-1121613139710.1186/1471-2164-6-112Research ArticleGH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily Naumoff Daniil G [email protected] Laboratory of Bioinformatics, State Institute for Genetics and Selection of Industrial Microorganisms, I-Dorozhny proezd, 1, Moscow 117545, Russia2005 30 8 2005 6 112 112 21 3 2005 30 8 2005 Copyright © 2005 Naumoff; licensee BioMed Central Ltd.2005Naumoff; 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
As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms.
Results
The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20).
Conclusion
The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α)8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.
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Background
On the basis of sequence similarity, glycoside hydrolases (or glycosidases, EC3.2.1.-) have been grouped into 96 families (GH1-GH100, except GH21, GH40, GH41, and GH60) by the Carbohydrate-Active Enzymes (CAZy) classification [1,2]. In the case of poly-domain proteins each catalytic domain is considered separately. A family was initially defined as a group of at least two sequences displaying significant amino acid similarity and with no significant similarity with other families [1]. Later, some related families of glycosidases have been combined into clans [3,4]. According to its definition, a clan is a group of families that are thought to have a common ancestry and are recognized by significant similarities in tertiary structure together with conservation of the catalytic residues and a catalytic mechanism [3]. Glycosidases catalyze hydrolysis of the glycosidic bond of their substrates via two general mechanisms, leading to either inversion or overall retention of the anomeric configuration at the cleavage point [4-6]. Currently, 14 clans (GH-A-GH-N) are described, and in total they contain 46 families [2]. Families of four clans (GH-A, GH-D, GH-H, and GH-K), as well as several other families, that have not been assigned to any clan, contain proteins with a similar (β/α)8-barrel fold of the catalytic domain [2]. Several glycosidases, that do not have any homologues, are included into a group of non-classified glycoside hydrolases [1,2]. In several instances, proteins from this group have been reclassified into new families when their homologues were found [7].
Two different clans have never been merged in the CAZy classification [2], even after their significant similarity has been established. Instead, related clans (and families) having statistically significant sequence similarity of the corresponding proteins were proposed to be grouped into superfamilies at a higher hierarchical level. For example, we have described the furanosidase (β-fructosidase) superfamily, that includes clans GH-F (inverting glycosidases) and GH-J (retaining glycosidases), as well as the GHLP (COG2152) family of enzymatically-uncharacterized proteins [8-11].
Nowadays, some families are very large. For example, GH13 family (clan GH-H) includes more than 2,000 representatives [2]. This large and poly-specific group of enzymes has been studied by many authors [12-19]. In particular, it was shown that splitting of this family into smaller subfamilies allowed to clarify the relationship of its members [12,13].
The majority of known glycosidases with the α-galactosidase activity [EC3.2.1.22] belong to families GH27 and GH36, that form clan GH-D [2,20]. This clan and family GH31 compose the α-galactosidase superfamily [21-24]. This superfamily has a distant relationship with clan GH-H [25,26], which we have proposed to name the α-glucosidase superfamily [24]. Both superfamilies contain proteins sharing the same enzymatic mechanism (retention), a similar (β/α)8-barrel fold of the catalytic domain [2], and use substrates only with the axial orientation of the glycosidic bond [4].
Gram-negative obligate anaerobe Bacteroides thetaiotaomicron ATCC29148 is a commensal bacterium found in the human colon where it ferments a wide variety of polysaccharides [27,28]. Its starch utilization system (sus) has been studied in detail [29-35]. One of the corresponding loci (Figure 1) includes divergently oriented regulatory gene susR and seven structural genes susA-susG [30-34]. Genes susC-susF encode outer membrane proteins are involved in starch binding. Glycosidases SusA (a neopullulanase, EC 3.2.1.135) and SusG (an α-amylase, EC 3.2.1.1) are members of family GH13 [29-32]. SusB is an unusual α-glucosidase [EC 3.2.1.20] that for a long time was considered a unique glycosidase with no homologues [29,30]. Therefore it was included in the group of non-classified glycoside hydrolases [2]. We have found a group of its homologues among hypothetical proteins encoded by open reading frames (ORFs), that recently were sequenced in the frame of several prokaryotic genome projects. We referred to this group of proteins as the GHX family [23,24]. In June 2004, 18 members of this family were recognized in the CAZy classification as the GH97 family of glycoside hydrolases. Currently (June 2005), family GH97 includes two α-glucosidases SusB from closely related bacteria B. thetaiotaomicron ATCC29148 and Tannerella forsythensis (Bacteroides forsythus) ATCC43037, as well as 22 hypothetical proteins encoded by ORFs [2].
Figure 1 Structure of Bacteroides thetaiotaomicron ATCC29148 genome fragment containing gene clusters for starch and hemicellulose utilization. Arrows indicate the direction of gene transcription. Red arrows correspond to glycosidase (GH) and glycosyltransferase (GT) genes: family belonging is indicated. Yellow arrows correspond to genes coding outer membrane proteins involved in starch binding (susC-susF) and their homologues. Green arrows correspond to genes of the transcriptional activator SusR and predicted transcriptional regulators homologous to AraC.
In this work we updated the GH97 family of glycosidases, performed its phylogenetic analysis, and established its evolutionary relationship with several other glycosidase families.
Results and discussion
Collecting sequences of family GH97
PSI-BLAST search of the non-redundant database with the Bacteroides thetaiotaomicron α-glucosidase SusB (97A1_BACTH, see Table I) as a query sequence yielded 32 protein sequences with the worst (the largest) E-value of 2 × 10-20 during the first round. Among them we found 10 paralogous proteins from B. thetaiotaomicron ATCC29148 and their 22 homologues from other species. Among 32 obtained proteins were found all 24 members of the GH97 family listed at the CAZy server [2]. Genomic BLAST revealed 13 additional homologous sequences. Based on the sequence similarity, we propose to enlarge the GH97 family by including all known homologues of SusB. As a result, currently this family includes 45 proteins. The majority of them represent Eubacteria (16 different species). Three other sequences correspond to Archaea (Haloarcula marismortui) and two uncultured bacteria. Four sequences are annotated in the NCBI database as eukaryotic (Anopheles gambiae) genome fragments. Only five out of 45 protein sequences (from Anopheles and an uncultured bacterium) are short fragments (Table I).
Table I Glycoside hydrolases analyzed in the work
Name Family, subfamily Organism Accession numbera Protein function (annotation) Lengthb
97A1_BACTH GH97, 97a Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAC44671
alpha-glucosidase SusB 738
97A2_BACTH GH97, 97a Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO79686
ORF: alpha-glucosidase 719
97A3_BACTH GH97, 97a Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO75790
ORF: alpha-glucosidase 671
97B1_BACTH GH97, 97b Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO76978
ORF: putative alpha-glucosidase 662
97B2_BACTH GH97, 97b Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO78400
ORF: putative alpha-glucosidase 650
97B3_BACTH GH97, 97b Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO77727
ORF: alpha-glucosidase 649
97B4_BACTH GH97, 97b Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO78269
ORF: putative alpha-glucosidase 674
97C1_BACTH GH97, 97c Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO78766
ORF: alpha-glucosidase 647
97C2_BACTH GH97, 97c Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO78769
ORF: putative alpha-glucosidase 638
97E1_BACTH GH97, 97e Bacteroides thetaiotaomicron VPI-5482 = ATCC29148
AAO75239
ORF: putative alpha-glucosidase 644
97A1_BACFR GH97, 97a Bacteroides fragilis YCH46
BAD47941
ORF: alpha-glucosidase 719
97A2_BACFR GH97, 97a Bacteroides fragilis YCH46
BAD48072
ORF: alpha-glucosidase 671
97B1_BACFR GH97, 97b Bacteroides fragilis YCH46
BAD50730
ORF: putative alpha-glucosidase 649
97B2_BACFR GH97, 97b Bacteroides fragilis YCH46
BAD50235
ORF: putative alpha-glucosidase 649
97A1_TANFO GH97, 97a Tannerella forsythensis (Bacteroides forsythus) ATCC43037 AAO33827 alpha-D-glucosidase SusB 708
97A1_PREIN GH97, 97a Prevotella intermedia 17 (TIGR_246198) ORF 733
97A1_PRERU GH97, 97a Prevotella ruminicola 23 (TIGR_264731) ORF 737
97B1_PRERU GH97, 97b Prevotella ruminicola 23 (TIGR_264731) ORF 645
97B2_PRERU GH97, 97b Prevotella ruminicola 23 (TIGR_264731) ORF 658
97C1_PRERU GH97, 97c Prevotella ruminicola 23 (TIGR_264731) ORF 621
97C2_PRERU GH97, 97c Prevotella ruminicola 23 (TIGR_264731) ORF 639
97C3_PRERU GH97, 97c Prevotella ruminicola 23 (TIGR_264731) ORF 645
97A1_SALRU GH97, 97a Salinibacter ruber DSM13855 (NC_006812) ORF 708
97A1_AZOVI GH97, 97a Azotobacter vinelandii AvOP
EAM07225
ORF: alpha-glucosidase 673
97A1_XANAX GH97, 97a Xanthomonas axonopodis pv. citri 306
AAM37448
ORF: alpha-glucosidase 693
97D1_XANAX GH97, 97d Xanthomonas axonopodis pv. citri 306
AAM38156
ORF: alpha-glucosidase 654
97A1_XANCA GH97, 97a Xanthomonas campestris pv. campestris ATCC33913
AAM41744
ORF: alpha-glucosidase 692
97D1_XANCA GH97, 97d Xanthomonas campestris pv. campestris ATCC33913
AAM42433
ORF: alpha-glucosidase 654
97A1_MICDE GH97, 97a Microbulbifer (Saccharophagus) degradans 2–40
ZP_00315606
ORF: hypothetical protein 684
97B1_MICDE GH97, 97b Microbulbifer (Saccharophagus) degradans 2–40
ZP_00317369
ORF: hypothetical protein 679
97C1_MICDE GH97, 97c Microbulbifer (Saccharophagus) degradans 2–40
ZP_00317507
ORF: hypothetical protein 674
97C2_MICDE GH97, 97c Microbulbifer (Saccharophagus) degradans 2–40
ZP_00315142
ORF: hypothetical protein 661
97A1_SHEON GH97, 97a Shewanella oneidensis MR-1
AAN55484
ORF: alpha-glucosidase 699
97A1_SHEBA GH97, 97a Shewanella baltica OS155
EAN43632
ORF: alpha-glucosidase 710
97A1_SHEFR GH97, 97a Shewanella frigidimarina NCIMB400
EAN73178
ORF: alpha-glucosidase 697
97A1_SHEDE GH97, 97a Shewanella denitrificans OS-217
EAN70289
ORF: alpha-glucosidase 727
97A1_SHEAM GH97, 97a Shewanella amazonensis SB2B
EAN38820
ORF: alpha-glucosidase 676
97A1_NOVAR GH97, 97a Novosphingobium aromaticivorans DSM12444
ZP_00303588
ORF: transketolase 682
97A1_SPHAL GH97, 97a Sphingopyxis alaskensis RB2256
EAN45679
ORF: alpha-glucosidase 680
97D1_CAUCR GH97, 97d Caulobacter crescentus CB15
AAK22781
ORF: putative alpha-glucosidase 670
97A1_ERYLI GH97, 97a Erythrobacter litoralis HTCC2594
EAL74063
ORF: alpha-glucosidase 681
97E1_RHOBA GH97, 97e Rhodopirellula baltica SH1 (Pirellula sp. 1)
CAD78916
ORF: alpha-glucosidase 645
97C1_LEIXY GH97, 97c Leifsonia xyli subsp. xyli CTCB07 (NC_006087)* ORF: similar to alpha-glucosidase 775*
97X1_SOLUS GH97 Solibacter usitatus Ellin6076
EAM58489
ORF: hypothetical protein 619
97A1_HALMA GH97, 97a Haloarcula marismortui ATCC43049 AAV45265 ORF: alpha-glucosidase 1144
97A1_ANOGA GH97, 97a Anopheles gambiae str. PEST (African malaria mosquito) (AAAB01006165) ORF 380*
97A2_ANOGA GH97, 97a Anopheles gambiae str. PEST (African malaria mosquito) (AAAB01064948) ORF 209*
97A3_ANOGA GH97, 97a Anopheles gambiae str. PEST (African malaria mosquito) (AAAB01020110) ORF 231*
97A4_ANOGA GH97, 97a Anopheles gambiae str. PEST (African malaria mosquito) (AAAB01068263) ORF 229*
97A1_UNBAC GH97, 97a uncultured murine large bowel bacterium BAC31B
AAX16382
ORF: alpha-glucosidase 720
97A2_UNBAC GH97, 97a uncultured bacterium (AY350337) ORF 106*
97A1_ENSEQ GH97, 97a environmental sequence (cf. Shewanella SAR-1) EAJ06144* ORF: unknown 703
97A2_ENSEQ GH97, 97a environmental sequence (cf. Shewanella SAR-2)
EAI69763
ORF: unknown 699
97A3_ENSEQ GH97, 97a environmental sequence
EAJ75652
ORF: unknown 714
97A4_ENSEQ GH97, 97a environmental sequence
EAI51202
ORF: unknown 713
97A5_ENSEQ GH97, 97a environmental sequence
EAI80962
ORF: unknown 702*
97A6_ENSEQ GH97, 97a environmental sequence EAH92811, EAI03708, EAD44407, EAG79875, EAH92819, EAI36772 ORF: unknown 711
97A7_ENSEQ GH97, 97a environmental sequence EAJ99185, EAD99255, EAH48404, EAH57728, EAD83763, EAH04981, EAC91563, EAH85977, EAD11728 ORF: unknown 710
97A8_ENSEQ GH97, 97a environmental sequence EAJ85380, EAH86891 ORF: unknown 669*
97C1_ENSEQ GH97, 97c environmental sequence EAD85224* ORF: unknown 218*
GH27_ORYSA GH27, 27a Oryza sativa japonica cultivar Nipponbare (rice)
BAB12570
alpha-galactosidase 417
GH36_LACPL GH36, 36A Lactobacillus plantarum ATCC8014
AAF02774
alpha-galactosidase MelA 738
GH31_ECOLI GH31 Escherichia coli K12
AAC76680
alpha-xylosidase YicI 772
aAccession numbers of protein sequences are given according to the NCBI database [72]. Numbers of nucleic sequences are given (in parentheses) if the corresponding protein sequences have not been deposited. In some cases (asterisked), protein sequences were edited by changing the start codon.
bProtein length was established as the number of amino acids in the corresponding precursor. Incomplete sequences (protein fragments) are asterisked.
PSI-BLAST searches with a few randomly selected divergent representatives of the GH97 family used as a query sequence during the first round always yielded the same 32 protein sequences as with 97A1_BACTH. An analysis of the order of the sequence appearance during the first round of searches by PSI-BLAST, depending on the query, allows us to distinguish five subfamilies (97a–97e) in the GH97 family with at least two known members in each of them (Table I). The obtained pairwise alignments were used for generating the protein multiple sequence alignment of family GH97. The most conserved parts of the alignment are shown on Figure 2.
Figure 2 Portion of the multiple sequence alignment of the sequences analyzed. Ten-letter name for each sequence is indicated in the leftmost column (for origin of the sequences see Table I). The alignment continuously spans three panels. Distances to the N- and C-termini and length of omitted fragments are indicated. Highly conserved residues are highlighted in sequences. Amino acid positions that are highly conserved within several subfamilies but varied in amino acid residues in different subfamilies are coloured. Subfamily belonging of sequences (for family GH97) are indicated in the most right. Amino acid residues, interacting with the substrate in the active center of GH27 and GH31 family glycosidases, are indicated by arrows at the bottom [50-54]. The arrow on the gray background corresponds to the Asp residue, playing the role of the nucleophile in glycosidases of families GH27 and GH31. Red asterisks over and under the alignment indicate three conserved positions (in red) probably corresponding to the nucleophile and proton donor in the glycosidases of family GH97 (see text). Alignment of GH27_ORYSA and GH31_ECOLI is structure-based. At the bottom of the figure, β-strands and α-helixes of the (β/α)8-barrel are indicated. The first part of the barrel (β1–β4) is shown according to the known structures of GH27 and GH31 family members [51, 54]. The second part of the barrel (α4–α8) is based on generalization of predictions for several GH97 family proteins by 3D-PSSM, GOR IV, and nnpredict programs.
The fragment of Leifsonia xyli CTCB07 genome [GenBank: NC_006087] revealed by Genomic BLAST has 2 stop codons in the region homologous to genes of GH97 family proteins. An analysis of the nucleic acid sequence allowed us to detect a frame shift (data not shown). The improved ORF encodes protein sequence (97C1_LEIXY), showing a significant sequence similarity with the other members of family GH97 along its whole length (Figure 2). However, it was impossible to determine the very beginning of the protein sequence including the start codon. This protein is a divergent representative of the GH97 family and it could not be classified into any subfamily on the basis of pairwise sequence comparison. 97C1_LEIXY and its closest homologue 97D1_CAUCR (E-value = 2 × 10-54) have only 30% of sequence identity.
A short gene fragment [GenBank: AY350337] from an uncultured bacterium was revealed by Genomic BLAST. It had been obtained and sequenced during PCR screening of human gut microflora [36]. The deduced protein sequence (97A2_UNBAC) corresponds to the C-terminal part of the others GH97 family proteins and has the highest similarity level with 97A1_BACTH (63% of sequence identity) and 97A1_TANFO (60%). It allows us to include this protein fragment into subfamily 97a (Table I).
PSI-BLAST search of the non-redundant protein database yielded a unique eukaryotic protein fragment [GenPept: EAL42226] homologous to GH97 family proteins. Screening of the database of eukaryotic nucleic acid sequences uncovered the corresponding DNA sequence [GenBank: AAAB01006165], as well as three other short sequences [GenBank: AAAB01064948, AAAB01020110, and AAAB01068263]. All of them had been sequenced during the mosquito Anopheles gambiae genome project [37]. These 4 sequences were aligned for the identification of overlapping regions. AAAB01064948 sequence is homologous to the central part of AAAB01006165 sequence having 54% of identity at the protein level. The ends of AAAB01020110 sequence are respectively homologous to one end of AAAB01006165 and AAAB01068263 sequences: 65% and 69% sequence identity at the protein level. Thus, these 4 sequences correspond to at least two different genes. In total, they cover a complete bacterial gene encoding of a protein of family GH97. Taking into account i) a high similarity level of the 4 deduced protein sequences with bacterial proteins (50–71% identity with 97A1_BACFR, 97A2_BACTH, 97A1_TANFO, and 97A1_BACTH), ii) the intron-free gene structure, iii) an inability to map the genes on the mosquito chromosomes, and iv) absence of GH97 family proteins in any other eukaryotic organism, we suggest the bacterial origin of these four gene fragments. The bacterial origin could have resulted from a contamination of Anopheles gambiae tissue used for preparing of genome library by mosquito Bacteroides-like gut microflora. The evidence for such kind of contamination was obtained when testing the 35,575 clones from A. gambiae cDNA library [38]. It was found that at least 808 sequences appeared to be bacterial contaminants.
In order to enlarge database of family GH97 we performed screening of the so-called "Environmental Samples data" [39]. It revealed 60 nucleic acid sequences from the Sargasso Sea that are homologous to genes of GH97 family proteins. However, the majority of them encode only short protein fragments and many of them have a very high level of sequence similarity. Among them we found only 5 full-size or almost complete genes (each encodes a protein consisting of more than 650 amino acid residues). Three additional "gene" sequences were obtained by combining overlapping gene fragments with almost identical sequences (at least 95% of sequence identity at the protein level). Hypothetical proteins (97A1_ENSEQ-97A8_ENSEQ) encoded by these 8 genes should be placed in the 97a subfamily, on the basis of sequence similarity (Table I). Moreover, the majority of the incomplete genes encode protein fragments belonging to the same subfamily. Only four [GenPept: EAE76000, EAE67019, EAH16525, and EAH96685] and two [GenPept: EAE21375 and EAG68085] protein fragments correspond to subfamilies 97b and 97c, respectively. One short fragment (137 amino acids; [GenPept: EAD85224]) cannot be unambiguously classified into any subfamily of the GH97 family. An analysis of the nucleic acid sequence encoding the latter protein fragment [GenBank: AACY01501371] allowed us to extend the protein fragment by using another start codon. The resulting protein sequence (97C1_ENSEQ; 218 amino acids) shows similarity with the sequences of the other members of family GH97 along its whole length. However, it was still impossible to include this protein fragment into any subfamily on the basis of pairwise sequence comparison.
Phylogenetic analysis of family GH97
To check the actual relationships of proteins within the GH97 family we performed a phylogenetic analysis using the obtained multiple sequence alignment. It is well known that phylogeny is the best basis for verification of subfamily structure of a protein family. In many works, where composition of a glycosidase family has been analyzed, the monophyletic status was used as the main argument for a subfamily description. Among others [40-44], this method has been applied to GH13 [12,13], GH27 [23,24], and GH36 [24] families of glycoside hydrolases.
In order to verify our subdivision of the GH97 family into subfamilies we checked the clustering of the family members in the phylogenetic tree. The maximum parsimony (MP; Figure 3A) and the neighbor-joining (NJ; Figure 3B) trees have very similar topology, suggesting the correct interpretation of the evolutionary events. When any subfamily of the GH97 family was considered as an outgroup, both MP and NJ trees showed that all other subfamilies appear to form monophyletic groups with a high bootstrap value (at least 95.4% of support at both trees). It should be noted that there is no pair of subfamilies that compose neighbor clusters on both trees with significant bootstrap support. This suggests approximately the same evolutionary distance between each pair of the subfamilies.
Figure 3 Phylogenetic trees of family GH97. The trees were reconstructed by the PHYLIP package. Each node was tested using the bootstrap approach and the number of supporting pseudoiterations (out of 1000) is indicated for each internal knot. Subfamily belongings of sequences are indicated, the value of bootstrap support for each subfamily is coloured in yellow. Red arrows indicate to the enzymatically-characterized proteins 97A1_BACTH and 97A1_TANFO (see text). The origin of sequences is given in Table I. (A) The maximum parsimony phylogenetic tree. The bootstrap values were determined using the maximum parsimony (PROTPARS) method. (B) The neighbor-joining phylogenetic tree. The number of amino acid substitutions per site is taken as a measure of branch length.
The archaeal protein 97A1_HALMA is a clear outlayer in the cluster of subfamily 97a at MP and NJ trees (Figure 3). The other members of this subfamily compose several subclusters, that include representatives either from Bacteroidetes or Proteobacteria phyla.
Unclassified protein 97C1_LEIXY is the closest neighbor of subfamily 97c cluster at MP and NJ trees (Figure 3) and therefore it can be considered as a divergent representative of this subfamily (Table I). Phylogenetic analysis of 97C1_ENSEQ protein fragment (data not shown) allowed us to place it into the same subfamily 97c.
An analysis of the GH97 family multiple sequence alignment revealed a number of amino acid positions that are highly conserved within several subfamilies but varied in amino acid residues in different subfamilies (Figure 2). Taken together, these signature sequence positions allow to predict the subfamily belonging of a protein sequence.
Relationship of family GH97 with some other glycosidase families
Depending on the GH97 query and the statistical significance threshold of E-value, during the second or third PSI-BLAST iterations, as a rule, we detected statistically significant similarities with α-galactosidases. They represent families GH27 and GH36 of clan GH-D (the α-galactosidase superfamily). More distant similarities were found with glycosidases of family GH31 (the α-galactosidase superfamily) and in some cases with enzymatically-uncharacterized proteins from COG0535. COG0535 has been annotated as a family of predicted Fe-S oxidoreductases, like the closest COG0641 [45]. Our BLAST searches show, that both COG families are related to the radical SAM superfamily of Fe-S enzymes [46], having (β/α)8-barrel fold [PDB: 1R30].
When we used some representatives of subfamily 97a (for example, 97A1_BACTH) as a query and an E-value cut-off of 0.01, it was possible to reveal statistically significant similarity with glycosidases of family GH20 (clan GH-K). A similarity with proteins of this family was detected after the second PSI-BLAST iteration, while the next one or two iterations revealed a distant relationship with members of COG0296 (family GH13 of clan GH-H). It should be noted that glycosidases from the clans GH-D, GH-H, and GH-K have a similar (β/α)8-barrel fold of their catalytic domain and the same molecular mechanism of the hydrolyzing reaction [2]. Thus, our results agree with the data of several authors [20,25,47-49] showing the relationship of glycosidases from GH13, GH27, GH31, and GH36 families. More detail analysis of these families and their relationship was done by Rigden [26].
Using the α-galactosidases from rice (GH27_ORYSA, family GH27) and Lactobacillus plantarum (GH36_LACPL, family GH36) as a query sequence for PSI-BLAST searches we found their homology with some representatives of the GH97 family (for example, 97B1_BACFR and 97B2_BACTH) after two or three iterations. However, a statistically significant sequence similarity of GH97 family proteins with α-galactosidases is restricted to a fragment of about 100–150 amino acid residues (Figure 2). This fragment corresponds to the N-terminal half of the catalytic (β/α)8-barrel domain of glycosidases from the α-galactosidase superfamily [50-54]. This half of the domain is known to be more conserved than the C-terminal half [26]. Therefore, we can assume that the catalytic domain of the GH97 family proteins also has a similar (β/α)8-barrel fold.
In order to check whether the whole (β/α)8-barrel domain is present in GH97 family proteins, we tried to reconstruct their secondary and tertiary structure. The SWISS-MODEL program failed to unambiguously predict the type of the tertiary structure. The 3D-PSSM, GOR IV, and nnpredict programs were used for prediction of the protein secondary structure. The results obtained suggest that the central part of the GH97 family protein sequences represents a typical and complete (β/α)8-barrel domain (Figure 2). The N- and C-terminal parts of the sequences, mainly consisting of β-strands, most probably form two additional non-catalytic domains with an unknown function. However, different programs produce contradictory results regarding the number and exact location of the β-strands (data not shown). The non-catalytic domains of glycosidases from the α-galactosidase and α-glucosidase superfamilies are also predominantly composed of β-strands. At least some of these domains are involved in oligomerization and carbohydrate binding [2,54].
3D-PSSM searches of the PDB database with several GH97 family proteins used as a query sequence yielded the highest level of similarity with the GH27 family glycosidases [PDB: 1KTB, 1R46, and 1UAS]. Among other best hits we have found representatives of several other (β/α)8-barrel fold glycoside hydrolase families: GH2 (clan GH-A), GH5 (GH-A), GH13 (GH-H), GH17 (GH-A), GH18 (GH-K), and GH20 (GH-K), as well as some other enzymes with (β/α)8-barrel fold, for example Bacillus subtilis inositol utilization protein IolI [PDB: 1I6N]. These results are in agreement with the hypothesis about common origin of all (β/α)8-barrel protein domains, that evolved from an ancestral (β/α)4 half-barrel by a tandem gene duplication followed by a fusion [55-60].
In all known glycosidases with the (β/α)8-barrel fold, the amino acid residues involved in the active center are located on the C-termini of the β-strands [61], a similar location of the active site was found in many other (β/α)8-barrel fold enzymes [60]. It is well known that two acidic groups (Asp and/or Glu) are almost always involved in the glycosidase active center, playing the roles of nucleophile and proton donor [4-6]. Their sequence location has been determined for several representatives of the GH27 and GH31 families [54,62-69].
The Asp residue, playing the role of nucleophile, is located on the C-terminus of the fourth β-strand of the barrel. This residue is highly conserved among proteins of the α-galactosidase superfamily [23,26]. The homologous residue in the GH97 family proteins is more variable, being Asp in all members of three subfamilies (97b, 97c, and 97d) and Gly in the other proteins (subfamilies 97a and 97e), including 97A1_BACTH and 97A1_TANFO (Figure 2). Since these two proteins display the α-glucosidase activity [29,30,70] we can conclude that a residue, set in another site, plays the role of nucleophile at least in some proteins of the GH97 family. It should be noted that we have found a residue on the C-terminus of the fifth β-strand in GH97 family sequences that is Gly in 97b, 97c, and 97d subfamilies, but Glu and Asp in subfamilies 97a and 97e respectively (Figure 2). Therefore, this residue can be suggested as a possible nucleophile in glycosidases of 97a and 97e subfamilies. As a rule, the catalytically essential residues are highly conserved among enzymatically active members of a glycoside hydrolase family, being either Asp, or Glu. The distance between the carboxylic groups of the nucleophile and the proton donor should be similar in order to keep the catalytic machinery. Thus, the difference in the predicted nucleophile residue between 97a and 97e subfamilies is unexpected. However, this does not exclude the existence of a glycosidase activity in proteins with Asp residue at the fifth β-strand (subfamily 97e). To illustrate, in the GH32 family the Asp residue was experimentally shown to be the nucleophile, while several proteins of this family have Glu residue at the homologous position and at least some of them are catalytically active [10,11].
The proton donor of families GH27 and GH31 is located on the C-terminus of the sixth β-strand of the (β/α)8-barrel domain. It is outside of the N-terminal half of barrel, which can be unambiguously aligned with proteins of the GH97 family. However, on the C-terminus of the sixth β-strand of the predicted (β/α)8-barrel of the GH97 family there is an Asp residue, which is highly conserved in all subfamilies of the family (Figure 2). We suggest this residue as a possible proton donor. Taking into account another structure of the active center and significant sequence similarity of only a half of the catalytic domain, the current data do not support an inclusion of the GH97 family into the α-galactosidase superfamily.
As far as we know, 97A1_BACTH and 97A1_TANFO are the only enzymatically-characterized proteins in the GH97 family [2]. All other members of this family have been found recently during genome projects and are encoded by ORFs. Genes of this family are represented only in a limited number of Eubacteria from phyla Actinobacteria (1 genus), Bacteroidetes (4 genera), Planctomycetes (1 genus), and Proteobacteria (3 and 4 genera from α- and γ-classes, respectively), as well as in a unique Archaea (Haloarcula marismortui). However, many of these bacteria have several paralogous genes. The most interesting case is that of B. thetaiotaomicron ATCC29148, which has α-glucosidase SusB (97A1_BACTH) and 9 putative paralogues representing four GH97 subfamilies (Table I), at least two of the paralogues (97C1_BACTH and 97C2_BACTH) are also expressed in vivo [28]. This human commensal microorganism is known as a bacterium with the highest number of glycosidase and glycosyltransferase genes [27,71]. Taken together, these facts we can suggest that evolution of GH97 family proteins has been associated with multiple duplications, gene elimination, and horizontal transfer.
Conclusion
The results of the sequence analysis allow us to distinguish five subfamilies in the GH97 family of glycoside hydrolases. The experimental data on the enzymatic activity are available only for two representatives of the GH97 family: α-glucosidases 97A1_BACTH and 97A1_TANFO [29,30,70]. However, we suppose that the other members of this family may also possess some glycosidase activities. Our data suggest that proteins of this family have a common evolutionary origin with glycosidases of the α-galactosidase superfamily. Many genes, encoding proteins of the GH97 family, are located in clusters with genes of glycoside hydrolases and other carbohydrate-active enzymes. For example, 97C1_BACTH and 97C2_BACTH (subfamily 97c) are encoded by genes of B. thetaiotaomicron located at a hemicellulose utilization locus together with eight other glycosidase genes (Figure 1). Taken together, these data support a recent suggestion to consider family GH97 (or GHX) as a new family of glycoside hydrolases [2,24]. The evolutionary relationship of GH97 proteins with glycosidases of the GH-D, GH-H, and GH-K (and probably GH-A) clans allows to extrapolate their common most important characteristics to glycoside hydrolases of the GH97 family. We can predict a similar (β/α)8-barrel fold of the catalytic domain and retaining mechanism of the glycoside bond hydrolysis for glycosidases of the GH97 family.
Methods
Protein and nucleic sequences were retrieved from the NCBI database [72]. All proteins analyzed in this work were designated by a ten-letter name (see Table I). The search for homologous proteins was done using the PSI-BLAST [73] and Genomic BLAST at the NCBI server. The statistical significance threshold for including a sequence in the model (E-value) used by PSI-BLAST in the next iteration was either 10-2 or 10-3, BLOSUM45 was used as a substitution matrix. Multiple sequence alignment was prepared manually using the program BioEdit [74] on the basis of BLAST pairwise alignments.
The multiple sequence alignment was used to implement classical phylogenetic inference programs, using either maximum parsimony or distance methods. Programs PROTPARS and NEIGHBOR from the PHYLIP package (version 3.6; [75]) were used. Moreover, programs SEQBOOT, PROTPARS, and CONSENSE and programs SEQBOOT, PROTDIST, NEIGHBOR, and CONSENSE were successively used to derive confidence limits, estimated by 1000 bootstrap replicates, for each node in the maximum parsimony and distance tree, respectively. The program TreeView Win32 (version 1.6.6; [76]) was used for drawing the trees.
An analysis of the order of the display sequence during searches by PSI-BLAST [73] was used for a preliminary division of a family into subfamilies. The latter was defined as a group of proteins that are displayed at the top of the list in a PSI-BLAST query results. Depending on particular criteria of the protein similarity used, the algorithm can split a family into a larger or smaller number of groups of proteins. Like in some of our previous works [10,23,24,77], in this study we define a subfamily as a group of proteins that have at least 30% sequence identity. Phylogenetic analysis was used in order to verify the obtained subfamilies and to clarify their boundaries. The monophyletic status was used as a criterion for the final definition of a subfamily.
The SWISS-MODEL modeling server [78] was used to predict the tertiary structure of proteins based on their amino acid sequences. The 3D-PSSM [79], GOR IV [80] and nnpredict [81] programs were used for prediction of the protein secondary structure. The 3D-PSSM program also was used to search the PDB database.
Added in proof
After submission of the manuscript, six new sequences of GH97 family proteins have been deposited at the NCBI database. Five of them (97A1_SHEBA, 97A1_SHEFR, 97A1_SHEDE, 97A1_SHEAM, and 97A1_SPHAL) belong to subfamily 97a (Table I). The sixth protein 97X1_SOLUS cannot be unambiguously classified into any subfamily of the GH97 family on the basis of pairwise sequence comparison, composition of the signature sequence positions, and phylogenetic analysis. Most probably it corresponds to a new subfamily.
Acknowledgements
I am grateful to Dr. Bernard Labedan (Université de Paris-Sud, France) for critical reading of an earlier version of the manuscript and a helpful discussion of the problem.
This work was supported by grants of the Russian President for young scientists (MK-118.2003.04 and MK-1461.2005.4).
==== Refs
Henrissat B A classification of glycosyl hydrolases based on amino acid sequence similarities Biochem J 1991 280 309 316 1747104
Coutinho PM Henrissat B Carbohydrate-Active Enzymes server
Henrissat B Bairoch A Updating the sequence-based classification of glycosyl hydrolases Biochem J 1996 316 695 696 8687420
Henrissat B Davies G Structural and sequence-based classification of glycoside hydrolases Curr Opin Struct Biol 1997 7 637 644 9345621 10.1016/S0959-440X(97)80072-3
McCarter JD Withers SG Mechanisms of enzymatic glycoside hydrolysis Curr Opin Struct Biol 1994 4 885 892 7712292 10.1016/0959-440X(94)90271-2
Davies G Henrissat B Structures and mechanisms of glycosyl hydrolases Structure 1995 3 853 859 8535779 10.1016/S0969-2126(01)00220-9
Henrissat B Bairoch A New families in the classification of glycosyl hydrolases based on amino acid sequence similarities Biochem J 1993 293 781 788 8352747
Naumov DG Doroshenko VG β-Fructosidases: a new superfamily of glycosyl hydrolases Mol Biol (Engl Tr) 1998 32 761 766 9914979
Naumoff DG Conserved sequence motifs in levansucrases and bifunctional β-xylosidases and α-L-arabinases FEBS Lett 1999 448 177 179 10217435 10.1016/S0014-5793(99)00369-5
Naumoff DG β-Fructosidase superfamily: homology with some α-L-arabinases and β-D-xylosidases Proteins 2001 42 66 76 11093261 10.1002/1097-0134(20010101)42:1<66::AID-PROT70>3.0.CO;2-4
Pons T Naumoff DG Martínez-Fleites C Hernández L Three acidic residues at the active site in the β-propeller architecture for the glycoside hydrolase families 32, 43, 62, and 68 Proteins 2004 54 424 432 14747991 10.1002/prot.10604
Oslancová A Janeček Š Oligo-1, 6-glucosidase and neopullulanase enzyme subfamilies from the α-amylase family defined by the fifth conserved sequence region Cell Mol Life Sci 2002 59 1945 1959 12530525
Janeček Š Svensson B MacGregor EA Relation between domain evolution, specificity, and taxonomy of the α-amylase family members containing a C-terminal starch-binding domain Eur J Biochem 2003 270 635 645 12581203 10.1046/j.1432-1033.2003.03404.x
Sarçabal P Remaud-Simeon M Willemot R Potocki de Montalk G Svensson B Monsan P Identification of key amino acid residues in Neisseria polysaccharea amylosucrase FEBS Lett 2000 474 33 37 10828446 10.1016/S0014-5793(00)01567-2
Berezina OV Lunina NA Zverlov VV Naumoff DG Liebl W Velikodvorskaia GA A cluster of Thermotoga neapolitana genes involved in the degradation of starch and maltodextins: the molecular structure of the locus Mol Biol (Engl Tr) 2003 37 801 809 14593916
Kuriki T Imanaka T The concept of the α-amylase family: structural similarity and common catalytic mechanism J Biosci Bioeng 1999 87 557 565 16232518 10.1016/S1389-1723(99)80114-5
Svensson B Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity, and stability Plant Mol Biol 1994 25 141 157 8018865 10.1007/BF00023233
Janeček Š Lévêque E Belarbi A Haye B Close evolutionary relatedness of α-amylases from Archaea and plants J Mol Evol 1999 48 421 426 10079280
van der Veen BA Uitdehaag JC Dijkstra BW Dijkhuizen L Engineering of cyclodextrin glycosyltransferase reaction and product specificity Biochim Biophys Acta 2000 1543 336 360 11150613
Dagnall BH Paulsen IT Saier JrMH The DAG family of glycosyl hydrolases combines two previously identified protein families Biochem J 1995 311 349 350 7575475
Naumoff DG Sequence analysis and classification of α-galactosidases International Summer School "From Genome to Life: Structural, Functional and Evolutionary Approaches" Cargèse, Corsica, France 40 July 15–27, 2002
Naumoff DG α-Galactosidase superfamily: phylogenetic analysis and homology with some α-glucosidases 5th Carbohydrate Bioengineering Meeting, University Hospital Groningen Groningen, The Netherlands 32 April 6–9, 2003
Naumoff DG Phylogenetic analysis of α-galactosidases from GH27 family Mol Biol (Engl Tr) 2004 38 388 399 15285616
Naumoff DG The α-galactosidase superfamily: sequence based classification of α-galactosidases and related glycosidases Proceedings of The Fourth International Conference on Bioinformatics of Genome Regulation and Structure, July 25–30, 2004 Novosibirsk Russia 1 315 318
Henrissat B Glycosidase families Biochem Soc Trans 1998 26 153 156 9649738
Rigden DJ Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36 and 66 share a common evolutionary origin with family 13 FEBS Lett 2002 523 17 22 12123797 10.1016/S0014-5793(02)02879-X
Xu J Bjursell MK Himrod J Deng S Carmichael LK Chiang HC Hooper LV Gordon JI A genomic view of the human-Bacteroides thetaiotaomicron symbiosis Science 2003 299 2074 2076 12663928 10.1126/science.1080029
Sonnenburg JL Xu J Leip DD Chen C-H Westover BP Weatherford J Buhler JD Gordon JI Glycan foraging in vivo by an intestine-adapted bacterial symbiont Science 2005 307 1955 1959 15790854 10.1126/science.1109051
Smith KA Salyers AA Characterization of a neopullulanase and an α-glucosidase from Bacteroides thetaiotaomicron 95-1 J Bacteriol 1991 173 2962 2968 1708385
D'Elia JN Salyers AA Contribution of a neopullulanase, a pullulanase, and an α-glucosidase to growth of Bacteroides thetaiotaomicron on starch J Bacteriol 1996 178 7173 7179 8955399
Reeves AR Wang GR Salyers AA Characterization of four outer membrane proteins that play a role in utilization of starch by Bacteroides thetaiotaomicron J Bacteriol 1997 179 643 649 9006015
Shipman JA Cho KH Siegel HA Salyers AA Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron J Bacteriol 1999 181 7206 7211 10572122
Cho KH Salyers AA Biochemical analysis of interactions between outer membrane proteins that contribute to starch utilization by Bacteroides thetaiotaomicron J Bacteriol 2001 183 7224 7230 11717282 10.1128/JB.183.24.7224-7230.2001
D'Elia JN Salyers AA Effect of regulatory protein levels on utilization of starch by Bacteroides thetaiotaomicron J Bacteriol 1996 178 7180 7186 8955400
Cho KH Cho D Wang GR Salyers AA New regulatory gene that contributes to control of Bacteroides thetaiotaomicron starch utilization genes J Bacteriol 2001 183 7198 7205 11717279 10.1128/JB.183.24.7198-7205.2001
Wei G Pan L Du H Chen J Zhao L ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts J Microbiol Methods 2004 59 91 108 15325756 10.1016/j.mimet.2004.06.007
Holt RA Subramanian GM Halpern A Sutton GG Charlab R Nusskern DR Wincker P Clark AG Ribeiro JM Wides R Salzberg SL Loftus B Yandell M Majoros WH Rusch DB Lai Z Kraft CL Abril JF Anthouard V Arensburger P Atkinson PW Baden H de Berardinis V Baldwin D Benes V Biedler J Blass C Bolanos R Boscus D Barnstead M Cai S Center A Chaturverdi K Christophides GK Chrystal MA Clamp M Cravchik A Curwen V Dana A Delcher A Dew I Evans CA Flanigan M Grundschober-Freimoser A Friedli L Gu Z Guan P Guigo R Hillenmeyer ME Hladun SL Hogan JR Hong YS Hoover J Jaillon O Ke Z Kodira C Kokoza E Koutsos A Letunic I Levitsky A Liang Y Lin JJ Lobo NF Lopez JR Malek JA McIntosh TC Meister S Miller J Mobarry C Mongin E Murphy SD O'Brochta DA Pfannkoch C Qi R Regier MA Remington K Shao H Sharakhova MV Sitter CD Shetty J Smith TJ Strong R Sun J Thomasova D Ton LQ Topalis P Tu Z Unger MF Walenz B Wang A Wang J Wang M Wang X Woodford KJ Wortman JR Wu M Yao A Zdobnov EM Zhang H Zhao Q Zhao S Zhu SC Zhimulev I Coluzzi M della Torre A Roth CW Louis C Kalush F Mural RJ Myers EW Adams MD Smith HO Broder S Gardner MJ Fraser CM Birney E Bork P Brey PT Venter JC Weissenbach J Kafatos FC Collins FH Hoffman SL The genome sequence of the malaria mosquito Anopheles gambiae Science 2002 298 129 149 12364791 10.1126/science.1076181
Gomez SM Eiglmeier K Segurens B Dehoux P Couloux A Scarpelli C Wincker P Weissenbach J Brey PT Roth CW Pilot Anopheles gambiae full-length cDNA study: sequencing and initial characterization of 35,575 clones Genome Biol 2005 6 R39 15833126 10.1186/gb-2005-6-4-r39
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA Wu D Paulsen I Nelson KE Nelson W Fouts DE Levy S Knap AH Lomas MW Nealson K White O Peterson J Hoffman J Parsons R Baden-Tillson H Pfannkoch C Rogers YH Smith HO Environmental genome shotgun sequencing of the Sargasso Sea Science 2004 304 66 74 15001713 10.1126/science.1093857
Zona R Chang-Pi-Hin F O'Donohue MJ Janeček Š Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis Eur J Biochem 2004 271 2863 2872 15233783 10.1111/j.1432-1033.2004.04144.x
Durand A Hughes R Roussel A Flatman R Henrissat B Juge N Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18 FEBS Journal 2005 272 1745 1755 15794761 10.1111/j.1742-4658.2005.04606.x
Ahn YO Mizutani M Saino H Sakata K Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1 J Biol Chem 2004 279 23405 23414 14976214 10.1074/jbc.M311379200
Shallom D Golan G Shoham G Shoham Y Effect of dimer dissociation on activity and thermostability of the α-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases J Bacteriol 2004 186 6928 6937 15466046 10.1128/JB.186.20.6928-6937.2004
Suzuki K Taiyoji M Sugawara N Nikaidou N Henrissat B Watanabe T The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases Biochem J 1999 343 587 596 10527937 10.1042/0264-6021:3430587
Tatusov RL Koonin EV Lipman DJ A genomic perspective on protein families Science 1997 278 631 637 9381173 10.1126/science.278.5338.631
Sofia HJ Chen G Hetzler BG Reyes-Spindola JF Miller NE Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods Nucleic Acids Res 2001 29 1097 1106 11222759 10.1093/nar/29.5.1097
Henrissat B Romeu A Families, superfamilies and subfamilies of glycosyl hydrolases Biochem J 1995 311 350 351 7575477
Margolles-Clark E Tenkanen M Luonteri E Penttilä M Three α-galactosidase genes of Trichoderma reesei cloned by expression in yeast Eur J Biochem 1996 240 104 111 8797842 10.1111/j.1432-1033.1996.0104h.x
Naumoff DG Sequence analysis of glycosylhydrolases: β-fructosidase and α-galactosidase superfamilies Glycoconj J 2001 18 109
Garman SC Hannick L Zhu A Garboczi DN The 1.9 Å structure of α-N-acetylgalactosaminidase: molecular basis of glycosidase deficiency diseases Structure 2002 10 425 434 12005440 10.1016/S0969-2126(02)00726-8
Fujimoto Z Kaneko S Momma M Kobayashi H Mizuno H Crystal structure of rice α-galactosidase complexed with D-galactose J Biol Chem 2003 278 20313 20318 12657636 10.1074/jbc.M302292200
Garman SC Garboczi DN The molecular defect leading to Fabry disease: structure of human α-galactosidase J Mol Biol 2004 337 319 335 15003450 10.1016/j.jmb.2004.01.035
Golubev AM Nagem RAP Brandão Neto JRNeustroev KN Eneyskaya EV Kulminskaya AA Shabalin KA Savel'ev AN Polikarpov I Crystal structure of α-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism J Mol Biol 2004 339 413 422 15136043 10.1016/j.jmb.2004.03.062
Lovering AL Lee SS Kim Y-W Withers SG Strynadka NCJ Mechanistic and structural analysis of a family 31 α-glycosidase and its glycosyl-enzyme intermediate J Biol Chem 2005 280 2105 2115 15501829 10.1074/jbc.M410468200
Höcker B Jürgens C Wilmanns M Sterner R Stability, catalytic versatility and evolution of the (βα)8-barrel fold Curr Opin Biotechnol 2001 12 376 381 11551466 10.1016/S0958-1669(00)00230-5
Höcker B Beismann-Driemeyer S Hettwer S Lustig A Sterner R Dissection of a (βα)8-barrel enzyme into two folded halves Nat Struct Biol 2001 8 32 36 11135667 10.1038/83021
Gerlt JA Babbitt PC Barrels in pieces? Nat Struct Biol 2001 8 5 7 11135656 10.1038/83048
Lang D Thoma R Henn-Sax M Sterner R Wilmanns M Structural evidence for evolution of the β/α barrel scaffold by gene duplication and fusion Science 2000 289 1546 1550 10968789 10.1126/science.289.5484.1546
Farber GK Petsko GA The evolution of α/β barrel enzymes Trends Biochem Sci 1990 15 228 234 2200166 10.1016/0968-0004(90)90035-A
Wierenga RK The TIM-barrel fold: a versatile framework for efficient enzymes FEBS Lett 2001 492 193 198 11257493 10.1016/S0014-5793(01)02236-0
Nagano N Porter CT Thornton JM The (β/α)8 glycosidases: sequence and structure analyses suggest distant evolutionary relationships Protein Eng 2001 14 845 855 11742103 10.1093/protein/14.11.845
Quaroni A Semenza G Partial amino acid sequences around the essential carboxylate in the active sites of the intestinal sucrase-isomaltase complex J Biol Chem 1976 251 3250 3253 776963
Hermans MMP Kroos MA van Beeumen J Oostra BA Reuser AJJ Human lysosomal α-glucosidase. Characterization of the catalytic site J Biol Chem 1991 266 13507 13512 1856189
Iwanami S Matsui H Kimura A Ito H Mori H Honma M Chiba S Chemical modification and amino acid sequence of active site in sugar beet α-glucosidase Biosci Biotechnol Biochem 1995 59 459 463 7766184
Kimura A Takata M Fukushi Y Mori H Matsui H Chiba S A catalytic amino acid and primary structure of active site in Aspergillus niger α-glucosidase Biosci Biotechnol Biochem 1997 61 1091 1098 9255970
Hart DO He S Chany CJ Withers SG Sims PF Sinnott ML Brumer H Identification of Asp-130 as the catalytic nucleophile in the main α-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase Biochemistry 2000 39 9826 9836 10933800 10.1021/bi0008074
Ly HD Howard S Shum K He S Zhu A Withers SG The synthesis, testing and use of 5-fluoro-alpha-D-galactosyl fluoride to trap an intermediate on green coffee bean α-galactosidase and identify the catalytic nucleophile Carbohydr Res 2000 329 539 547 11128583 10.1016/S0008-6215(00)00214-7
Okuyama M Okuno A Shimizu N Mori H Kimura A Chiba S Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of α-glucosidase from Schizosaccharomyces pombe Eur J Biochem 2001 268 2270 2280 11298744 10.1046/j.1432-1327.2001.02104.x
Kashiwabara S Azuma S Tsuduki M Suzuki Y The primary structure of the subunit in Bacillus thermoamyloliquefaciens KP1071 molecular weight 540,000 homohexameric α-glucosidase II belonging to the glycosyl hydrolase family 31 Biosci Biotechnol Biochem 2000 64 1379 1393 10945254 10.1271/bbb.64.1379
Hughes CV Malki G Loo CY Tanner ACR Ganeshkumar N Cloning and expression of α-D-glucosidase and N-acetyl-β-glucosaminidase from the periodontal pathogen, Tannerella forsythensis (Bacteroides forsythus) Oral Microbiol Immunol 2003 18 309 312 12930523 10.1034/j.1399-302X.2003.00091.x
Coutinho PM Stam M Blanc E Henrissat B Why are there so many carbohydrate-active enzyme-related genes in plants? Trends Plant Sci 2003 8 563 565 14659702 10.1016/j.tplants.2003.10.002
Wheeler DL Barrett T Benson DA Bryant SH Canese K Church DM DiCuccio M Edgar R Federhen S Helmberg W Kenton DL Khovayko O Lipman DJ Madden TL Maglott DR Ostell J Pontius JU Pruitt KD Schuler GD Schriml LM Sequeira E Sherry ST Sirotkin K Starchenko G Suzek TO Tatusov R Tatusova TA Wagner L Yaschenko E Database resources of the National Center for Biotechnology Information Nucleic Acids Res 2005 33 D39 45 15608222 10.1093/nar/gki062
Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389
Hall TA Bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt Nucleic Acids Symp Ser 1999 41 95 98
Felsenstein J PHYLIP – Phylogeny Inference Package (Version 3.2) Cladistics 1989 5 164 166
Page RDM TREEVIEW: An application to display phylogenetic trees on personal computers Comput Appl Biosci 1996 12 357 358 8902363
Naumoff DG Livshits VA Molecular structure of the Lactobacillus plantarum sucrose utilization locus: comparison with Pediococcus pentosaceus Mol Biol (Engl Tr) 2001 35 15 22 11234378
Peitsch MC ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling Biochem Soc Trans 1996 24 274 279 8674685
Kelley LA MacCallum RM Sternberg MJ Enhanced genome annotation using structural profiles in the program 3D-PSSM J Mol Biol 2000 299 499 520 10860755 10.1006/jmbi.2000.3741
Garnier J Gibrat JF Robson B GOR method for predicting protein secondary structure from amino acid sequence Methods Enzymol 1996 266 540 553 8743705
Kneller DG Cohen FE Langridge R Improvements in protein secondary structure prediction by an enhanced neural network J Mol Biol 1990 214 171 182 2370661 10.1016/0022-2836(90)90154-E
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1161615015510.1186/1471-2164-6-116Research ArticleComparison of theoretical proteomes: Identification of COGs with conserved and variable pI within the multimodal pI distribution Nandi Soumyadeep [email protected] Nipun [email protected] Andrew M [email protected] Alok [email protected] Centre for Computational Biology and Bioinformatics, School of Information Technology, Jawaharlal Nehru University, New Delhi 110067, India2 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India2005 9 9 2005 6 116 116 9 3 2005 9 9 2005 Copyright © 2005 Nandi et al; licensee BioMed Central Ltd.2005Nandi 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
Theoretical proteome analysis, generated by plotting theoretical isoelectric points (pI) against molecular masses of all proteins encoded by the genome show a multimodal distribution for pI. This multimodal distribution is an effect of allowed combinations of the charged amino acids, and not due to evolutionary causes. The variation in this distribution can be correlated to the organisms ecological niche. Contributions to this variation maybe mapped to individual proteins by studying the variation in pI of orthologs across microorganism genomes.
Results
The distribution of ortholog pI values showed trimodal distributions for all prokaryotic genomes analyzed, similar to whole proteome plots. Pairwise analysis of pI variation show that a few COGs are conserved within, but most vary between, the acidic and basic regions of the distribution, while molecular mass is more highly conserved. At the level of functional grouping of orthologs, five groups vary significantly from the population of orthologs, which is attributed to either conservation at the level of sequences or a bias for either positively or negatively charged residues contributing to the function. Individual COGs conserved in both the acidic and basic regions of the trimodal distribution are identified, and orthologs that best represent the variation in levels of the acidic and basic regions are listed.
Conclusion
The analysis of pI distribution by using orthologs provides a basis for resolution of theoretical proteome comparison at the level of individual proteins. Orthologs identified that significantly vary between the major acidic and basic regions maybe used as representative of the variation of the entire proteome.
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Background
A protein's Isoelectric point (pI) – the pH at which a protein has no net charge – is the basis for its isolation using isoelectric focussing and along with Molecular Mass (Mr) is exploited in two dimensional gel electrophoresis used to seperate a cell's protein content. Bjellquist [1,2] has shown that a the pI of a denatured linear protein can be calculated with high accuracy using the pK values of the amino acids responsible for charge. Using these calculations, it is posible to create an image of an organisms theoretical proteome, by plotting the theoretical pI against their theoretical Mr. The distribution of pI in these plots have a multimodal distribution. Early results on bacteria demonstrated a bimodal distribution with peaks centered around pH 5.5 and pH 9 [3]. This bimodality was explained as being caused by the fact that as proteins are least soluble at their pI, they have evolved to have pI's away from neutral pH – which was assumed to be the intracellular pH. Schwartz et al [4], further showed the presence of a trimodal pI distribution in Eukaryotes, and observed a correlation of pI to intracellular localisation. Cytoplasmic, nuclear and membrane proteins seemed to lie largely in the acidic, neutral and basic portions of the trimodal distribution, respectively.
Exhaustive work on virtual proteomes has been completed recently by two groups. Weiller et al [5] have proved that the multimodal distribution of protein pI is present in randomly generated sequences, and is a function of allowed combinations of amino-acid pKa values, rather than a cause of sequence evolution. A trimodal distribution is present in the virtual proteome of most organisms, with minima at 7.4 and 8.0. Knight et al [6] have shown that the variation in proteomes though the trimodal distribution is largely maintained – is influenced by the ecological niche of the organism.
Environmental influences of the proteome are known[7]. Amino acid usage is influenced by the G+C content of a genome [8]. Acidic residues predominate over basic residues in halophilic bacteria [9,10], and compositional properties are further distinguished in thermophilic and mesophilic bacteria – with a preference for salt-bridges (residues with opposite charges) and long-chain hydrophobic residues in the former for increased stability [11].
All studies so far have used the gross properties of the proteome, or broad functional groups (e.g membrane proteins), and not attempted to resolve the multimodal distribution on the basis of individual proteins. This analysis could provide answers to the apparant conflict that though the pI multimodal distribution is caused by the properties of amino acids and not evolutionary factors, environmental influences induce variation in the sizes of each cluster in the distribution. By mapping the variation of pI in orthologs, one can in principal identify proteins whose pI is conserved as well as those whose pI does not seem to be responsible for its function, and whose variation maybe used as markers for an organism's environment.
The COG (Cluster of Orthologous Groups) database [12,13] lists orthologs present across completed genomes. In this study, we consider the subset of an organism's proteome, as specified by the COG database, which contain orthologs present in other genomes – providing a basis to study the variation of pI of individual proteins across genomes.
Results and discussion
Comparison of virtual proteomes among different Bacteria organisms
The predicted proteomes, using values of Mr and pI calculated from the protein ortholog sequence, all displayed a trimodal distribution, with minima at 7.4 and 8.1. For convenience, the three major peaks demarkated by these minima are referred to as the acidic cluster (pI less than 7.4), "neutral" cluster (pI between 7.4 and 8.1) and basic cluster (pI greater than 8.1). This observation of a trimodal distribution is consistent with earlier results calculated from the complete genome [4-6], showing that orthalogs maybe used as representative samples of the complete genome. Figure 1 shows representative plots of proteomes using whole genomes and orthalog sub-sets for Escherichia coli K12 and Helicobacter pylori, Buchenara and Halobacterium. As only orthologs are used, it is possible to compare the pI of two genomes, by using a scatter plot. Diagonal points show invariant pI between the organisms, while a shift in pI is visible as an off-diagonal point. As the trimodal distribution is dominated by the large acidic and basic clusters, A shift in the pI from one cluster to the other is visable on the upper left and lower right quadrants of the plot. Figure 2 shows the pairwise comparison of the pI and Mr for the proteomes of two variants of E. coli (K-12 and 0157), between E. coli K12 and H. pylori which have different environmental pI – and extremophiles Buchenara and Halobacterium. Closely related variants of the same species do not have much variation in both pI and Mr, however the effect of a changed pH environment – specifically the acidic stomach for H. pylori and the basic intestine for E. coli – causes a large proportion of orthologs to change pI to compensate for the external pH change. Organisms that have extreme proteome pI distributions are Halobacterium – which has a large acidic cluster and Buchnera – a large negative cluster. As expected, most orthologs shared by the two organisms show a shift in their pI from one cluster to the other, however a baseline of pI conservation is maintained, implying that the pI maybe a conserved property for a few orthologs. The Mr is much more highly conserved, with the scatter plot clustered along the diagonal.
Figure 1 Ortholog (left) and whole genome (right) isoelectric point (pI) frequency distributions for (A) Escherichia coli K 12, (B) Helicobacter pylori (C) Buchenera APS and (D) Halobacterium sps NRC-1
Figure 2 Pairwise comparison of isoelectric point and molecular mass of orthologs. (A) Escherichia coli K12 and 0517; (B) Escherichia coli K12 and Helicobacter pylori (C) Halobacterium sps NCR-1 and Buchnera sps APS
Variation in pI among functional categories of COGS
The COG database is functional classified into eighteen categories [12] (Table 1, shows a listing of these functions, which the single letter code used in the figures). The distribution of pI values across all organisms for each function, summarised by the mean and standard deviation, is shown in Figure 3. The mean values for each function is close to neutral pH, with a deviation spreading across both clusters. Again the use of orthologs provides the means to correlate conservation of pI between pairs of organisms. The mean pairwise-correlation of ortholog pI corresponding to each function was also computed (Figure 4A), along with the corresponding pairwise correlation of ortholog Mr (Figure 4B). We have used Kruskal-Wallis multiple comparison testing [14] to identify groups that significantly deviate from the expected distribution. Five functional groups deviate significantly, three towards the basic cluster and two towards the acidic cluster (Table 1).
Table 1 Variation in pI in functionally classified groups of COGs. The table lists functional groups of COGs as defined in the COG website [22] along with the results of the Dunn Multiple Comparison test. Ri – mean rank for the group, Rt – mean rank for sample representing the complete distribution. Significant values are those where |Rt - Ri| > 467.35 which is calculated for α = 0.2
Ri Ri - Rt
Metabolism
C Energy production and conversion 2563.72 276.96
E Amino acid transport and metabolism 2792.99 47.69
F Nucleotide transport and metabolism 2191.12 649.56
G Carbohydrate transport and metabolism 2958.98 -118.3
H Coenzyme metabolism 2736.4 104.28
I Lipid metabolism 2587.87 252.81
Q Secondary metabolism biosynthesis, transport and catabolism 2479.07 361.62
Information storage and processing
J Translation, ribosomal structure and biogenesis 3399.31 -558.63
K Transcription 2977.93 -137.25
L DNA replication, recombination and repair 3530.56 -689.88
Cellular process
M Cell Envelope biogenesis, Outer membrane 3287.13 -446.45
N Cell motility and secretion 2722.08 118.6
O Post translation modification, protein turnover, chaperones 2445.01 395.67
P Inorganic ion transport and metabolism 3482.93 -642.25
T Signal transduction mechanisms 2219.23 621.45
D Cell devision and chromosome partitioning 2836.43 4.25
Poorly characterized
R General function prediction only 3189.18 -348.5
S Function unknown 2918.88 -78.2
Rt = 2840.68
Figure 3 Frequency distribution for COG mean pI (grey vertical bars). The variation of pI (mean and standard deviation) for functionally classified groups of COGs is overlayed. Functional groups denoted by single letters is expanded in Table 1
Figure 4 Mean correlation of pI (A), Mr (B) and sequence distance (C) for all organism pairs for the functional groups of COGS. Functional groups are denoted by single letters, expanded in Table 1
Membrane proteins are known to have a preference for the basic cluster, caused by the larger proportion of basic charged residues to compensate for the negatively charged membrane bilayer [4,6]. A functional requirement for either basic or acidic charges could thus influence the protein's pI. Orthologs, by definition, perform the same function in different organisms, and if preference for either basic or acidic charged residues is related to this function, this should be reflected in the protien's pI having a bias for the respective cluster. However, the conservation of pI maybe accidental, especially if the proteins are highly conserved. We have calculated the pairwise distance of all proteins within a COG as a measure of their sequence similarity. The distributions of distance for each functional group is shown in Figure 4C.
Proteins associated with the group "J", involved with translation, ribosomal structure and biogenesis, are dominated by highly conserved ribosomal proteins – and this high level of conservation and intolerance to mutation is reflected in an invariance of pI. This conservation is also reflected in the higher correlation for this group of proteins in their Mr. Other groups which are polarised to either the acidic or basic modes of the pI distribution do not show such a high level of conservation, and pI conservation is dictated by the nature of their function.
Analysis of pI variation for individual COGS
The general function of a group of proteins and their level of conservation may dictate a preference for a specific range of pI, as had been shown earlier for membrane proteins, and for functional groups of COGs in the previous section of this paper. Individual proteins maybe identified that have a preference for either the acidic or basic cluster. The scatter plot of the pI of all ortholog proteins used in this analysis is sorted by COG mean pI and plotted in Figure (5). The allowed trimodal regions are clearly visable. However at both the left and right extremes, the scatter plot show that COGs do exist with a preference for the acidic or basic cluster respectively. We have computed the frequency distribution for each COG in the acidic, neutral and basic clusters. On analysis, no COG is conserved in the neutral cluster, the largest frequency being 0.5 for COGs 1689, 3016, 3783 and 3801, however their frequency of occurance among all organisms is less than ten percent. This is an expected result as the pI distribution is caused by an interplay of acidic and basic residues which acount for the large acidic and basic clusters. The "neutral" cluster is not caused by the absense or balance of charged residues but because allowed pK combinations of charged residues are minimum at pH 7.1 and 8.4 [5].
Figure 5 Isoelectric point distribution of orthologs sorted by mean isoelectric point value. Black dot – mean value for COG, grey bar – standard deviation for COG, red point – pI value of individual genes classified under the COG. The sorted list of COG's used for the X-axis is available as an additional file.
Proteins conserved in the acidic and basic clusters however have a preponderance of acidic and basic residues respectively, which maybe required for their function. A complete list of COGs whose protein pI are conserved in these clusters is listed as an additional file. We have scaled the frequency of conservation by the frequency of occurance, so that only COGs which are maximally represented across the organisms in the dataset are used as markers of each cluster. These are listed in Table 2 and Table 3 respectively. The dominant groups of proteins which seem to require an acidic pI are the amino acid tRNA synthetases. Among those proteins whose pI is highly conserved in the basic cluster are a large number of ribosomal proteins. Although highly conserved and intolerant to mutation, ribosomal proteins interweave with negatively charged RNA to form the ribosome, and being positively charged will be a requirement for strong electrostatic interactions.
Table 2 COGs conserved in the acidic cluster of the multimodal distribution. P(a) and P(b) are the frequency of occurance in the acidic and basic clusters respectively, F is the function group. The COGs are listed with their P(v) which is score of their variability between the acidic and basic clusters of the pI distribution weighted by their frequency of occurance across all genomes in the dataset. pI values of four organisms – the extremophiles Buchnera (environment bacteriocyte), Halobacterium (highly acidic – high salt), and E. coli and H. pylori – intestinal bacteria with different environmental pH.
COGs with pI conserved in Acidic Cluster
COG P(a) P(b) P(v) F Role Hbs Eco Hpy Buc
COG2890 0.80 0.18 0.35 J Predicted rRNA or tRNA methylase 4.23 4.99 5.61 9.38
COG0021 0.81 0.00 0.05 G Transketolase - 5.61 6.23 7.98
COG0215 0.82 0.11 0.24 J Cysteinyl-tRNA synthetase 4.47 5.29 5.88 9.33
COG0016 0.82 0.14 0.32 J Phenylalanyl-tRNA synthetase alpha subunit 4.14 5.71 6.35 9.51
COG0449 0.82 0.04 0.08 M Glucosamine 6-phosphate synthetase, contains amidotransferase and phosphosugar isomerase 5.08 5.52 6.05 9.52
COG0206 0.82 0.08 0.16 D Cell division GTPase 4.35 4.63 5.26 5.08
COG0436 0.82 0.04 0.10 E PLP-dependent aminotransferases 4.41 6.15 7.41 -
COG0468 0.83 0.15 0.31 L RecA/RadA recombinase 4.44 5.06 5.47 -
COG0125 0.83 0.17 0.35 F Thymidylate kinase 4.32 5.26 8.97 10.01
COG0084 0.84 0.03 0.09 L Mg-dependent DNase - 5.44 5.43 8.81
COG0504 0.84 0.07 0.18 F CTP synthase (UTP-ammonia lyase) 4.47 5.58 7.55 9.01
COG0495 0.84 0.11 0.27 J Leucyl-tRNA synthetase 4.18 5.14 7.51 9.53
COG0124 0.86 0.12 0.27 J Histidyl-tRNA synthetase 4.29 5.57 5.54 9.28
COG0443 0.86 0.02 0.04 O Molecular chaperone 3.95 5.08 5.02 7.44
COG0024 0.86 0.08 0.22 J Methionine aminopeptidase 4.05 5.55 5.89 8.70
COG0209 0.87 0.03 0.10 F Ribonucleotide reductase alpha subunit 4.39 5.89 5.73 9.11
COG0142 0.87 0.06 0.13 H Geranylgeranyl pyrophosphate synthase 4.31 4.96 5.94 9.58
COG0441 0.88 0.10 0.23 J Threonyl-tRNA synthetase 4.35 5.76 5.93 8.86
COG0112 0.88 0.10 0.19 E Glycine hydroxymethyltransferase 4.30 5.95 6.41 9.22
COG0525 0.88 0.12 0.23 J Valyl-tRNA synthetase 4.09 5.19 6.12 9.38
COG0060 0.89 0.11 0.23 J Isoleucyl-tRNA synthetase 4.13 5.63 6.15 9.22
COG0073 0.89 0.08 0.19 R EMAP domain 4.04 5.29 5.42 8.90
COG0172 0.89 0.09 0.21 J Seryl-tRNA synthetase 4.60 5.30 6.70 9.42
COG0018 0.89 0.09 0.20 J Arginyl-tRNA synthetase 4.18 5.29 5.94 9.72
COG0143 0.89 0.09 0.20 J Methionyl-tRNA synthetase 4.16 5.39 6.00 9.27
COG1109 0.89 0.09 0.21 G Phosphomannomutase 4.43 5.55 6.18 9.03
COG0006 0.90 0.04 0.11 E Xaa-Pro aminopeptidase 4.42 5.35 5.73 -
COG0442 0.91 0.09 0.18 J Prolyl-tRNA synthetase 4.45 5.11 5.90 9.51
COG0126 0.91 0.07 0.14 G 3-phosphoglycerate kinase 4.29 5.06 6.18 9.37
COG0012 0.91 0.09 0.17 R Predicted GTPase 4.15 4.86 5.50 9.07
COG0149 0.91 0.02 0.09 G Triosephosphate isomerase 4.15 5.55 7.58 9.23
COG0492 0.92 0.08 0.16 O Thioredoxin reductase 4.27 5.24 6.02 9.36
COG0072 0.93 0.07 0.14 J Phenylalanyl-tRNA synthetase beta subunit 4.33 5.26 6.63 8.30
COG0085 0.94 0.02 0.08 K DNA-directed RNA polymerase beta subunit/140 kD subunit (split gene in Mjan, Mthe, Aful) 4.77 5.14 6.14 7.81
COG0231 0.94 0.02 0.08 J Translation elongation factor P/translation initiation factor eIF-5A 4.78 5.28 5.34 9.40
COG0459 0.95 0.01 0.04 O Chaperonin GroEL (HSP60 family) 4.16 4.84 5.51 5.04
COG0013 0.95 0.05 0.09 J Alanyl-tRNA synthetase 4.29 5.51 5.96 9.42
COG0592 0.96 0.04 0.09 L DNA polymerase sliding clamp subunit (PCNA homolog) 3.97 5.20 5.45 8.93
COG0202 0.98 0.02 0.05 K DNA-directed RNA polymerase alpha subunit/40 kD subunit 4.11 4.95 4.95 5.03
COG0148 0.98 0.00 0.00 G Enolase 4.38 5.28 5.42 6.93
COG0480 0.99 0.01 0.03 J Translation elongation and release factors (GTPases) 4.41 5.41 5.23 7.61
Table 3 COGs conserved in the Basic cluster of the multimodal distribution. Table headers are the same as in Table 2.
COGs with pI conserved in Basic Cluster
COG P(a) P(b) P(v) F Role Hbs Eco Hpy Buc
COG0101 0.11 0.82 0.27 J Pseudouridylate synthase (tRNA psi55) 5.52 8.68 9.74 9.66
COG0582 0.05 0.83 0.12 L Integrase 5.31 9.70 9.61 -
COG0080 0.15 0.85 0.30 J Ribosomal protein L11 3.72 9.64 9.55 10.01
COG0477 0.09 0.87 0.20 E Permeases of the major facilitator superfamily 6.54 9.32 9.14 9.65
COG0477 0.09 0.87 0.20 E Permeases of the major facilitator superfamily 6.54 9.32 9.14 9.65
COG0477 0.09 0.87 0.20 E Permeases of the major facilitator superfamily 6.54 9.32 9.14 9.65
COG0477 0.09 0.87 0.20 E Permeases of the major facilitator superfamily 6.54 9.32 9.14 9.65
COG0541 0.07 0.88 0.19 N Signal recognition particle GTPase 4.27 9.52 9.40 9.82
COG0255 0.09 0.89 0.21 J Ribosomal protein L29 4.69 9.98 9.70 10.45
COG0092 0.07 0.93 0.14 J Ribosomal protein S3 3.87 10.27 10.25 10.40
COG0198 0.07 0.93 0.14 J Ribosomal protein L24 4.37 10.21 9.86 10.57
COG0051 0.04 0.93 0.11 J Ribosomal protein S10 5.15 9.68 9.30 9.85
COG0089 0.02 0.95 0.07 J Ribosomal protein L23 4.13 9.94 10.00 10.00
COG0256 0.05 0.95 0.09 J Ribosomal protein L18 5.13 10.42 10.16 10.94
COG0087 0.02 0.98 0.05 J Ribosomal protein L3 5.57 9.90 10.24 10.60
COG0088 0.02 0.98 0.05 J Ribosomal protein L4 4.83 9.72 9.43 10.19
COG0091 0.02 0.98 0.05 J Ribosomal protein L22 5.08 10.23 11.26 10.85
COG0098 0.02 0.98 0.05 J Ribosomal protein S5 4.92 10.11 9.90 10.51
COG0185 0.02 0.98 0.05 J Ribosomal protein S19 5.20 10.52 10.36 10.73
COG0200 0.02 0.98 0.05 J Ribosomal protein L15 4.85 11.19 10.52 11.62
COG0201 0.02 0.98 0.05 N Preprotein translocase subunit SecY 5.56 9.89 9.87 9.72
COG0049 0.02 0.98 0.04 J Ribosomal protein S7 5.51 10.37 10.17 10.33
COG0081 0.02 0.98 0.04 J Ribosomal protein L1 4.13 9.64 9.61 9.86
COG0094 0.02 0.98 0.04 J Ribosomal protein L5 4.66 9.49 9.74 9.89
COG0096 0.02 0.98 0.04 J Ribosomal protein S8 4.85 9.44 9.79 9.74
COG0097 0.02 0.98 0.04 J Ribosomal protein L6 4.20 9.71 9.77 10.07
COG0099 0.02 0.98 0.04 J Ribosomal protein S13 4.52 10.78 10.23 10.54
COG0100 0.02 0.98 0.04 J Ribosomal protein S11 5.72 11.33 10.33 11.18
COG0102 0.02 0.98 0.04 J Ribosomal protein L13 4.50 9.91 9.82 9.93
COG0184 0.02 0.98 0.04 J Ribosomal protein S15P/S13E 4.83 10.40 10.00 10.17
COG0186 0.02 0.98 0.04 J Ribosomal protein S17 4.75 9.64 10.01 10.14
COG0522 0.02 0.98 0.04 J Ribosomal protein S4 and related proteins 4.81 10.05 10.08 10.00
COG0199 0.02 0.98 0.04 J Ribosomal protein S14 5.86 11.16 11.01 11.38
COG0048 0.00 1.00 0.00 J Ribosomal protein S12 9.91 10.88 10.70 11.09
COG0090 0.00 1.00 0.00 J Ribosomal protein L2 9.51 10.93 10.36 10.77
COG0093 0.00 1.00 0.00 J Ribosomal protein L14 9.60 10.43 10.45 10.43
COG0103 0.00 1.00 0.00 J Ribosomal protein S9 9.39 10.94 10.70 11.40
COG0197 0.00 1.00 0.00 J Ribosomal protein L16/L10E 9.28 11.23 10.44 10.88
A majority of COGs however show distributions across both the basic and acidic clusters. Knight et al, have shown a correlation to a change in proteome patterns with the organism's ecological niche. Since the proteome always exists in a trimodal distribution, it will only vary from organism to organism in the relative amounts of proteins which are present in each of the three clusters of the pI distribution. We have computed the probability of being in both the acidic and basic clusters weighted by the frequency of occurance in the organisms under consideration in order to identify COGs which are highly represented and show no particular preference for either cluster. This list of COGs, with a joint probability greater than a cutoff value of 0.6 is tabulated in table 4. For reference, the individual values of genes corresponding to the four organisms is also listed, and in a majority of cases show good agreement with the shift in an organism's total theoretical proteome towards either the basic or acidic clusters. Except for some ribosomal proteins, which appear on the list because of their high frequency of occurance, all other COGs are membrane based proteins, which would have direct contact with the external environment. These COGs best represent an organism's shift from expected levels of the acidic and basic clusters of the multimodal distribution, and it is possible that they maybe used as markers to predict an organisms ecological niche, with particular reference to its environmental pH in free living microorganisms.
Table 4 Highly occuring COGs with pI varying across both the acidic and basic clusters. Table headers are the same as in Table 2.
COG P(a) P(b) P(v) F Role Hbs Eco Hpy Buc
COG0760 0.29 0.31 0.60 O Parvulin-like peptidyl-prolyl isomerase - 6.83 9.04 9.65
COG0725 0.31 0.29 0.60 P ABC-type molybdate transport system, periplasmic component 4.90 7.53 9.70 -
COG0042 0.44 0.25 0.60 R Predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family 4.82 6.04 9.20 -
COG0275 0.28 0.46 0.60 M Predicted S-adenosylmethionine-dependent methyltransferase - 5.92 8.99 9.93
COG0258 0.69 0.30 0.61 L 5'-3' exonuclease (including N-terminal domain of PolI) 4.06 5.74 9.06 9.59
COG0640 0.38 0.28 0.61 K Predicted transcriptional regulators 4.49 8.48 - -
COG0444 0.37 0.27 0.61 E ABC-type dipeptide/oligopeptide/nickel transport system, ATPase componen 4.36 6.64 8.29 -
COG0009 0.61 0.30 0.61 J Putative translation factor (SUA5) 4.43 5.36 - 9.52
COG0226 0.39 0.30 0.62 P ABC-type phosphate transport system, periplasmic component 4.04 8.37 - -
COG1475 0.41 0.26 0.62 K Predicted transcriptional regulators 4.46 6.78 8.39 -
COG0190 0.51 0.30 0.62 H 5,10-methylene-tetrahydrofolate dehydrogenase/Methenyl tetrahydrofolate cyclohydrolase 4.36 5.61 8.74 9.70
COG1057 0.31 0.33 0.62 H Nicotinic acid mononucleotide adenylyltransferase - 5.38 8.80 9.68
COG0482 0.42 0.28 0.62 J Predicted tRNA methyltransferase, contains the PP-loop ATPase domain - 4.89 8.82 9.50
COG1121 0.29 0.35 0.62 P ABC-type Mn/Zn transport systems, ATPase component 4.61 9.44 - 9.21
COG0359 0.31 0.48 0.62 J Ribosomal protein L9 - 5.82 8.80 10.29
COG0858 0.46 0.29 0.62 J Ribosome-binding factor A - 5.58 7.55 9.88
COG0356 0.30 0.25 0.62 C F0F1-type ATP synthase a subunit - 6.18 6.34 7.64
COG0501 0.29 0.41 0.63 O Zn-dependent protease with chaperone function 7.18 7.10 7.86 6.91
COG0350 0.31 0.46 0.64 L Methylated DNA-protein cysteine methyltransferase 5.67 7.13 9.38 -
COG1136 0.53 0.29 0.64 R ABC-type transport systems, involved in lipoprotein release, ATPase components 6.85 6.66 9.03 7.53
COG0816 0.26 0.36 0.64 L Predicted endonuclease involved in recombination - 6.21 9.05 9.81
COG1159 0.36 0.26 0.64 R GTPases - 6.54 8.86 9.78
COG0712 0.31 0.29 0.65 C F0F1-type ATP synthase delta subunit (mitochondrial oligomycin sensitivity protein) - 4.92 7.56 9.83
COG1160 0.33 0.46 0.65 R Predicted GTPases - 5.62 9.15 9.92
COG0463 0.31 0.55 0.66 M Glycosyltransferases involved in cell wall biogenesis 4.80 8.55 8.87 -
COG0324 0.34 0.30 0.66 J tRNA delta(2)-isopentenylpyrophosphate transferase - 5.58 9.56 9.58
COG0617 0.32 0.36 0.66 J tRNA nucleotidyltransferase/poly(A) polymerase - 7.84 9.14 9.35
COG1385 0.35 0.32 0.67 S Uncharacterized BCR - 6.80 9.08 9.71
COG1825 0.34 0.34 0.67 J Ribosomal protein L25 (general stress protein Ctc) - 9.60 9.41 9.91
COG0358 0.63 0.30 0.67 L DNA primase (bacterial type) 4.35 5.62 8.97 9.48
COG0593 0.42 0.32 0.69 L ATPase involved in DNA replication initiation - 6.97 8.35 9.55
COG0052 0.64 0.33 0.69 J Ribosomal protein S2 4.06 6.38 6.82 9.61
COG0616 0.49 0.32 0.69 N Periplasmic serine proteases (ClpP class) 4.93 7.44 9.41 9.66
COG0212 0.39 0.35 0.70 H 5-formyltetrahydrofolate cyclo-ligase 4.42 6.10 9.98 -
COG0470 0.59 0.34 0.70 L ATPase involved in DNA replication 4.47 6.46 8.70 9.39
COG1132 0.33 0.44 0.70 Q ABC-type multidrug/protein/lipid transport system, ATPase component 4.43 6.91 9.23 9.60
COG0750 0.33 0.52 0.71 M Predicted membrane-associated Zn-dependent proteases 1 4.91 6.41 9.18 -
COG0341 0.36 0.40 0.71 N Preprotein translocase subunit SecF 4.53 5.46 8.73 -
COG0475 0.35 0.32 0.71 P Kef-type K+ transport systems, membrane components - 5.13 9.12 -
COG0566 0.43 0.35 0.72 J rRNA methylases - 7.15 9.18 -
COG0438 0.35 0.46 0.73 M Predicted glycosyltransferases 4.96 8.21 8.66 -
COG0223 0.43 0.36 0.73 J Methionyl-tRNA formyltransferase - 6.01 8.87 9.80
COG0237 0.60 0.36 0.74 H Dephospho-CoA kinase 1 4.59 5.64 8.91 10.26
COG0668 0.36 0.42 0.76 M Small-conductance mechanosensitive channel 5.29 7.56 8.87 9.50
COG0532 0.57 0.34 0.77 J Translation initiation factor 2 (GTPase) 4.33 5.76 6.93 9.38
COG0484 0.35 0.41 0.82 O Molecular chaperones (contain C-terminal Zn finger domain) 4.54 7.94 8.12 9.17
COG0164 0.39 0.44 0.83 L Ribonuclease H 4.41 6.91 8.95 -
COG0020 0.38 0.47 0.85 I Undecaprenyl pyrophosphate synthase 5.11 6.29 8.97 9.45
COG0681 0.41 0.49 0.86 N Signal peptidase 4.99 6.46 8.34 9.51
COG0244 0.40 0.53 0.87 J Ribosomal protein L10 3.80 9.04 9.36 9.98
COG0130 0.45 0.43 0.88 J Pseudouridine synthase 5.37 5.59 - 9.74
Mean 4.76 6.64 8.75 9.47
Standard Deviation 0.7 1.14 0.73 0.69
Extrapolating results obtained from using the theoretical proteome must be viewed with caution as predicted pI values are for unfolded proteins obtained from sequence and not the native folded proteins in the cellular microenvironment, which remain unknown. We have resisted attempting to correlate observations of an ortholog's thoeretical pI with its function, unless clearly obvious, for this reason. The theoretical proteome is also generated from the total set of proteins present in an organism's genome, while only a subset maybe expressed at any given time and will vary in response to external stimuli. An organism may also respond to evolutionary pressure such as environmental pH by increasing the number of copies of a charged protein, a ploy frequently adopted in drug resistance. The effect of a shift in the relative levels of the acidic and basic peaks maybe replicated by an increase in copy number of proteins belonging to the relevant cluster. As theoretical proteome studies are not weighted by the copy number of the individual proteins, for lack of relevant data related to the quantity of individual proteins for the entire proteome, these observations are impossible to make.
Conclusion
Analysis of ortholog pI across forty two microorganism genomes which contain reprentatives of free living archea and bacteria are analysed to identify orthologs which are invariant in pI as well as those amenable to changes in pI. Orthologs with a high frequency of occurance and variation in pI are shortlisted, and maybe used as markers in future studies which attempt to map proteome properties to variation in the organisms ecological niche.
Methods
Genome sequences and the COG database generated with 43 organisms with sequences and alignments were sourced from NCBI [15]. Sacharomyces cerevisae was removed from the dataset so that it contained only archeae and bacteria which were single-celled organisms. This was intentionally to include only genomes of organisms which would have their membrane directly in contact with the external environment and included a range of organisms with diverse ecological niches.
The isoelectric point is determined from an equation subtracting the sum of all negative charges (of all acidic residues) from the sum of all positive charges (of all basic residues), by varying the pH by bisectional nesting of intervals on the pH scale until the interval is smaller than a given level (0.001), as described in [2]. Fixed values for pK are considered to calculate the isoelectric point, and are the same as used in the molecular weight/isoelectric point program at [16].
The molecular weights and isoelectric point of all the predicted open reading frames from the completed genomes of a number of prokaryotes were generated. All ORFs that are classified by respective COG identities and assigned to one of the 18 functional groups [12] were used in comparitive analysis between genomes.
Correlation of molecular weights and pI were performed, using scripts written in house, for all COGs classified to a particular function for each pair of organisms. Pairwise distances were calculated for all pairs of proteins belonging to the specified COG. Sequences assigned to each COG were aligned with ClustalW [17] using default parameters, and the resulting multiple alignment used to generate a distance matrix with the program PROTDIST, using default parameters (Jones-Taylor-Thornton model, where the distance is scaled in units of the expected fraction of amino acids changed), from the Phylip package [18]. Perl scripts used to automate this protocol were written using BioPerl Modules [19].
Individual molecular weight and isoelectric point along with pairwise distances were stored in a MySQL database v 4.1.9, running on a Fedora Core Linux dual Xeon Server, to enable easy retrieval. Statistical properties on the data (mean, variance and correlation coefficients) were calculated using standard methods after sectioning the data at the levels of COGs and functional groups. Figures plotted in this paper were made using Sigmaplot (Systat Software Inc, Richmond, California, USA).
Statistical significance of functional groups was assessed using non-parametric Kruskal-Wallis tests and Dunn's Multiple Comparison [20] tests (alpha = 0.2), after randomly sampling three hundred sequences from each group, and from the total population to form the sample sets.
To score the COGs both for variation and conservation, probabilities were estimated from frequencies in the dataset. The frequency of occurance of a COG i, F(i), calculated as
Where no is the number of organisms in which the COG is present and nt is the number of organisms in the sample dataset (42).
where i is the cog, j is the cluster – either acidic, neutral or basic, and r the range corresponding to the cluster – 0–7.4 for acidic, 7.4–8.1 for basic, and 7.4–14 for basic. nr is the number of proteins in the given cluster, ni total number of proteins corresponding to i. Pi (j) is the conservation score for the cluster j, and is the joint probabiity of conservation and occurance of the COG i.
We estimate the joint probability of variation between the acidic and basic clusters for a COG i, Pi(v), as the probability of variation into its frequency of occurance calculated as
where na and nb are the number of proteins belonging to the COG i in the acidic and basic cluster of the distribution respectively, ni being the total number of proteins classified under COG i .
Membrane proteins were predicted using TOPPRED [21].
Authors' contributions
Mr Soumyadeep Nandi did all computation and database work, Mr. Nipun Mehra started this work, and generated preliminary results, Dr. Andrew Lynn guided the implementation of the project and wrote the manuscript, Prof. Alok Bhattacharya provided overall guidance and interpretation of results.
Supplementary Material
Additional file 1
List of COGS sorted by mean pI. File with rows containing COG, mean, standard deviation and pI of individual ortholog proteins that were used in the calculation described in this paper. The rows are sorted by mean pI (column 2) and used to plot figure 5 of the manuscript. The file is tab-delimited and will open in any spreadsheet program.
Click here for file
Additional file 2
COGs with pI conserved in acid and basic clusters. Text file containing the comprehensive list of COGs which are conserved in either the acidic or basic cluster of the multimodal distribution.
Click here for file
Acknowledgements
The authors thank Department of Biotechnology, Government of India for financial support.
==== Refs
Bjellqvist B Hughes GJ Pasquali C Paquet N Ravier F Sanchez JC Frutiger S Hochstrasser D The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences Electrophoresis 1993 14 1023 31 8125050 10.1002/elps.11501401163
Altland K Becher P Rossmann U Bjellqvist B Isoelectric focusing of basic proteins: the problem of oxidation of cysteines Electrophoresis 1988 9 474 85 3243245 10.1002/elps.1150090906
Van Bogelen RA Schiller EE Thomas JD Neidhardt FC Diagnosis of cellular states of microbial organisms using proteomics Electrophoresis 1999 20 2149 2159 10493120 10.1002/(SICI)1522-2683(19990801)20:11<2149::AID-ELPS2149>3.0.CO;2-N
Schwartz R Ting CS King J Whole proteome pI values correlate with subcellular localization of proteins for organisms within the three domains of life Genome Res 2001 11 703 709 11337469 10.1101/gr.GR-1587R
Weiller GF Caraux G Sylvester N The modal distribution of protein isoelectric points reflects amino acid properties rather than sequence evolution Proteomics 2004 4 943 9 15048976 10.1002/pmic.200200648
Knight CG Kassen R Hebestreit H Rainey PB Global analysis of predicted proteomes: functional adaptation of physical properties Proc Natl Acad Sci U S A 2004 101 8390 5 15150418 10.1073/pnas.0307270101
Brocchieri L Environmental signatures in proteome properties Proc Natl Acad Sci 2004 101 8257 8258 15159533 10.1073/pnas.0402797101
Tekaia F Yeramian E Dujon B Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis Gene 2002 297 51 60 12384285 10.1016/S0378-1119(02)00871-5
Kennedy SP Ng WV Salzberg SL Hood L DasSarma S Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence Genome Res 2001 11 1641 50 11591641 10.1101/gr.190201
Karlin S Brocchieri L Trent J Blaisdell BE Mrazek J Heterogeneity of genome and proteome content in bacteria, archaea, and eukaryotes Theor Popul Biol 2002 61 367 90 12167359 10.1006/tpbi.2002.1606
Kumar S Nussinov R How do thermophilic proteins deal with heat? Cell Mol Life Sci 2001 58 1216 33 11577980
Tatusov RL Koonin EV Lipman DJ A Genomics Perspactive on Protein Families Science 1997 278 631 7 9381173 10.1126/science.278.5338.631
Tatusov RL Fedorova ND Jackson JD Jacobs AR Kiryutin B Koonin EV Krylov DM Mazumder R Mekhedov SL Nikolskaya AN Rao BS Smirnov S Sverdlov AV Vasudevan S Wolf YI Yin JJ Natale DA The COG database: an updated version includes eukaryotes BMC Bioinformatics 2003 4 41 12969510 10.1186/1471-2105-4-41
Kruskal WH Wallis WA Use of ranks in one-criterion variance analysis Journal of the American Statistical Association 1952 47 583 621 Addendum 1953, 48:907–911
FTP sites for COG and Genome data used in this analysis [] and []
Compute pI/MW tool at ExPASy proteomics server of the Swiss Institute of Bioinformatics
Thompson JD Higgins DG Gibson TJ CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Research 1994 22 4673 4680 7984417
Felsenstein J PHYLIP: Phylogeny inference package Cladistics 1989 5 164 166
Stajich JE Block D Boulez K Brenner SE Chervitz SA Dagdigian C Fuellen G Gilbert JGR Korf I Lapp H Lehväslaiho H Matsalla C Mungall CJ Osborne BI Pocock MR Schattner P Senger M Stein LD Stupka E Wilkinson MD Birney E The Bioperl Toolkit: Perl Modules for the Life Sciences Genome Research 2002 12 1611 1618 12368254 10.1101/gr.361602
Dunn DJ Multiple Comparisons Using Rank Sums Technometrics 1964 5 241 252
Claros MG von Heijne G TopPred II: an improved software for membrane protein structure predictions Comput Appl Biosci 1994 7704669
COG help file showing functional categories of orthologs
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==== Front
BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1191615938510.1186/1471-2164-6-119Research ArticleComputational evidence for hundreds of non-conserved plant microRNAs Lindow Morten [email protected] Anders [email protected] Bioinformatics Centre, Institute of Molecular Biology, University of Copenhagen, Denmark2005 13 9 2005 6 119 119 14 4 2005 13 9 2005 Copyright © 2005 Lindow and Krogh; licensee BioMed Central Ltd.2005Lindow and Krogh; 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
MicroRNAs (miRNA) are small (20–25 nt) non-coding RNA molecules that regulate gene expression through interaction with mRNA in plants and metazoans. A few hundred miRNAs are known or predicted, and most of those are evolutionarily conserved. In general plant miRNA are different from their animal counterpart: most plant miRNAs show near perfect complementarity to their targets. Exploiting this complementarity we have developed a method for identification plant miRNAs that does not rely on phylogenetic conservation.
Results
Using the presumed targets for the known miRNA as positive controls, we list and filter all segments of the genome of length ~20 that are complementary to a target mRNA-transcript. From the positive control we recover 41 (of 92 possible) of the already known miRNA-genes (representing 14 of 16 families) with only four false positives.
Applying the procedure to find possible new miRNAs targeting any annotated mRNA, we predict of 592 new miRNA genes, many of which are not conserved in other plant genomes. A subset of our predicted miRNAs is additionally supported by having more than one target that are not homologues.
Conclusion
These results indicate that it is possible to reliably predict miRNA-genes without using genome comparisons. Furthermore it suggests that the number of plant miRNAs have been underestimated and points to the existence of recently evolved miRNAs in Arabidopsis.
==== Body
Background
MicroRNAs (miRNAs), 20–25 nucleotides in length, are involved in negative post transcriptional regulation in most multi-cellular organisms (for a review see e.g. [1,2]). The generality and importance of this recently discovered regulatory mechanism is gradually becoming apparent, and here we present computational evidence for new miRNAs indicating that their numbers are more abundant than previously believed, and argue that they play a major role in evolution.
Most of the miRNAs identified so far are conserved in other species, some remarkably well[3]. Previous computational screens for miRNA have relied on this evolutionary conservation to identify a few hundred putative miRNAs in vertebrates[4], C. elegans[5], and plants [6-8], and many have been experimentally confirmed (reviewed in [9]). However, these screens miss all miRNAs that have diverged since the last common ancestor of the genomes under comparison. A recent study using a combined bioinformatic and high-throughput experimental approach have identified 53 miRNAs not conserved beyond primates[10]. In plants, where comparisons have been between the distantly related A. thaliana (thale cress) and O. sativa (rice) genomes that diverged some 200 million years ago[11], it is probable that there are miRNAs which have escaped detection. Of the 112 Arabidopsis miRNA-genes currently registered[12], only 56 are conserved in the monocot rice (see methods section), indicating the existence of a substantial number of unconserved miRNA-genes. miRNA and short interfering RNAs (siRNA) are very similar in function, but different in biogenesis. According to the current nomenclature[13] both microRNAs (miRNAs) and short interfering RNAs (siRNAs) are 20–25 nucleotides long single stranded molecules that arise from processing of double stranded RNA (dsRNA) precursors. They are distinguished by the type of dsRNA they are excised from. While siRNAs come from long exogenous or endogenous dsRNA molecules (very long hairpins or RNA duplexes), mature miRNAs come from the stem region of shorter hairpins.
The mature miRNA or siRNA forms part of the RNA induced silencing complex (RISC) that binds to mRNAs. miRNA/siRNAs that bind with almost perfect complementarity to an mRNA often results in the cleavage of its target. Currently it seems that the higher the degree of complementarity to a target mRNA, the larger chance of that target being degraded. miRNAs with imperfect complementarity to a 3' untranslated region of a mRNA have been shown to inhibit translation of the mRNA[14,15]
When the base pairing between the miRNA and the target is incomplete it is non-trivial to identify targets for a miRNA [16-19]. In plants, however, most of the known miRNAs pair almost perfectly with one or more mRNAs, making it straightforward to identify likely plant targets (miRNAs often have more than one target). Using this observation it is possible to predict miRNA candidates in Arabidopsis that exhibit near perfect base pairing with the targets, without relying on homology to other organisms[20]. Here this idea is extended and refined to yield a highly specific screen that finds plant miRNAs in numbers much larger than previously thought.
Results and discussion
Identification of non-conserved miRNAs
The general approach is outlined in figure 1. Initially, a mRNA is compared with the genomic sequence to identify matching regions of 20–27 nucleotides with at most 2 mismatches (allowing 3 mismatches produced more than 10 000 matches per mRNA). These are called micromatches, and the genomic part is referred to as a genomic match. An average mRNA gives rise to about 1000 such micromatches, the vast majority (often all) of which we assume are spurious non-miRNA hits. However, it is possible, without comparing to other genomes, to filter the micromatches and achieve highly specific and fairly sensitive predictions of miRNA genes (Figure 1).
Figure 1 Procedure for miRNA prediction. The number of matches between a mRNA and a segment of the genome (micromatches) after each step is shown in parenthesis. mRNAs are compared with the genomic sequence to identify matching regions of 20–27 nucleotides with at most 2 mismatches. Matches overlapping annotated exons, repeats or low-complexity regions are discarded. Additionally, the miRNA:mRNA-duplexes must be stable and the potential miRNAs must have a structure similar to known miRNAs to be included in the base set predictions. The multi-target set is a more reliable subset of those that have more than one target. See text for more details.
Six filters were used to identify a base set of genomic sequences as candidate miRNAs (with percentages of the initial micromatches that were remaining after each filter given in brackets): (1) they had high sequence complexity (26.9%); (2) they had no overlap with annotated exons on the same or the opposite strand (3.3%); (3) they had no overlap with repeat sequences defined by RepeatMasker (2.6%); (4) the putative miRNA:mRNA duplex should be relatively stable[17,21] with a calculated free energy of less than -34 kcal/mol (0.20%); (5) they had no more than identical 10 copies in the genome (0.19%), to eliminate repeated sequences not detected by standard repeat-masking; and (6) the miRNA was contained within a precursor structure that was similar to those observed in known Arabidopsis miRNA precursors, i.e. was predicted to be largely contained (at least 16 paired bases) within the stem of a double stranded stem-loop structure whose stem was predicted to have a free energy less than -60 kcal/mol, with at least 4 paired bases flanking the putative miRNA, and an intervening loop larger than 9 but less than 130 bases (0.0002%).
Although the base set predictions have a low number of false positives (see below), they can be even more refined to identify a subset of the predictions with extra confidence, because the probability of more than one mRNA matching a falsely predicted miRNA is minimal, unless the matching mRNA-targets are close homologs (in which case the multiple targets do not add much extra confidence). Most of the known miRNA in Arabidopsis are thought to have multiple targets often within the same family of homologous proteins[22]. If a known miRNA only has targets in a highly conserved protein family this filter can however be expected to falsely eliminate them.
In order to check the validity of our approach we took the mRNA targets of the known miRNAs and set out to see if using these as queries we would be able to correctly identify the known miRNA-genes. Of the 112 precursor sequences registered in RFAM (ver 5.1), we were able to map 92 perfectly to the current RefSeq assembly (TIGR ver 5.0) of the Arabidopsis genome; the remaining precursors were excluded from the positive control set. Likely targets for Arabidopsis miRNAs have previously been predicted allowing for up to 3 mismatches[23]. Repeating this procedure we find that our known miRNAs match 142 different annotated mRNA*. These are the positive control targets (refered to a 'known targets') and many have been experimentally confirmed[24,25]. Initially, the 142 mRNAs in the positive control set yielded 359,976 micromatches after removal of low complexity sequences. However, the filtering procedure reduces this dramatically to 45 different loci (41 of which are already known) representing 16 different families (14 known). Assuming that the 'unknown' loci we find are false positives the procedure has 91% specificity and 45% sensitivity on the level of loci identified. Using the refinement step requiring more than one non-homologouos target only true positives are found, but at the expense of halving the sensitivity to 22%. The validity of the estimates of specificity and sensitivity is discussed below.
Hundreds of novel miRNAs
Applying the micromatcher procedure to all 28860 mRNAs annotated in Arabidopsis identifies 592 miRNA candidate loci (480 families) in the base set (Additional file 1). In the final step this is reduced to a set of 90 (70 new) when more than one non-homologouos target per miRNA is required. This is called the multi-target set and is a subset of the base set.
All miRNA gene predictions, their targets (with some basic annotation) and the predicted secondary structure of the precursor are available as supplementary data [Additional file 1], and at our website[26]
Using public databases we were able to acquire evidence for the expression of a small number of the predictions, 9 in the base set overlap with RNA molecules recently sequenced in a large scale cloning effort of Arabidopsis small RNA4, 109 have significant matches to Arabidopsis ESTs and 52 of the predicted precursors contain a 20-mer sequence tag from the Arabidopsis MPSS database[27].
Evolutionary conservation of the predicted miRNA-genes
From an evolutionary point of view, it would seem to be a lot easier to adapt 20 bases in a miRNA for a new target than to evolve a protein for a specific regulatory task.
For mammals it has been suggested that the more targets a microRNA has the more likely it is to be conserved[28] because of the additional constraints of having to match multiple targets.
Indeed also for plants: comparison of our predictions in Arabidopsis to two other plant species reveals that the more targets a miRNA is predicted to have, the more likely it is to be conserved (Figure 2). Although no Brassica species is yet completely sequenced and we had to use a conjunction of all single sequence Brassica entries from GenBank, significantly more of the predicted miRNAs are conserved in Brassica than in rice, indicating that many miRNA-genes have diverged beyond recognition since the divergence of monocots and dicots approximately 200 million years ago.
Figure 2 Duplex energy is a strong discriminant between true and false micromatches. The procedure was started with 142 mRNAs targeted by known miRNAs. Micromatches were filtered for low-complexity, overlap with exons and repeats. Then the remaining micromatches were divided in two bins: true positives (green trace) that overlap with known miRNA genes and false positives (red trace) that do not.
Thus, we speculate that the highly conserved miRNAs are likely to be central regulators, often of many target mRNAs (imposing the evolutionary constraint to stay conserved), and are more likely to be highly expressed. Whereas more recently evolved miRNA would have fewer targets, and a more localized spatiotemporal expression, making them less likely to be detected by cloning efforts.
Since evolutionary conservation is part of many of the previous discovery procedures, it is likely that the set of known miRNAs is biased towards those that are conserved, and our data suggest that in fact, miRNAs evolve fast and are less conserved than e.g. protein-coding genes.
It has been proposed that some miRNAs originate from inverted duplication of target sequences, exemplified by the single locus miRNAs miR-161 and miR-163, which have precursors that show extended homology to the target mRNAs also outside the mature miRNA sequence[29]. However, our structural filters require that the match between miRNA and target is in the range 20–25, effectively eliminating such miRNA with extended homology.
Comparison to other studies
Of the predicted 592 precursors in the base set, 29 overlap with the 92 predictions made by Bonnet et al.[30], and 4 of those by Wang et al.[8]. Thus, the different methods complement each other: The present method based on matching targets and miRNA is capable of finding non-conserved miRNAs, whereas the interspecies comparisons[8,31] can find miRNAs without obvious targets.
The idea to use potential targets to find miRNA-genes has recently been employed in two other studies. Xie et al. [32] started by finding frequently occurring subsequences of human 3' UTR sequences conserved in other mammals and successfully searched the genome for new miRNA genes.
Moreover Adai and coworkers[33] published results in Arabidopsis using potential targets to find new miRNA-genes. However, our approach differs significantly from theirs in the way the matches (that we term micromatches) are analysed and the kind of conclusions that can be drawn: Adai et al. looks for a 'cluster' of miRNA-genes that target the same sequence of a mRNA, and then aligns the candidates in such a cluster, scoring the alignment high if it shows a characteristic pattern where the miRNA and miRNA* are more conserved than the intertwining sequence. Thus, their method is limited to finding miRNAs that occur more than once in the genome, presumably as a result of duplication events. Moreover as a postfilter, Adai et al. require conservation in rice to generate their short-list used for experimental validation. Also, Adai et al. do not make any estimation of the specificity of their computational procedure and are consequently unable to speculate about the number of miRNAs.
In contrast our method is independent of whether a candidate has been duplicated in the genome or is conserved across species. Instead our aggressive filtering on the structural properties of the precursor enables us to make highly specific prediction (judging from the results using targets for known miRNAs as queries).
The multi-target miRNAs have a total of 528 different mRNA targets, which are involved in a variety of functions, but there is a notable over-representation of proteins with transcription factor activity and receptor binding activity as well as involvement in developmental processes (false discovery rate < 0.001, see Additional file 2). The predicted miRNA-genes are generally found scattered throughout the genome (Table 2). Unlike in mammals where 90 out of 232 miRNA-genes are within introns of protein coding genes [34], there is only one previously discovered Arabidopsis microRNA situated in an intron. This trend of plant microRNAs to be outside protein-coding genes also holds for our baseset predictions and even stronger for the multiple target predictions (Table 2).
Table 2 The distribution of predicted miRNA-genes in relation to genomic features. IGR, intergenic region. The ratio of the number of bases annotated as intergenic vs. intron is 3.1 in the genome as a whole.
Position of predicted miRNA genes
Base set >1 target
Total number of loci 592 90
In introns (sense strand) 24 3
in introns (antisense) 18 2
In intergenic regions (both strands) 550 85
Within 500 bases upstream of gene 26 3
Within 500 bases downstream of gene 52 7
Ratio IGR/introns 14 18
Although estimating the sensitivity and specificity on the basis of the ability to correctly identify the small set of known miRNAs carries the danger of biasing, the presently most important concern must be not to massively overpredict new miRNA-genes. In constructing the filters we have therefore aim at high specificity at the expense of sensitivity. While false positives undoubtfully remain, the fact that the predictions share the properties of functional overrepresentation and bias of genomic location (properties not selected for in the filters) with known miRNAs provides independent indication that we indeed do not massively overpredict new miRNA-genes.
It is becoming evident that many regions between protein coding genes are transcribed (e.g. [35,36]). Indeed given the cases of miRNAs that have been suggested to regulate other miRNAs[37] or RNAs that guide methylation DNA[38], it would be interesting to extend our filtered intragenomic match approach to identify other possible miRNAs whose targets are not mRNAs.
Conclusion
The present analysis predicts 71 new Arabidopsis miRNA genes with very few false positives (estimated specificity is 100%) and over five hundred with an estimate of 9% false predictions. The procedure misses some real miRNAs, such as those encoded in untranslated regions of genes, those with very many targets (classified as repeats by our method), and those not fulfilling our strict structural constraints, and we believe that the real number could be several thousands. Although, the predictions should eventually be confirmed in the lab, our data suggest that the Arabidopsis genome encodes substantially more miRNA genes than previously thought, and that the number of miRNAs is comparable to the number of protein transcription factors. Our results also indicate that many miRNA are specific to small groups of related species and we speculate that they could play a part in speciation. Finally we find it unlikely that these conclusions are specific to plants, and we hypothesize that they extend to most other multicellular organisms.
Methods
Sequences
Arabidopsis genome and annotation were the RefSeq sequences based on the 5.0 version released by TIGR. Known miRNAs were from the 5.1 release of the microRNA registry[39].
The micromatcher procedure
Finding all micromatches
For each annotated spliced mRNA we exhaustively searched the genome for micromatches of length at least 20 with maximum 2 mismatches (no gaps allowed) using the suffixarray based program vmatch[40] (This search took 6 days on an Intel Xeon 2.2 Ghz machine running Linux).
Note about the positive control set of mRNAs: To select the positive control mRNA-targets we allow for 3 mismatches over the whole length of the mature miRNA; this potentially includes in the positive control set mRNAs that will be unable to recover the matching miRNA allowing only 2 mismatches over a length of 20 bases (the criterion used later). This discrepancy can lead to a too pessimistic estimation of the performance of procedure.
Lowcomplexity filter
Genomic micromatches not fulfilling a simple low complexity filter were discarded: 1) all four bases had to be present at least once, and 2) at most 11 of the three most frequent dinucleotides in the sequence were allowed.
Duplex stability
Using the program RNAcofold (Vienna RNA package[41]) the free energy change when a microRNA-candidate binds to a target site was calculated. Micromatches where this duplex energy is larger than -34 kcal/mol were discarded.
Long matches
Micromatches longer than 26 residues were discarded. To ascertain that a micromatch was not part of a longer match, the two parts of the micromatch extended by 50 bases to each side were aligned with bl2seq (two sequence NCBI blast), and those with a match longer than 26 were discarded.
Overlaps with known features and repeats
A micromatch was discarded if it had any bases in common with annotated exons (including matches to the reverse strand of the exon) or repeats as determined by RepeatMasker[42] run with Arabidopsis specific repeat libraries (RepBase Update 8.12, RM database version 20040306).
Copy number
Additionally to traditional repeat-masking that relies on the identification of known repeats, we made an additional pragmatic repeat filter: We simply determined the number of times all candidate sequences occurs in the entire genome, and removed candidates with a copy number higher than 10.
Filtering on properties of the possible precursor
In order to predict a possible precursor molecule, two genomic sequences around each micromatch were extracted: One starting 10 bases 5' of the micromatch and extending 240 bases 3' of the micromatch, and one with the extension lengths reversed. Each of these was treated independently in the following analysis. First the potential precursor sequence was folded with RNAfold[43] to find the minimum free energy structure These values are comparable, because all sequences are of almost equal length. Candidates with a folding free energy larger than -60 kcal/mol are discarded. This is a highly permissive filter. The mature miRNA has to be fully contained in a double stranded region of the precursor. The complementary part of the miRNA in this stem is denoted miRNA*. It is demanded that all base pairs between the miRNA and the miRNA* are pairing in the same direction opposite each other. The number of paired bases in the mature miRNA is required to be 16 or more.
In the known miRNA precursors, the stem is always longer than just the length of the mature miRNA. To find how far the stem of a candidate extends from the mature miRNA, we count how far inward towards the loop or outwards toward the ends of RNA-string the stem extends using the following algorithm: Moving out from the terminal basepair between miRNA and miRNA* a score of 1 is assigned for each base pair encountered and a score of -1 for each unpaired base. The extension is stopped when the current score is less than 5 lower than the maximum score so far. The last base pair is considered the terminus of the stem. Candidates with extensions less than 4 bases on either side of the mature miRNA were discarded. It was also required that the shortest number of bases between the miRNA and miRNA* were larger than 9 and less than 130.
Taken together these structural criteria constitute a highly selective, but somewhat conservative filter.
Matches to ESTs and ASRP
BLASTN was used to search all Arabidopsis ESTs downloaded from GenBank on September 27, 2004. Hits longer than 70 nucleotides with more than 95% identity between a predicted precursor and an EST were considered positive. Sequences cloned and sequenced as part of the Arabidopsis Small RNA Project (ASRP)[44], were downloaded from [45]. All matches at least 15 long with at most one mismatch with our predicted mature miRNA-sequences were found using vmatch[46].
Conservation in other genomes
To determine how many of our predictions were conserved in other plant genomes, we blasted the predicted Arabidopsis precursors against the rice-genome and brassica sequence downloaded from [47]. A miRNA prediction was taken to be conserved if it had a significant (e-value < 0.01) blast hit containing the mature miRNA with no more than 2 mismatches and the homolog had flanking sequence capable of folding back on the mature miRNA with at least 15 base pairs between the miRNA and miRNA*.
The number of non-homologous targets for a putative miRNA
For all candidate microRNAs in the baseset matching more than one mRNA, we found the number of different non-homologous targets by performing single linkage clustering on the aminoacid sequences of the corresponding mRNAs using the program 'blastclust' from NCBI. Two proteins were considered homologous if they had more than 70% identity across at least 50% of the length.
Clustering of micromatches into genomic loci
Micromatches with genomic start position within 4 nucleotides were logically grouped into the same locus.
Clustering of similar miRNA sequences into families
We used the program vmatch[48] to align and perform single linkage clustering of the predicted mature miRNA sequences. Candidate pairs aligning over at least 17 bases, allowing an edit distance of 1 were grouped in the same family.
Functional analysis of targets
We obtained gene ontology annotation (GOSLIM) from [49]. From each GOSLIM category we constructed a 2 × 2 contingency table counting the number of targets vs non-targets with or without the GOSLIM annotation. We used R[50] to calculate p-values with Fisher's Exact Test and employed the package 'qvalue'[51] to correct for multiple testing setting a false discovery rate level at 0.001. The results are included as [Additional file 2], along with the R-code used.
Authors' contributions
ML and AK designed the study. ML wrote the programs. ML and AK drafted the manuscript. Both authors read and approved the final manuscript.
Figure 3 Multi target predictions tend to be better conserved. The precursor sequences of the predictions were used as queries for a blast search against rice (downloaded from tigr.org, March 2004) or brassica (downloaded from arabidopsis.org, August 2004), respectively. Columns show the proportion of miRNA predictions in Arabidopsis that were found to be conserved. Numbers refer to the actual number of conserved miRNA predictions.
Table 1 Summary of the results, starting with 136 mRNA targets to known miRNAs or all mRNAs, respectively. Numbers in parenthesis indicate the number of already known (RFAM) miRNA genes or families.
micromatches miRNA genes found distinct families distinct targets
Query: known targets
Baseset 176 45(41) 16(14) 51
>1 non-homologous target 63 20(20) 12(12) 34
Query: all mRNAs
Baseset 927 592 480 656
>1 target-homologous target 255 90 73 205
Supplementary Material
Additional File 1
Predicted miRNA genes. List of predicted miRNA-genes, their predicted targets, genomic location and graphics showing predicted structure of the precursors.
Click here for file
Additional File 2
Functional analysis of the predicted miRNA targets. Analysis of overrepresented Gene Ontology terms among the mRNAs predicted to be targeted by miRNAs.
Click here for file
Acknowledgements
We wish to thank anonymous reviewers for helpful comments and suggestions.
==== Refs
He L Hannon GJ MicroRNAs: small RNAs with a big role in gene regulation Nat Rev Genet 2004 5 522 531 15211354 10.1038/nrg1379
Tomari Y Zamore PD Perspective: machines for RNAi Genes Dev 2005 19 517 529 15741316 10.1101/gad.1284105
Floyd SK Bowman JL Gene regulation: ancient microRNA target sequences in plants Nature 2004 428 485 486 15057819 10.1038/428485a
Lim LP Glasner ME Yekta S Burge CB Bartel DP Vertebrate microRNA genes Science 2003 299 1540 12624257 10.1126/science.1080372
Lim LP Lau NC Weinstein EG Abdelhakim A Yekta S Rhoades MW The microRNAs of Caenorhabditis elegans Genes Dev 2003 17 991 1008 12672692 10.1101/gad.1074403
Jones-Rhoades MW Bartel DP Computational Identification of Plant MicroRNAs and Their Targets, Including a Stress-Induced miRNA Mol Cell 2004 14 787 799 15200956 10.1016/j.molcel.2004.05.027
Bonnet E Wuyts J Rouze P Van de PY Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes Proc Natl Acad Sci USA 2004 101 11511 11516 15272084 10.1073/pnas.0404025101
Wang XJ Reyes JL Chua NH Gaasterland T Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets Genome Biol 2004 5 R65 15345049 10.1186/gb-2004-5-9-r65
Baulcombe D RNA silencing in plants Nature 2004 431 356 363 15372043 10.1038/nature02874
Bentwich I Avniel A Karov Y Aharonov R Gilad S Barad O Identification of hundreds of conserved and nonconserved human microRNAs Nat Genet 2005 37 766 770 15965474 10.1038/ng1590
Nelson DR Schuler MA Paquette SM Werck-Reichhart D Bak S Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot Plant Physiol 2004 135 756 772 15208422 10.1104/pp.104.039826
Griffiths-Jones S The microRNA Registry Nucleic Acids Res 2004 32 D109 D111 14681370 10.1093/nar/gkh023
Ambros V Bartel B Bartel DP Burge CB Carrington JC Chen X A uniform system for microRNA annotation RNA 2003 9 277 279 12592000 10.1261/rna.2183803
Doench JG Petersen CP Sharp PA siRNAs can function as miRNAs Genes Dev 2003 17 438 442 12600936 10.1101/gad.1064703
Doench JG Sharp PA Specificity of microRNA target selection in translational repression Genes Dev 2004 18 504 511 15014042 10.1101/gad.1184404
Doench JG Sharp PA Specificity of microRNA target selection in translational repression Genes Dev 2004 18 504 511 15014042 10.1101/gad.1184404
Enright AJ John B Gaul U Tuschl T Sander C Marks DS MicroRNA targets in Drosophila Genome Biol 2003 5 R1 14709173 10.1186/gb-2003-5-1-r1
Stark A Brennecke J Russell RB Cohen SM Identification of Drosophila MicroRNA Targets PLoS Biol 2003 1 E60 14691535 10.1371/journal.pbio.0000060
Lewis BP Shih IH Jones-Rhoades MW Bartel DP Burge CB Prediction of mammalian microRNA targets Cell 2003 115 787 798 14697198 10.1016/S0092-8674(03)01018-3
Adai A Johnson C Mlotshwa S rcher-Evans S Manocha V Vance V Computational prediction of miRNAs in Arabidopsis thaliana Genome Res 2005 15 78 91 15632092 10.1101/gr.2908205
Lewis BP Shih IH Jones-Rhoades MW Bartel DP Burge CB Prediction of mammalian microRNA targets Cell 2003 115 787 798 14697198 10.1016/S0092-8674(03)01018-3
Rhoades MW Reinhart BJ Lim LP Burge CB Bartel B Bartel DP Prediction of plant microRNA targets Cell 2002 110 513 520 12202040 10.1016/S0092-8674(02)00863-2
Rhoades MW Reinhart BJ Lim LP Burge CB Bartel B Bartel DP Prediction of plant microRNA targets Cell 2002 110 513 520 12202040 10.1016/S0092-8674(02)00863-2
Llave C Xie Z Kasschau KD Carrington JC Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA Science 2002 297 2053 2056 12242443 10.1126/science.1076311
Aukerman MJ Sakai H Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes Plant Cell 2003 15 2730 2741 14555699 10.1105/tpc.016238
Meyers BC Lee DK Vu TH Tej SS Edberg SB Matvienko M Arabidopsis MPSS. An online resource for quantitative expression analysis Plant Physiol 2004 135 801 813 15173564 10.1104/pp.104.039495
Mattick JS Makunin IV Small regulatory RNAs in mammals Hum Mol Genet 2005 14 R121 R132 15809264 10.1093/hmg/ddi101
Allen E Xie Z Gustafson AM Sung GH Spatafora JW Carrington JC Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana Nat Genet 2004 36 1282 1290 15565108 10.1038/ng1478
Bonnet E Wuyts J Rouze P Van de PY Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes Proc Natl Acad Sci USA 2004 101 11511 11516 15272084 10.1073/pnas.0404025101
Bonnet E Wuyts J Rouze P Van de PY Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes Proc Natl Acad Sci USA 2004 101 11511 11516 15272084 10.1073/pnas.0404025101
Xie X Lu J Kulbokas EJ Golub TR Mootha V Lindblad-Toh K Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals Nature 2005 434 338 345 15735639 10.1038/nature03441
Adai A Johnson C Mlotshwa S rcher-Evans S Manocha V Vance V Computational prediction of miRNAs in Arabidopsis thaliana Genome Res 2005 15 78 91 15632092 10.1101/gr.2908205
Rodriguez A Griffiths-Jones S Ashurst JL Bradley A Identification of Mammalian microRNA Host Genes and Transcription Units Genome Res 2004
Cheng J Kapranov P Drenkow J Dike S Brubaker S Patel S Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution Science 2005 308 1149 1154 15790807 10.1126/science.1108625
Stolc V Samanta MP Tongprasit W Sethi H Liang S Nelson DC Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays Proc Natl Acad Sci USA 2005 102 4453 4458 15755812 10.1073/pnas.0408203102
Lai EC Wiel C Rubin GM Complementary miRNA pairs suggest a regulatory role for miRNA:miRNA duplexes RNA 2004 10 171 175 14730015 10.1261/rna.5191904
Mathieu O Bender J RNA-directed DNA methylation J Cell Sci 2004 117 4881 4888 15456843 10.1242/jcs.01479
Griffiths-Jones S The microRNA Registry Nucleic Acids Res 2004 32 D109 D111 14681370 10.1093/nar/gkh023
Kurtz S The Vmatch large scale sequence analysis software Ref Type: Computer Program 4-12-2003
Hofacker IL Vienna RNA secondary structure server Nucleic Acids Res 2003 31 3429 3431 12824340 10.1093/nar/gkg599
Hofacker IL Vienna RNA secondary structure server Nucleic Acids Res 2003 31 3429 3431 12824340 10.1093/nar/gkg599
Xie Z Johansen LK Gustafson AM Kasschau KD Lellis AD Zilberman D Genetic and functional diversification of small RNA pathways in plants PLoS Biol 2004 2 E104 15024409 10.1371/journal.pbio.0020104
Kurtz S The Vmatch large scale sequence analysis software Ref Type: Computer Program 4-12-2003
Kurtz S The Vmatch large scale sequence analysis software Ref Type: Computer Program 4-12-2003
Berardini TZ Mundodi S Reiser L Huala E Garcia-Hernandez M Zhang P Functional annotation of the Arabidopsis genome using controlled vocabularies Plant Physiol 2004 135 745 755 15173566 10.1104/pp.104.040071
Storey JD A direct approach to false discovery rates Journal of the Royal Statistical Society, Series B 2002 64 479 498 10.1111/1467-9868.00346
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1241616228610.1186/1471-2164-6-124Research ArticleComparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts Rensink Willem Albert [email protected] Yuandan [email protected] Jia [email protected] Stacy [email protected] Shu [email protected] C Robin [email protected] The Institute for Genomic Research, 9712 Medical Center Dr., Rockville MD, 20850, USA2005 14 9 2005 6 124 124 13 5 2005 14 9 2005 Copyright © 2005 Rensink et al; licensee BioMed Central Ltd.2005Rensink 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 Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs) for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale.
Results
All available ESTs and Expressed Transcripts (ETs), 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana), were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices.
Conclusion
Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.
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Background
The Solanaceae family encompasses a number of species of agronomic and ornamental importance. With regards to cultivation for food consumption, in 2003, potato was the world's fifth largest crop in world-wide production acreage and the solanaceous vegetables tomato, eggplant, and pepper ranked 11th, 19th, and 22nd, respectively [1]. Species grown for ornamental purposes include petunia and Nicotiana species. While not consumed for food, these horticultural species are a substantial component of the US agronomic economy. For example, petunia represents greater than $148M output per year in the US [2]. Tobacco represents another crop of significant economical importance with $1.6B in crop value in 2003 [3]. A close relative of tobacco, Nicotiana benthamiana, has been utilized as an experimental model for viral research and disease resistance studies. Coupled with the robust ability of virus induced gene silencing to silence transcripts [4], N. benthamiana has emerged as a model species for disease resistance research.
The Solanaceae have been bred and developed for a variety of purposes. Potato has been bred for tubers (modified stems) while tomato, pepper, and eggplant have been bred for enhanced fruit production. Likewise, petunia has been bred and selected for floral phenotypes while tobacco has been bred for leaf size. While these modern varieties are accentuated for particular morphological features, these species share common taxonomic features of the Solanaceae such as alternate leaves, flower parts in five, and fruit as a berry or capsule. Compared with other plant families such as the Poaceae, the range of genome sizes of solanaceous species is fairly narrow, ranging from 900 to 4600 Mb per haploid genome [5]. Early studies of the Solanaceae genome revealed conservation of gene content among potato, tomato, tobacco, petunia, and eggplant. These studies employed relatively small scale cross-hybridization studies using cDNA and random genomic DNA clones [6] in which a set of 20 tomato cDNA clones were hybridized with a panel of solanceous species including Lycopersicon, Solanum, Datura, Petunia, and Nicotiana. For the cDNA clones, there was strong hybridization across the Solanaceae; however, with the genomic clones (50 in total), there was a reduced degree of cross-hybridization with the non-Lycopersicon species. These data suggested conservation among the coding sequences while the non-coding sequences had undergone substantial divergence.
Conserved gene content prompts the question of conserved gene order, i.e. synteny across the Solanaceae. A number of solanaceous species have a base chromosome number of 12 including the main vegetable crop species potato, tomato, pepper and eggplant. Using markers developed from tomato, a strong degree of co-linearity between potato and tomato has been demonstrated with the differences attributable to paracentric inversions occurring between these two species [7,8]. Using the same approach in pepper, 18 homologous linkage blocks between tomato and pepper could be identified [9]. In eggplant, tomato markers yet again revealed syntenic regions among tomato and eggplant [10]. While these synteny studies utilized anonymous DNA clones as markers, comparative mapping of phenotypes such as fruit morphology [11], pigmentation [12] and disease resistance [13] revealed syntenous mapping of these traits across the Solanaceae.
These early studies relied heavily on cDNA and random genomic clones. The advent of high throughput sequencing projects such as Expressed Sequence Tags (ESTs) [14] has resulted in the generation of hundreds of thousands of sequences for solanaeous species. For this study, a total of 441,154 ESTs were collected from the public database (dbEST) representing the solanaceous species tomato (162,621), potato (189,864), pepper (29,894), tobacco (26,497), and N. benthamiana (26,918). The available solanaceous ESTs, along with Expressed Transcripts (ETs), available in Genbank, can be clustered into gene indices [15] that represent a non-redundant set of transcripts and facilitate analysis of redundant EST collections. Using potato and tomato gene indices, a comparative analysis of tomato and potato ESTs revealed that approximately 80% of the potato ESTs had a significant sequence match with a tomato EST at the nucleotide level (E value cutoff of 10-10) [16].
In this study, we report the construction and comparative analyses of gene indices for six solanaceous species (tomato, potato, tobacco, pepper, petunia and N. benthamiana). These gene indices represent a total of 116,207 non-redundant sequences which we have utilized to assess sequence conservation among the Solanaceae on a genomic scale. We significantly extended previous studies on sequence similarity and conservation among these species as well as documented more thoroughly the characteristics of the coding portion of the Solanaceae genome. Using computational methods, we have identified putative orthologs among these species and generated a phylogenetic tree to ascertain the relationship and sequence divergence among these species. In addition to these computational approaches, we assessed the similarity of expression profiles in mature leaves to experimentally validate the sequence conservation of these species using heterologous hybridization to potato cDNA microarrays. The comparison of the solanaceous transcripts to the predicted proteomes of the near-complete genome sequences of Arabidopsis, rice, as well as to 21 other plant gene indices resulted in the identification of solanaceous transcripts without putative homologs, suggesting that a portion of these transcripts have a high likelihood of being unique to the Solanaceae. These analyses provide insight into the overall sequence conservation among eudicots (Arabidopsis and Solanaceae) as well as between the Solanaceae and the monocots (i.e., rice).
Results
Assembly of sequences into gene indices for potato, tomato, petunia, tobacco, pepper, and N. benthamina
A total of 446,248 sequences for six different Solanaceae family members were retrieved from Genbank, including dbEST. All sequences were derived from multiple libraries. The differences in relative expression levels of the various transcripts will result in a large number of redundant transcripts within these libraries. In order to analyze the transcriptome on the single transcript level, all sequences for each species were assembled into a gene index resulting in a total of 116,207 unique sequences over all six species. Abundant sequences could be assembled into longer, more accurate consensus transcripts termed tentative consensus (TC) sequences. Less abundant or lowly expressed transcripts could not be assembled into larger contigs resulting in singletons in the assemblies, termed singleton ESTs or singleton ETs. A summary of the composition of each gene index is shown in Table 1. The potato gene index contained the highest number of EST and ET sequences (190,851) and petunia the lowest number (8,690). For these species, the number of singleton sequences remaining after assembly is an indication of the level of sequencing and the diversity of the libraries selected for sequencing. Potato (44%), tomato (48%), and N. benthamiana (49%) have the lowest number of singleton sequences indicating a better coverage of the respective transcriptome when compared to the higher number of singletons in tobacco (82%), pepper (67%), and petunia (64%). Additional EST sequencing may reduce the number of singletons as it will allow for collapsing of singletons into contigs with increased coverage and representation of the transcriptome.
Table 1 Summary of gene indices of potato, tomato, pepper, tobacco, Nicotiana benthamiana and petunia. EST: expressed sequence tag; ET: expressed transcript; TC: tentative consensus; sEST: singleton EST; sET: singleton ET. TCs are the assembled clusters of redundant and overlapping EST and ET sequences. The total unique sequences for each gene index are created by combining the TCs, sETs, and sESTs.
Total EST Total ET TC sEST sET Total Unique Sequences Average length
Potato 189864 987 21063 17077 99 38239 796
Tomato 162621 1587 16268 15392 178 31838 690
Pepper 29894 259 4172 8788 43 13003 542
N. benthamiana 26918 76 3799 3707 48 7554 536
Petunia 8336 354 1416 2883 167 4466 908
Tobacco 26497 1831 3023 17385 699 21107 571
Assessment of the transcript sampling
The sequences used for the construction of the gene indices were generated from various diverse libraries that cover different treatments and stages of development (Table 2). The tissue sources used for library construction and sequencing largely reflected the various agronomic usages and research foci of the different Solanaceae species. For petunia and tomato, most of the sequences were generated from flower libraries as well as fruit libraries, reflecting the research interests in flower and fruit development for petunia and tomato. In contrast, for potato a large number of sequences were generated from stolons and tubers (Table 2). From all species, sequences from leaves were available, in some instances challenged with various stressors. As described below, 76–78% of the potato and tomato sequences have significant matches with each other although the sources of the libraries were very different, i.e. flower and fruit vs. tuber and stolon. For the Nicotiana species, tobacco and N. benthamiana, most sequences were generated from mixed tissue or callus libraries. This resulted in higher unique transcript discovery rates as judged from the ratio of the total number of sequences versus the number of unique sequences (Table 1). It should be noted that seed libraries, which may contain additional distinct transcripts, were not used for the sequencing in any of the six species examined in this study.
Table 2 Tissue representation of EST sequences among the gene indices. For each species, the origin of the library was determined and the total number of sequences from each source calculated. a. For the potato ESTs, 62,931 of the Mixed/Other ESTs were derived from a series of stolon and tuber cDNA libraries. b. For the N. benthamiana ESTs, 18,817 of the Mixed/Other ESTs were derived from a single cDNA library constructed by pooling mRNA from abiotic and biotic stressed leaves, roots, and callus.
Leaf Root Flower Fruit Callus/Susp. Culture Mixed/Other NA Total
Potato 26874 15634 5282 0 22040 119735a 299 189864
Tomato 20724 15000 45684 31684 22187 27178 164 162621
Pepper 5639 5165 3436 15640 0 0 14 29894
N. benthamiana 8099 0 0 0 0 18817b 2 26918
Petunia 72 0 6618 1646 0 0 0 8336
Tobacco 561 9 218 0 19578 5924 207 26497
Analysis of the GC content of Solanaceae gene indices
We analyzed the GC content (ratio of guanine and cytosine) of all of the sequences. It has been shown that Poaceae have GC rich genomes and the transcripts cover a broad range of GC content, whereas eudicot genomes have a lower GC content and transcripts have narrow symmetrical distribution of GC content [17]. The GC content range of the transcripts of the gene indices of the six Solanaceae species was determined (Figure 1). To provide a reference, the GC content range of the Arabidopsis (eudicot) and rice (monocot) gene indices was determined as well. The observed GC content of the Solanaceae gene indices is very similar and in accordance with Arabidopsis. All have a very symmetrical distribution. The average GC content for the majority of transcripts ranges from 40–45%, which is similar to what has been reported previously [18]. The only exception to this distribution were the tobacco transcripts which showed a slightly different profile with an overall lower GC content, in contrast to that previously reported [18] and other Solanaceae species examined in this study.
Figure 1 Analysis of the GC content of the six Solanaceae gene indices, Arabidopsis, and rice. The average GC content range was calculated for each transcript for the Solanaceae gene indices as well as Arabidopsis and rice.
Functional annotation of the gene indices
Automated annotation of the gene indices was performed as part of the assembly pipeline. In addition, Gene Ontology (GO) terms which provide a more global representation of the gene functions in a controlled vocabulary [19] were assigned to the consensus transcripts of the gene indices. The functional annotations of GO were further reduced using GO-Slim terms, which provide a more accurate GO assignment by assigning a higher level annotation in the GO hierarchy. The GO slim assignments for the six Solanaceae gene indices are shown in Figure 2. A total of 51,830 sequences within the six Solanaceae gene indices were assigned GO-Slim terms. The largest functional categories were catalytic activity (14–17%), hydrolase activity (11–14%) and transferase (11–12%). Overall, the relative composition of the sampled transcriptome over the various functional categories was very similar among the Solanaceae species. In addition, for every species, representative clones could be annotated to every functional GOSlim category, further supporting the representative coverage of the transcriptome throughout all six species. These data indicate that, although the number of sequences and cDNA library sources differ between the six gene indices, the relative functional composition of the transcripts sampled is very similar, further validating the genomic scale comparisons of this study.
Figure 2 Assignment of Gene Ontology terms to the Solanaceae gene indices. Plant GOSlim terms were assigned to the six Solanaceae gene indices in the categories indicated.
Sequence conservation among six Solanaceae species
Previous reports of sequence conservation within the Solanaceae were based on a relatively small number of genes [6]. The availability of six gene indices allowed for the first genomic scale comparisons of sequence similarity between multiple solanaceous species. Pair-wise sequence comparisons of all gene indices were performed using BLASTN [20] and an E value cutoff of 10-10 was used as a minimum cutoff for significant sequence similarity at the nucleotide level. The results are shown in Figure 3. The number of similar sequences between different gene indices is dependent on the number of sequences available, the depth of sequencing from the various libraries, and the tissue diversity represented in the EST collections. For example, comparison of tomato and potato, the largest gene indices, revealed that 76–78% of the sequences had a match in the respective gene index. For the smaller gene indices, such as N. benthamiana, 81% of the sequences had matches in potato, whereas the reciprocal comparison revealed only 29% similar sequences which can be attributed to the lower number of sequences present in the N. benthamiana gene index. As expected, increasing the stringency (E value 10-25) resulted in a lower percentage of matches (data not shown). The similarities at the nucleotide level were paralleled at the protein level as revealed by TBLASTX searches (data not shown).
Figure 3 Percentage of BLASTN matches among Solanaceae gene indices. Each gene index (query database) was searched against each Solanaceae gene index (color bars). A BLAST score E value cutoff of 10-10 was used for significant sequence matches. Shown is the percentage of transcripts in each Solanaceae gene index.
Sequence comparisons of the Solanaceae species were further refined by the identification of putative orthologs at the nucleotide level among the Solanaceae gene indices. Orthologs are defined as genes with a common ancestor before speciation and which have retained their biological function. The approach we used to identify orthologs [21] utilizes a reciprocal best hit method and was applied to the six Solanaceae gene indices (Table 3). For potato and tomato, the percentage of sequences (39–47%) for which putative orthologs could be identified was lower than the percentage of sequences with significant matches (76–78%), indicating that the identification of orthologs is a more stringent approach for the identification of transcripts with a conserved function. Overall, with the exception of tobacco, 47–60% of the sequences had a reciprocal best match within one of the Solanaceae gene indices and could be classified as a putative ortholog. The clusters of orthologous genes are available in supplemental Table 1 [see Additional file 1].
Table 3 Identification of orthologs among solanaceous species. Number and percentages of reciprocal best hit pairs determined by BLAST searches (E value cutoff 10-10) were listed and the percentages of the total unique sequences of the species (first column) were calculated.
Total N. benthamiana Pepper Petunia Potato Tobacco Tomato
N. benthamiana 7554 2275 (30%) 1050 (14%) 3738 (49%) 2372 (31%) 3520 (47%)
Pepper 13003 2275 (17%) 1688 (13%) 7061 (54%) 3273 (25%) 6762 (52%)
Petunia 4466 1050 (24%) 1688 (38%) 2689 (60%) 1618 (36%) 2591 (58%)
Potato 38239 3738 (10%) 7061 (18%) 2689 (7%) 6337 (17%) 14911 (39%)
Tobacco 21107 4120 (20%) 3273 (16%) 1618 (8%) 6337 (30%) 5869 (28%)
Tomato 31838 3520 (11%) 6762 (21%) 2591 (8%) 14911 (47%) 5859 (18%)
Arabidopsis and rice were included to identify orthologs among the six Solanaceae species, rice, and Arabidopsis. Due to the higher sequence divergence of these two species, a lower number of orthologs can be expected, however, a total of 308 transcripts could be identified with reciprocal matches over all eight species. A phylogenetic tree was constructed based on the sequence alignment of the concatenated sequences from these 308 transcripts (see Figure 4). As these 308 transcripts are expected to be functionally conserved, their sequence divergence was used to assess the overall sequence divergence between the six Solanaceae species, Arabidopsis, and rice (Figure 4). Potato and tomato, as well as tobacco and N. benthamiana (both Nicotiana species), form closely related groups in the tree. Both petunia and the Nicotiana species are outliers among the Solanaceae, whereas pepper is more closely related to tomato and potato. As expected, Arabidopsis and rice form the outliers in the tree. These results further illustrate the process of sequence divergence during speciation of the Solanaceae.
Figure 4 Sequence divergence among solanaceous species. Orthologous genes (308) were identified among all eight species indicated. The phylogenetic tree was constructed using the neighbor joining method of the PHYLIP package.
Identification of transcripts likely unique to Solanaceae
Sequence information generated for a large number of plant species is primarily available in the form of EST collections while for Arabidopsis [22] and rice [23-25], near-complete genome sequences are available. To identify transcripts likely to be unique to the Solanaceae, the solanaceous transcripts were compared to 21 other gene indices as well as the predicted proteomes of rice and Arabidopsis to provide a representative sampling of plant genes. Like the Solanaceae, Arabidopsis is a eudicot whereas rice is a monocot. The Arabidopsis genome has been re-annotated since its completion [26] and the refinement of the annotation of rice is an ongoing process [27]. It is unlikely that a substantial number of novel new genes will be identified in either of these two species with the continuing annotation efforts, therefore comparison to these genomes is indicative of the number of Solanaceae transcripts not present in these two model species. From the comparison to the proteomes of Arabidopsis and rice (Figure 5), it appeared that there are a number of potentially novel or highly diverged transcripts among the Solanaceae family compared to Arabidopsis and rice. The percentage of sequences from potato, tomato, tobacco, N. benthamiana and petunia with significant matches (BLASTX using an E value cutoff of 10-5) in Arabidopsis varied between 70% (potato) and 79% (N. benthamiana). For rice, the percentages were slightly lower, between 67% (potato) and 78% (N. benthamiana), consistent with the eudicot nature of the Solanaceae. The sole exception to this high degree of conservation with these two model species is tobacco with only 42% of the tobacco gene index sequences matching an Arabidopsis protein and 41% matching a rice protein. As indicated in Figure 1, tobacco also has also a lower percentage of homologous sequences among other Solanaceae species examined in this study indicating the presence of unusual sequences within the available tobacco ESTs and ETs.
Figure 5 Comparison of the six Solanaceae gene indices to Arabidopsis (blue) and rice (green). Shown is the percentage of sequences of the Solanaceae gene indices with matches, BLAST score E value cut-off of 10-5 in Arabidopsis and rice
To further identify transcripts likely to be unique to the Solanaceae, all transcripts with no sequence similarity to Arabidopsis or rice were searched against 21 plant gene indices [28]. From the initial 116,207 transcripts, a total of 29,588 transcripts did not have any significant matches in Arabidopsis, rice, or the other 21 gene indices (see Table 4). With the exception of tobacco, the number of Solanaceae unique transcripts ranged between 15% for N. benthamiana and 22% for potato. The large number of transcripts without matches in these other plant species suggests that the Solanaceae contains unique sequences although this number may decrease as additional plant sequences become available in the future and more comparative analyses are performed. Overall the average length of these transcript assemblies was comparable to the overall average transcript assembly length; 420 bases compared to 531 bases for the singleton sequences, which were highly enriched in the Solanaceae-specific sequence data set. Transcripts with no matches in any of the 23 plant species or among Solanaceae are available in supplemental Table 2 [see Additional file 3].
Table 4 Identification of Solanaceae specific transcripts. Number of transcripts identified in the Solanaceae gene indices with no matches in Arabidopsis, rice or any of the 21 plant gene indices; * including Arabidopsis and rice.
Total Transcripts with no matches in Arabidopsis or rice Transcripts with no matches in plant Gene Indices *
Potato 38239 10951 (29%) 8465 (22%)
Tomato 31838 7756 (24%) 6209 (20%)
Pepper 13003 3287 (25%) 2699 (21%)
Tobacco 21107 11671 (55%) 10251 (49%)
Petunia 4466 1064 (24%) 855 (19%)
N. benthamiana 7554 1481 (20%) 1109 (15%)
Next, we determined the number of transcripts unique to each of the six Solanaceae gene indices. Using TBLASTX, two different BLAST score cut-off E values were used to identify transcripts with no significant sequence homology within the Solanaceae. Using an E value cut-off of 10-5, 26% of the transcripts in any of the six Solanaceae gene indices had no match among the Solanaceae gene indices; using the more stringent E value cut-off of 10-10, 21% of the sequences had no match (see Table 5). Of these transcripts, 19% (E value cut-off of 10-5) or 16% (E value cut-off of 10-10) also did not have significant sequence homology in Arabidopsis, rice, or any of the 21 other plant gene indices; thus, these transcripts appear unique to each of the six Solanaceae gene indices based on these comparisons. The largest number of unique transcripts (38%) was found in tobacco in contrast to the 8–13% unique transcripts found in the other five solanaceous gene indices. These results indicate that in addition to a large number of conserved sequences among the Solanaceae, each species contained a subset of sequences likely to be unique to each species. As these transcripts also did not have significant homology to 21 other plant species for which sequence data is available, it is unlikely this can be attributed to differences in transcript sampling or the availability of a relatively low number of total sequences.
Table 5 Identification of Solanaceae species-specific transcripts. The left panel shows the number of sequences without matches in any of the Solanaceae gene indices. The right panel shows the number of sequences for each species without matches to Arabidopsis, rice, or any plant gene index, including Solanaceae.
Unique among Solanaceae gene index Solanaceae specific Transcripts
TBLASTX (10-5) TBLASTX (10-10) TBLASTX (10-5) TBLASTX (10-10)
Potato 8878 (23%) 6967 (18%) 5796 (15%) 4825 (13%)
Tomato 6131 (19%) 4796 (15%) 3792 (12%) 3151 (10%)
Pepper 2483 (19%) 1921 (15%) 1865 (14%) 1531 (12%)
Tobacco 10580 (50%) 9131 (43%) 8949 (42%) 8076 (38%)
Petunia 825 (18%) 609 (14%) 631 (14%) 512 (11%)
N. benthamiana 1032 (14%) 757 (10%) 740 (10%) 571 (8%)
Total 29929 (26%) 24181 (21%) 21773 (19%) 18666 (16%)
Expression profiling of solanaceous species
To experimentally validate the level of sequence conservation among the Solanaceae, global expression profiles in mature leaves were compared using microarrays. Potato microarrays containing ~12,000 potato cDNA clones were used to compare global gene expression patterns among the six Solanaceae species. As all probes on the microarray were derived from potato, we first assessed whether the potential sequence divergence of these probes would affect signal intensities. All probes on the potato array were searched against the other five Solanaceae gene indices and grouped based on BLASTN similarity score (5% bins) ranging from <60% to 95–100% sequence identity. Total RNA isolated from mature leaves of tomato, pepper, tobacco, petunia and N. benthamiana (query samples) was labeled with Cy3 and hybridized to the potato cDNA microarrays with RNA isolated from mature potato leaves that had been labeled with Cy5 (reference sample). The sequence similarity between the different Solanaceae species allowed for the detection of transcripts from the various species on the potato microarray for over 80% of the probes on the microarray. Normalized signal intensities were calculated for each element and the median intensity for each group of probes based on the BLASTN similarity score was plotted (Figure 6). Overall, the signal intensity increased for probes with a higher sequence similarity among the Solanaceae species, including potato. If this trend was attributable to the potential sequence divergence of the probes, it would be expected that the trend for potato would be different, as the potato RNA provides a perfect match to the probes on the array. Thus, the potential sequence divergence of the probes was not the limiting factor in reliable detection of expression levels for these heterologous hybridizations. This suggests that more highly conserved genes were expressed at relatively higher levels than more diverged genes because the group of probes with the higher sequence similarity all showed a higher median expression intensity. More conserved genes most likely represent "housekeeping" genes that can be expected to be generally expressed at higher levels. Alternatively, these probes may contain conserved motifs and therefore the probes on the microarray will cross-hybridize to multiple transcripts resulting in the higher signal intensities observed on the microarray for these elements. The number of clones that could be detected on the microarray was dependent on the species used as target. For the more diverged species, such as petunia, a lower number of clones were detected on the microarray (data not shown). Overall, we found similar expression levels in leaves across the six Solanaceae species used in this study (not shown), indicating that indeed sequence conservation may represent a functional similarity as well. In conclusion, the analyses of microarray data indicated that for the core genes conserved among Solanaceae with significant sequence similarity to potato, reliable gene expression values can be derived from microarrays with potato cDNA probes.
Figure 6 Expression analysis of six solanaceous species. Probes on the microarray were grouped according to the sequence similarity with potato and plotted against the median normalized signal intensity of each group. Shown is the average of two experiments of the median intensity of each group.
Discussion
A high degree of sequence conservation among Solanaceae family members had been suggested previously based on small scale assays and analysis. Here, we report for the first time, a large scale comparison of six Solanaceae family members. Although the analyses in this study confirmed the high degree of sequence conservation, they also revealed a large number of Solanaceae specific transcripts and sequence divergence among Solanaceae.
Transcript sampling for the Solanaceae gene indices
To date, only a limited amount of genomic sequence data is (publicly) available for the Solanaceae. Therefore, the EST sequence data assembled in this study was used to assess the diversity of transcripts among the Solanaceae. The assessment of the annotation by GO terms of the six gene indices indicated an overall similar functional composition of the transcripts. In addition, the analysis of GC content was consistent with Arabidopsis and among the Solanaceae, with tobacco being the exception. These data show that the sequences used in this study provide a valid representation of the various solanaceous genomes. The wide range of different library sources of the sequences did not affect the number of sequence matches among the different Solanaceae species, indicating the absence of a high percentage of tissue specific transcripts. This can be explained by the close developmental relationship between most plant organs as flowers can be considered modified leaves and stolons as modified stems. A low number of tissue specific transcripts were also observed in Arabidopsis using Massive Parallel Signature Sequencing [29]. Among five different libraries of callus, inflorescence, leaves, roots and siliques, less than 0.25% of the transcripts showed tissue specificity [29]. Also in maize, using cDNA microarrays, only 7% of the genes were expressed in a highly tissue specific manner among seven different organs of maize [30]. In contrast, the assessment of the frequency of the EST sequences can be used for the comparative analysis to evaluate differential expression. This approach has been used for tomato and potato [16,31], but can only be successfully employed with a large number of diverse libraries and deep sequencing as most tissue specific transcripts may be expressed at low levels and therefore be relatively rare and not be sampled by sequencing.
A single microarray platform was successfully applied for heterologous hybridization of Solanaceae species. For transcripts with significant sequence similarity to the potato probes on the cDNA microarray, reliable expression data could be obtained. Similar hybridization characteristics were found using heterologous hybridization to a fish cDNA microarray [32]; the number of elements that could be detected on the microarray was correlated with the phylogenetic distance. Cross-species hybridization was also shown for human and bovine orthologous genes on a human cDNA microarray [33]. The global expression data indicated that the conserved transcripts were expressed similarly among leaf tissue of the six Solanaceae species examined.
Solanaceae species contain unique transcripts
Overall, a high degree of sequence conservation among the Solanaceae was observed in accordance with previous small scale studies [6]; for up to 81% of the gene index sequences, significant matches at the nucleotide level could be found within the Solanaceae, consistent with the level of sequence conservation observed at the protein level. Using a more stringent approach of orthology revealed that for the largest gene indices of potato and tomato, a putative ortholog could be identified at the nucleotide level for 47% of the unique transcripts in the gene index. In addition, comparison of the Solanaceae gene indices to Arabidopsis, rice, and 21 other gene indices revealed transcripts without matches to these non-solanaceous species as well as transcripts without matches to individual Solanaceae species. Depending on the stringency of alignment, 16–19% of the transcripts did not have a match among the plant sequences examined. A similar approach was used to identify transcripts specific for legumes [34]. These results show that between these closely related species there was still substantial sequence divergence, which was supported by the sequence divergence among 308 orthologous transcripts of six Solanaceae, Arabidopsis and rice. The available EST sequences only provide a snapshot of the genome, thus the number of unique transcripts may be lower but still be substantial as the transcript sampling among the Solanaceae proved to be a representative sampling. The large number of EST sequences available for tomato and potato were likely to contain the most abundant transcripts, so a large number of transcripts without sequence homology is likely to remain with increased EST sequencing until more sequence data is generated.
The outlier for most analyses appeared to be tobacco with a low number of significant matches among Solanaceae, Arabidopsis, and rice. No obvious explanation could be found for this but it is unlikely that tobacco will contain a much higher plant specific gene content. Matsuoka et al. [35] report on the EST sequencing of a cell suspension library of tobacco, which was the origin of a large portion of the tobacco gene index. In this study, a low number of tobacco sequences matched sequences from other plant species, consistent with our analyses. The GO assignments and the identification of orthologs indicated that the tobacco sequence sample did contain similar transcripts as the other five Solanaceae gene indices, validating the general conclusions for the Solanaceae species in this study, including tobacco.
The finding of a large number of transcripts without matches among the Solanaceae species will complicate the efforts of establishing a single reference genome for the Solanaceae by sequencing a single representative species. Although a large level of synteny exists between the Solanaceae, it is unclear how novel genes evolved and whether there is a large difference in gene content among the Solanaceae. Fortunately, for three Solanaceae species (tomato, potato and tobacco), genome sequencing projects are in progress. The availability of three draft genome sequences will allow for the detailed analysis of genome conservation and understanding of the genes involved in the different phenotypes within the Solanaceae.
Conclusion
In summary, this study documents for the first time the genomic scale comparison of the available coding sequences (ESTs and ETs) from six Solanaceae species. Sequence comparisons at the nucleotide level among potato, tomato, pepper, eggplant, tobacco and N. benthamiana, including ortholog analysis, confirmed a high level of sequence conservation. In addition, phylogenetic analysis and comparative analyses with Arabidopsis, rice and 21 other gene indices revealed sequence divergence during speciation as evidenced by transcripts likely unique among the Solanaceae and unique to individual Solanaceae species. Global expression profiling showed similar expression patterns of conserved genes in mature leaves among the six solanaceous species.
Methods
Computational methods
Gene indices were constructed essentially as described [15]. In summary, all available sequences for potato, tomato, pepper, eggplant and petunia were collected from Genbank and sequences with over 94% sequence identity over 40 or more bases with unmatched overhangs of 30 bases in length were placed in clusters using the Paracel Transcript Assembler to generate tentative consensus sequences (TC) and singleton ESTs and ETs. The TCs were searched against a non-redundant protein database to provide a putative annotation for the TC, with a minimum of 30% identity over 20% of the length of the translated TC. All gene indices are available at [28]. The 21 gene indices used for searches against the Solanaceae gene indices were: Ice plant (v4.0), Cocao (v1.0), Cotton (v6.0), Grape (v4.0), Barley (v9.0), Sugar beet (v1.0), Brassica napus (v1.0), Sunflower (v3.0), Lettuce (v2.0), Lotus (v3.0), Wheat (v10.0), Maize (v15.0), Medicago truncatula (v8.0), Onion (v1.0), Pinus (v5.0), Poplar (v2.0), Rye (v3.0), Sorghum bicolor (v8.0), Sugarcane (v2.1), Soybean (v12.0) and Spruce (v1.0). GO terms were transitively annotated based on sequence similarity (E value cutoff of 10-10) to Arabidopsis proteins (Release 5, [26] which has been manually curated for molecular function GO terms. The Plant/GOSlim reduced ontologies were used [36].
Each of the six gene indices was pair-wise matched against the other gene indices using WU-BLAST [37] with BLASTN and TBLASTX options. BLAST scores were filtered for significant hits using an E value cut-off as indicated in the text. Each of the six gene indices were searched against the predicted rice and Arabidopsis proteome using BLASTX and the top hit was picked for each entry of the gene indices using an E value cutoff of 10-5. Putative orthologs among the six Solanaceae species, rice and Arabidopsis were identified essentially as described [21]. In summary, the non-redundant sets of eight gene indices were compiled and searched against each other using BLASTN. The reciprocal best hit pairs with a cutoff E value 10-10 were clustered to generate the ortholog groups. 308 clusters which contain at least one transcript from each of the 8 species were selected and one representative sequence for each species was chosen for each group by counting the reciprocal matches in the clusters. Multiple sequence alignments for each of the 308 clusters were performed and sequences in both ends without consensus matches were removed. Sequences from each species were concatenated together in the same order and aligned to each other using CLUSTAL W [38]. A neighbor joining tree was generated using PHYLIP (Phylogeny Inference Package) (Felsenstein, J. 2004, distributed by the author. Department of Genome Sciences, University of Washington, Seattle).
Microarray hybridizations and data analysis
Potato cDNA microarrays were constructed as described [39]. Potato, tobacco, tomato, petunia, pepper and N. benthamiana plants were grown in Percival growth chambers (Percival Scientific, Inc. Perry, IA) at 25°C and 16 h light for 4–6 weeks. Total RNA was extracted from mature leaves using the Qiagen RNAesy kit (Qiagen, Valencia, CA) and labeled as described previously [39]. Hybridization and washing was performed essentially as described [39]. After the final washing step and spin-drying of the slide, slides were scanned using an Axon scanner at maximum laser power (Axon Instruments, Union City, CA) at both 532 and 635 nm. The PMT values for both wavelengths were adjusted to capture a similar number of normalized counts for each channel.
The TIFF images were quantified using Genepix 5.0 (Axon Instruments, Union City, CA). The software automatically flags spots that cannot be found in one of the channels; these are flagged and excluded from further analysis. Spots containing > 30% saturated pixels in either channel or a diameter <70 μm were flagged and not used for subsequent analysis. Local background was subtracted from the signal value (mean pixel intensity). The data were normalized using the quantile method in the limma package [40] of BioConductor [41]. Flagged spots were given a weight of 0 using the weight function within the package which excludes these spots from affecting the normalization. All analyses used the average of the two on-slide replicates. If one of the two replicates was flagged, the remaining value was used for analysis.
The microarray data are available at the TIGR Potato functional genomics and Solanaceae resources web pages [42] and through the Gene Expression Omnibus (GEO) [43] under platform accession GPL1901.
Authors' contributions
WAR coordinated and designed the study, performed the microarray data analysis and drafted the manuscript. DL constructed the Gene Indices, performed the analysis of orthologs, GC content and constructed the phylogenetic tree. JL performed all the BLAST searches and analyses. SI carried out the microarray hybridizations. SO performed the assignment of GO terms. CRB performed the library composition analysis of the Gene Indices, participated in coordination and design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Supplemental Table 1 (split into two files) containing the Solanaceae ortholog clusters, for each cluster the TC numbers from each gene index are listed that form an ortholog cluster.
Click here for file
File 2
Additional File 3
Supplemental Table 2 containing the Solanaceae specific transcripts, for each of the six solanaceous species the TC numbers are listed without matches in 23 other plant species (see Table 4), transcripts unique to Solanaceae (Table 5, right panel, TBLASTX E-05) and transcripts unique to each species (Table 5, left panel, TBLASTX E-05).
Click here for file
Acknowledgements
Funding for this work was provided through a grant from the National Science Foundation Plant Genome Research Program (DBI-0218166).
==== Refs
Food and Agricultural Organization of The United Nations, FAOSTAT 2005
United States Department of Agriculture (USDA), National Agricultural Statistics Service, Floriculture Crops 2005
United States Department of Agriculture (USDA), National Agricultural Statistics Service, Crop Production 2005
Lu R Martin-Hernandez AM Peart JR Malcuit I Baulcombe DC Virus-induced gene silencing in plants Methods 2003 30 296 303 12828943 10.1016/S1046-2023(03)00037-9
Arumuganathan K Earle ED Nuclear DNA Content of Some Important Plant Species Plant Molecular Biology Reporter 2004 9 208 218
Zamir D Tanksley S Tomato genome is comprised largely of fast-evolving, low copy-number sequences Mol Gen Genet 1988 213 254 261 10.1007/BF00339589
Bonierbale MW Plaisted RL Tanksley SD RFLP Maps Based on a Common Set of Clones Reveal Modes of Chromosomal Evolution in Potato and Tomato Genetics 1988 120 1095 1103 17246486
Tanksley SD Ganal MW Prince JP de Vicente MC Bonierbale MW Broun P Fulton TM Giovannoni JJ Grandillo S Martin GB . High density molecular linkage maps of the tomato and potato genomes Genetics 1992 132 1141 1160 1360934
Livingstone KD Lackney VK Blauth JR van Wijk R Jahn MK Genome mapping in capsicum and the evolution of genome structure in the solanaceae Genetics 1999 152 1183 1202 10388833
Doganlar S Frary A Daunay MC Lester RN Tanksley SD A comparative genetic linkage map of eggplant (Solanum melongena) and its implications for genome evolution in the solanaceae Genetics 2002 161 1697 1711 12196412
Doganlar S Frary A Daunay MC Lester RN Tanksley SD Conservation of gene function in the solanaceae as revealed by comparative mapping of domestication traits in eggplant Genetics 2002 161 1713 1726 12196413
Thorup TA Tanyolac B Livingstone KD Popovsky S Paran I Jahn M Candidate gene analysis of organ pigmentation loci in the Solanaceae Proc Natl Acad Sci U S A 2000 97 11192 11197 11027328 10.1073/pnas.97.21.11192
Grube RC Radwanski ER Jahn M Comparative genetics of disease resistance within the solanaceae Genetics 2000 155 873 887 10835406
Adams MD Soares MB Kerlavage AR Fields C Venter JC Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library Nat Genet 1993 4 373 380 8401585 10.1038/ng0893-373
Quackenbush J Cho J Lee D Liang F Holt I Karamycheva S Parvizi B Pertea G Sultana R White J The TIGR Gene Indices: analysis of gene transcript sequences in highly sampled eukaryotic species Nucleic Acids Res 2001 29 159 164 11125077 10.1093/nar/29.1.159
Ronning CM Stegalkina SS Ascenzi RA Bougri O Hart AL Utterbach TR Vanaken SE Riedmuller SB White JA Cho J Pertea GM Lee Y Karamycheva S Sultana R Tsai J Quackenbush J Griffiths HM Restrepo S Smart CD Fry WE Van der HR Tanksley S Zhang P Jin H Yamamoto ML Baker BJ Buell CR Comparative analyses of potato expressed sequence tag libraries Plant Physiol 2003 131 419 429 12586867 10.1104/pp.013581
Carels N Bernardi G Two classes of genes in plants Genetics 2000 154 1819 1825 10747072
Carels N Hatey P Jabbari K Bernardi G Compositional properties of homologous coding sequences from plants J Mol Evol 1998 46 45 53 9419224
Ashburner M Ball CA Blake JA Botstein D Butler H Cherry JM Davis AP Dolinski K Dwight SS Eppig JT Harris MA Hill DP Issel-Tarver L Kasarskis A Lewis S Matese JC Richardson JE Ringwald M Rubin GM Sherlock G Gene ontology: tool for the unification of biology. The Gene Ontology Consortium Nat Genet 2000 25 25 29 10802651 10.1038/75556
Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389
Lee Y Sultana R Pertea G Cho J Karamycheva S Tsai J Parvizi B Cheung F Antonescu V White J Holt I Liang F Quackenbush J Cross-referencing eukaryotic genomes: TIGR Orthologous Gene Alignments (TOGA) Genome Res 2002 12 493 502 11875039 10.1101/gr.212002
Initiative AG Analysis of the genome sequence of the flowering plant Arabidopsis thaliana Nature 2000 408 796 815 11130711 10.1038/35048692
Goff SA Ricke D Lan TH Presting G Wang R Dunn M Glazebrook J Sessions A Oeller P Varma H Hadley D Hutchison D Martin C Katagiri F Lange BM Moughamer T Xia Y Budworth P Zhong J Miguel T Paszkowski U Zhang S Colbert M Sun WL Chen L Cooper B Park S Wood TC Mao L Quail P Wing R Dean R Yu Y Zharkikh A Shen R Sahasrabudhe S Thomas A Cannings R Gutin A Pruss D Reid J Tavtigian S Mitchell J Eldredge G Scholl T Miller RM Bhatnagar S Adey N Rubano T Tusneem N Robinson R Feldhaus J Macalma T Oliphant A Briggs S A draft sequence of the rice genome (Oryza sativa L. ssp. japonica) Science 2002 296 92 100 11935018 10.1126/science.1068275
Yu J Hu S Wang J Wong GK Li S Liu B Deng Y Dai L Zhou Y Zhang X Cao M Liu J Sun J Tang J Chen Y Huang X Lin W Ye C Tong W Cong L Geng J Han Y Li L Li W Hu G Huang X Li W Li J Liu Z Li L Liu J Qi Q Liu J Li L Li T Wang X Lu H Wu T Zhu M Ni P Han H Dong W Ren X Feng X Cui P Li X Wang H Xu X Zhai W Xu Z Zhang J He S Zhang J Xu J Zhang K Zheng X Dong J Zeng W Tao L Ye J Tan J Ren X Chen X He J Liu D Tian W Tian C Xia H Bao Q Li G Gao H Cao T Wang J Zhao W Li P Chen W Wang X Zhang Y Hu J Wang J Liu S Yang J Zhang G Xiong Y Li Z Mao L Zhou C Zhu Z Chen R Hao B Zheng W Chen S Guo W Li G Liu S Tao M Wang J Zhu L Yuan L Yang H A draft sequence of the rice genome (Oryza sativa L. ssp. indica) Science 2002 296 79 92 11935017 10.1126/science.1068037
Yu J Wang J Lin W Li S Li H Zhou J Ni P Dong W Hu S Zeng C Zhang J Zhang Y Li R Xu Z Li S Li X Zheng H Cong L Lin L Yin J Geng J Li G Shi J Liu J Lv H Li J Wang J Deng Y Ran L Shi X Wang X Wu Q Li C Ren X Wang J Wang X Li D Liu D Zhang X Ji Z Zhao W Sun Y Zhang Z Bao J Han Y Dong L Ji J Chen P Wu S Liu J Xiao Y Bu D Tan J Yang L Ye C Zhang J Xu J Zhou Y Yu Y Zhang B Zhuang S Wei H Liu B Lei M Yu H Li Y Xu H Wei S He X Fang L Zhang Z Zhang Y Huang X Su Z Tong W Li J Tong Z Li S Ye J Wang L Fang L Lei T Chen C Chen H Xu Z Li H Huang H Zhang F Xu H Li N Zhao C Li S Dong L Huang Y Li L Xi Y Qi Q Li W Zhang B Hu W Zhang Y Tian X Jiao Y Liang X Jin J Gao L Zheng W Hao B Liu S Wang W Yuan L Cao M McDermott J Samudrala R Wang J Wong GK Yang H The Genomes of Oryza sativa: a history of duplications PLoS Biol 2005 3 e38 15685292 10.1371/journal.pbio.0030038
Wortman JR Haas BJ Hannick LI Smith RKJ Maiti R Ronning CM Chan AP Yu C Ayele M Whitelaw CA White OR Town CD Annotation of the Arabidopsis genome Plant Physiol 2003 132 461 468 12805579 10.1104/pp.103.022251
Yuan Q Ouyang S Wang A Zhu W Maiti R Lin H Hamilton J Haas B Sultana R Cheung F Wortman J Buell CR The institute for genomic research osa1 rice genome annotation database Plant Physiol 2005 138 18 26 15888674 10.1104/pp.104.059063
The Institute for Genomic Research (TIGR), Plant Gene Indices 2005
Meyers BC Vu TH Tej SS Ghazal H Matvienko M Agrawal V Ning J Haudenschild CD Analysis of the transcriptional complexity of Arabidopsis thaliana by massively parallel signature sequencing Nat Biotechnol 2004 22 1006 1011 15247925 10.1038/nbt992
Fernandes J Brendel V Gai X Lal S Chandler VL Elumalai RP Galbraith DW Pierson EA Walbot V Comparison of RNA expression profiles based on maize expressed sequence tag frequency analysis and micro-array hybridization Plant Physiol 2002 128 896 910 11891246 10.1104/pp.010681
Fei Z Tang X Alba RM White JA Ronning CM Martin GB Tanksley SD Giovannoni JJ Comprehensive EST analysis of tomato and comparative genomics of fruit ripening Plant J 2004 40 47 59 15361140 10.1111/j.1365-313X.2004.02188.x
Renn SC Aubin-Horth N Hofmann HA Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray BMC Genomics 2004 5 42 15238158 10.1186/1471-2164-5-42
Adjaye J Herwig R Herrmann D Wruck W Benkahla A Brink TC Nowak M Carnwath JW Hultschig C Niemann H Lehrach H Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays BMC Genomics 2004 5 83 15511299 10.1186/1471-2164-5-83
Graham MA Silverstein KA Cannon SB VandenBosch KA Computational identification and characterization of novel genes from legumes Plant Physiol 2004 135 1179 1197 15266052 10.1104/pp.104.037531
Matsuoka K Demura T Galis I Horiguchi T Sasaki M Tashiro G Fukuda H A comprehensive gene expression analysis toward the understanding of growth and differentiation of tobacco BY-2 cells Plant Cell Physiol 2004 45 1280 1289 15509851 10.1093/pcp/pch155
The Gene Ontology 2005
Washington University BLAST 2005
Thompson JD Higgins DG Gibson TJ CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 1994 22 4673 4680 7984417
Rensink WA Iobst S Hart A Stegalkina S Liu J Buell CR Gene expression profiling of potato responses to cold, heat, and salt stress Funct Integr Genomics 2005 In press
Smyth GK Linear models and empirical Bayes methods for assessing differential expression in microarray experiments Statistical Applications in Genetics and Molecular Biology 2004 3 Article 3 16646809
BioConductor 2005
The Institute for Genomic Research (TIGR), Potato Functional Genomics & Solanaceae Resources 2005
Gene Expression Omnibus 2005
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1281617151710.1186/1471-2164-6-128Research ArticleGenome annotation of Anopheles gambiae using mass spectrometry-derived data Kalume Dário E [email protected] Suraj [email protected] Raghunath [email protected] Jun [email protected] Mobolaji [email protected] Nirbhay [email protected] Akhilesh [email protected] McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry and Oncology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, DK-5230, Denmark3 Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA4 Institute of Bioinformatics, Discoverer Unit 1, 7th Floor International Tech Park Ltd., Whitefield Road, Bangalore – 560 066, India2005 19 9 2005 6 128 128 30 1 2005 19 9 2005 Copyright © 2005 Kalume et al; licensee BioMed Central Ltd.2005Kalume et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
A large number of animal and plant genomes have been completely sequenced over the last decade and are now publicly available. Although genomes can be rapidly sequenced, identifying protein-coding genes still remains a problematic task. Availability of protein sequence data allows direct confirmation of protein-coding genes. Mass spectrometry has recently emerged as a powerful tool for proteomic studies. Protein identification using mass spectrometry is usually carried out by searching against databases of known proteins or transcripts. This approach generally does not allow identification of proteins that have not yet been predicted or whose transcripts have not been identified.
Results
We searched 3,967 mass spectra from 16 LC-MS/MS runs of Anopheles gambiae salivary gland homogenates against the Anopheles gambiae genome database. This allowed us to validate 23 known transcripts and 50 novel transcripts. In addition, a novel gene was identified on the basis of peptides that matched a genomic region where no gene was known and no transcript had been predicted. The amino termini of proteins encoded by two predicted transcripts were confirmed based on N-terminally acetylated peptides sequenced by tandem mass spectrometry. Finally, six sequence polymorphisms could be annotated based on experimentally obtained peptide sequences.
Conclusion
The peptide sequences from this study were mapped onto the genomic sequence using the distributed annotation system available at Ensembl and can be visualized in the context of all other existing annotations. The strategy described in this paper can be used to correct and confirm genome annotations and permit discovery of novel proteins in a high-throughput manner by mass spectrometry.
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Background
The recent completion of Anopheles gambiae genome sequence [1] provided an architectural scaffold for mapping, identifying, selecting, and exploiting malaria insect vector genes for future studies. An. gambiae genome consists of 3 pairs of chromosome, designated as 2R/2L, 3R/3L and X. The Y chromosome is yet to be completely sequenced and assembled because of the high number of transposable element fragments. Thus far, approximately 85% of the genome has been assembled with the total genome size being 278 Mbp. About 15,189 genes are annotated in the An. gambiae genome, of which 11,757 are derived from prediction programs [2]. Currently, there are approximately 700 known An. gambiae proteins that are annotated in the databases. The annotation of the An. gambiae genome sequence has been an ongoing process since it was completed in 2002 [1]. The assembled genome is publicly available through NCBI (National Center for Biotechnology Information) and EBI (European Bioinformatics Institute)/Ensembl . It is important to note that the existing genome annotation is mainly based on de novo gene predictions in addition to a small number of experimentally obtained transcripts. Because of the magnitude of sequence data, automatic annotation is a necessity. However, this results in different types of errors, some of which can be overcome by combining manual annotation and experimental evidence. In this regard, mass spectrometry is a powerful tool that can contribute to the identification of novel genes and assist in confirmation of predicted transcripts as well as correction of incorrect assignments from automated gene annotations [3]. The use of mass spectrometry to assist the validation of genome annotation has been previously demonstrated in prokaryotes [4], yeast [5], plants [6] and humans [7]. However, two of these studies [5,7] did not directly search mass spectrometry-derived data against the genomic databases – rather, a post hoc integration of peptide sequences with the genomic sequence was carried out. This approach is not preferable for annotating genomes because if there is any region of a genome that has no transcript associated with it (e.g. introns and intergenic regions), it will not be identified.
In this study, we carried out a proteomic analysis of salivary gland proteins from An. gambiae and searched this data against the An. gambiae genome database. We were able to validate 73 transcripts, which were predicted from the genome. We also corrected several erroneous predictions (e.g. missed exons) and identified one gene that was not predicted at all. To share our results with the biomedical community, we have taken advantage of the Distributed Annotation System [8] provided by Ensembl. We have mapped all the peptides identified in this study such that they can be visualized by anyone using the Ensembl genome browser. It is hoped that availability of additional proteomic data will aid in further refining the annotation of genomic data.
Results and discussion
Curent status of the An. gambiae genome sequence and annotation
A preliminary annotation of protein coding regions of the An. gambiae genome has recently been published. Using automated gene prediction programs, two groups have annotated a total of 15,189 predicted transcripts [1]. This included a total of 7,840 predicted transcripts that were unique to Otto, the gene prediction pipeline used by the Celera group and 1,375 predicted transcripts that were unique to the Ensembl annotation [1]. Altogether, 5,974 transcripts were predicted by both analyses. It must be cautioned, however, that these are preliminary annotations for the An. gambiae genome. A validation of predicted transcripts could be accomplished through the use of direct peptide sequence data such as that obtained by tandem mass spectrometry in our study. In this study, we have used the strategy outlined in Figure 1 to map the peptides identified by mass spectrometry onto the existing Ensembl annotations of the Anopheles genome. We will illustrate seven different situations in which mass spectrometry data assisted us in the genome annotation: a) peptide sequences that matched exons in known transcripts; b) peptide sequences that matched exons in novel transcripts; c) peptide sequences that matched regions of the genome where no genes were predicted at all; d) matching of peptide sequences regions annotated as untranslated regions (UTRs); e) matching of peptide sequences to regions annotated as introns of known or novel genes; f) data on N-terminal acetylation sites for mapping the amino terminus of the mature protein; and, g) sequence polymorphisms that could represent coding single nucleotide polymorphisms (cSNPs).
Figure 1 A workflow depicting the steps involved in mass spectrometry data analysis for genome annotation purposes. In this case, the tryptic peptide mixture derived from digestion of Anopheles gambiae salivary gland proteins was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The mass spectrometry data was searched against the NCBI non-redundant protein database to identify known or novel transcripts from An. gambiae. The data was also searched against the An. gambiae genome database to identify novel protein-coding genes. A careful bioinformatics analysis was performed to use peptide data for correcting genomic annotations.
Use of distributed annotation system at Ensembl for sharing our genome annotation with the community
DAS [8] allows integration of annotation efforts from various sources and facilitates visualization of annotations interactively in a browser. It serves as a means for decentralization of annotation efforts. A DAS reference server providing access to the An. gambiae genome is available through Ensembl. Therefore, we decided to take advantage of sharing our annotations of the An. gambiae genome with the community using this resource. We uploaded all the peptides identified in our effort onto the Ensembl DAS server. They can now be viewed as peptide sequence hits that mapped to specific genomic sequences along with all other annotations provided by Ensembl (ESTs, transcript, proteins etc.). To view the peptide data, a user has to select the 'MS data JHU' option under 'DAS Sources' pull down menu in the 'Detailed View' panel in ContigView page of Ensembl. Figure 2 shows a screenshot of the DAS view of ten peptides that were sequenced by our laboratory under the 'MS data JHU' track in the negative strand. As indicated in the screenshot, there were no peptide matches in the positive strand. We mapped a total of 370 peptides corresponding to 73 known or predicted transcripts. The peptides are marked as rectangles and are designated as JHU_xxxx where JHU stands for Johns Hopkins University and xxxx is the serial number assigned to the peptide. Clicking on the peptide accession numbers takes the user to a page hosted by our laboratory where additional details such as the peptide sequence are provided .
Figure 2 A screenshot depicting the mapping of mass spectrometry-derived peptide sequence data onto the genome sequence in 'ContigView' of Ensembl Genome Browser. The red rectangles on 'MS data JHU' track are peptide sequences obtained through tandem mass spectrometry. The brown colored rectangles on the 'Ensembl trans.' are the Ensembl known transcripts. Ten peptides are shown to match two exons in the known transcript encoding D7-related protein 1. The prefix JHU refers to Johns Hopkins University and is followed by a unique identifier for each peptide. Clicking on the peptide accession number links to a page containing additional information including its sequence.
Use of peptide sequences to validate known transcripts at the protein level
Transcripts annotated as known transcripts are generally those for whom a full length cDNA exists. However, it is possible for transcripts to exist without being translated, as pseudogenes are also known to be transcribed [9,10]. Here, we describe the use of peptide sequences obtained by tandem mass spectrometry to assist in the annotation and confirmation of exons of known transcripts. Figure 2 demonstrates how we were able to obtain peptide evidence for two of three exons of a known transcript encoding D7-related protein 1 whose cDNA was recently isolated [11,12]. Additional file 1 lists 172 peptides that led to validation of exons from 23 known transcripts.
Use of peptide sequences to validate exons in novel transcripts
Novel transcripts generally refer to those obtained by gene prediction programs and might or might not have corresponding cDNA evidence. Often, a large number of transcripts are predicted from genome annotation pipelines and it would be helpful if one could easily determine if a transcript was not only expressed but also if the translated protein was present. For instance, in the case of the An. gambiae genome, there is a large degree of non-overlap in predicted transcripts between two different sets of annotations. We used the peptides that we identified and searched against the transcripts predicted by Ensembl. Figure 3A illustrates an MS/MS spectrum that led to identification of a peptide as TTLVNMQFGQLVAHDMGLR (Table 2- protein [ENSANG:P00000000593]), which matched the third annotated exon of a novel transcript predicted to encode a novel member of the peroxidase family of proteins. This peptide also matched another transcript as well ([ENSANG:P00000028058]). In cases where a peptide matched more than one genomic sequence, we have annotated all such regions, as we are unable to unambiguously determine the genomic sequence that codes for the peptide. Figure 4 shows validation of all 3 exons of this predicted transcript. Additional file 2 lists a total of 198 peptides that were used to validate the presence of proteins encoded by 50 novel transcripts.
Figure 3 MS/MS spectra in four different instances that were used for annotation of An. gambiae genome. (A) MS/MS spectrum of a peptide, whose sequence validated an exon of a novel transcript encoding a peroxidase family of proteins ([ENSANG:P00000000593]); (B) MS/MS spectrum of a peptide used to identify a novel protein-coding gene. (C) MS/MS spectrum of a peptide that maps to a predicted UTR of a known transcript encoding Antigen 5-related 1 protein. (D) MS/MS spectrum corresponding to a peptide that is acetylated at its N-terminus. The acetyl moiety is denoted by Ac.
Table 1 Proteins with the identified cSNP.
Protein Accession # Protein name Peptide sequence Amino acid change
[ENSANG:P00000029569] Similar to SGS4 SFASDGTDVTVR A→S
[ENSANG:P00000025580] D7-related 3 protein precursor CNAEAEKVHTSSK D→H
[ENSANG:P00000012716] Putative 5'-nucleotidase precursor VPYDTKYDTVEGDYPLVVK I→V
[ENSANG:P00000000593] Peroxidase precursor LLPAEYGDGVSVPR Y→S
[ENSANG:P00000017522] TRIO protein SQNPASPAGSLGGKDVVSK L→Q
T→A
Table 2 List of peptide sequences shown in Figures 3 and 6B.
Protein Accession # Protein name Peptide sequence Figure #
[ENSANG:P00000000593] Peroxidase family of proteins TTLVNMQFGQLVAHDMGLR 3A
- Novel protein-coding gene AAAYCADPSLLFAR 3B
[ENSANG:P00000021028]/ [ENSANG:P00000023200] Antigen 5-related 1 protein FPGLCNASEEPR 3C
[ENSANG:P00000012700] Similar to calmodulin1 ADQLTEEQIAEFKEAFSLFDKDGDGTITTK 3D
[ENSANG:P00000018280] D7 protein family FVDLSR
TLNEANSR
VIDCIFR
IYAAMPQIK
KVIDCIFR
IPVQHEAYK
KIWGGYNKK
LYHGTVEGAAK
GESFFAYCAK
NYELSGSSQFK
KLYHGTVEGAAK
NYELSGSSQFKK
CYEDHLPAGSSR
ALDPEQALYVYK
QKGESFFAYCAK
SERIPVQHEAYK
GRNYELSGSSQFK
LEPNDAVTHCYAK
VYEGPEQVKEEMK
ALDPEQALYVYKR
GRNYELSGSSQFKK 6B
Figure 4 A screenshot depicting validation of a novel transcript using 4 mass spectrometry-derived peptide sequences. The figure shows a novel transcript ([ENSANG:T00000000593]) on chromosome 3L with the red rectangles on 'MS Data' track corresponding to peptide sequences obtained through mass spectrometry.
Identification of a novel protein-coding gene through peptide sequence data
In our analysis, we found several high quality MS/MS spectra that did not match any protein or predicted transcripts. These could arise from novel gene that were not predicted, or be due to a modified peptide. Figure 3B shows one such MS/MS spectrum that corresponds to the peptide sequence AAAYCADPSLLFAR (Table 2- Novel protein-coding gene). When the data was searched against the An. gambiae genome database, we found in addition to that peptide, another one that aligned perfectly to a region in the genome which had no known or predicted genes (Figure 5). Both of these peptide matches are unique in the An. gambiae genome. From these data, we conclude that we have identified a novel protein-coding gene although more detailed studies will be required to ascertain the exact structure of this gene.
Figure 5 A screenshot depicting identification of a novel gene. Two peptide sequences AAAYCADPSLLFAR and MVVDGTFLR were mapped onto the forward strand of chromosome 2R, whose scaffold coordinates are 3012881–3012922 (JHU_431) and 3012836–3012862 (JHU_432), respectively. There are no known or novel transcripts where these two peptides matched as shown
Correction of transcripts using peptide sequence data
Because of the limitations of gene prediction programs, exons can be missed entirely or their boundaries might be annotated incorrectly. This implies that errors in exon identification can also lead to erroneous assignments of coding exons as non-coding exons (untranslated regions) [13,14]. Figure 3C shows an MS/MS spectrum of a peptide corresponding to the sequence, FPGLCNASEEPR (Table 2- protein [ENSANG:P00000021028]/ [ENSANG:P00000023200]), which matched an annotated 3' UTR of Antigen 5-related 1 protein whose cDNA was described recently [12]. Figure 6A shows that the peptide FPGLCNASEEPR (JHU_0096) and two other peptides (JHU_0097 and JHU_0098) are located just downstream of the stop codon in one of the known transcripts. A closer examination of the Ensembl transcript in which this region was annotated as a UTR ([ENSANG:T00000021028]) revealed that it was erroneously marked as a UTR as there was no stop codon where the annotated coding region apparently ended. This explains why we were able to find the above-mentioned peptides. Further, we found that there was another transcript deposited in GenBank in which this region was annotated correctly as coding region (GenBank accession # Y17702). This other GenBank transcript was not seen in the genome browser view as it was not present in the Ensembl database. We hope that studies such as ours will help identify such errors and facilitate their correction in various databases. Figure 6B shows several peptides (Table 2- protein [ENSANG:P00000018280]) that matched an annotated intron in a novel transcript encoding a novel member of the D7 protein family. This is likely due to a false negative prediction for an exon in this genomic region.
Figure 6 A screenshot depicting the correction of annotation using peptide sequence data. Panel A – Ensembl known gene [ENSANG:G00000018539] has two transcripts. Three peptides (JHU_0096, JHU_0097 and JHU_0098) align to the untranslated regions of both transcripts in this region. Panel B – Peptides are mapped onto the intronic regions of the Ensembl novel transcript [ENSANG:T00000018280]
Use of mass spectrometry data for annotation of translational start sites
Proteins generally undergo proteolytic cleavage of their N-termini by aminopeptidases, in vivo, which results in removal of one or more amino acids from the N-termini [15]. In most cases, this is followed by addition of an acetyl moiety to the amino terminus of the processed protein often referred to as a 'blocked' N-terminus, which cannot be sequenced easily by the traditional Edman method. In this study, we found two acetylated peptides that matched two different predicted transcripts. The peptide sequence Ac-ADQLTEEQIAEFKEAFSLFDKDGDGTITTK, whose MS/MS spectrum is shown in Figure 3D (Ac refers to the acetyl moiety), corresponds to a novel transcript (Table 2- protein [ENSANG:P00000012700]). The predicted protein is orthologous to rabbit calmodulin (93% identity), which has been observed to contain a blocked amino terminus [16]. We found another N-terminally acetylated peptide sequence, Ac-STVDKEELVQK, which corresponds to another novel transcript ([ENSANG:P00000009311]) predicted to encode a protein very similar (96% identity) to a chaperone found in D. melanogaster. In both of the above-mentioned cases, the amino terminal methionine residue was cleaved and the newly exposed amino terminus was acetylated. Thus, we were able to validate the assignment of the translation initiation codons for these two predicted transcripts which is not always straightforward as it has been shown that, contrary to popular beliefs, the most 5' AUG codon is not used for translation initiation in a large proportion of cases [17,18]. We should also note that our strategy was not designed to enrich for N-termini of proteins. If such strategies were to be used in conjunction with mass spectrometry, a large number of N-termini could be assigned in a single experiment.
Identification of sequence polymorphisms
In An. gambiae genome, 444,963 single nucleotide polymorphisms have been reported [1]. In this study, we identified six sequence polymorphisms. We carried out a search of the mass spectrometry-derived data against Ensembl Anopheles gambiae protein database using the X! Tandem algorithm. We found four cSNPs in four different proteins ([ENSANG:P00000029569], [ENSANG:P000000000593], [ENSANG:P00000012716], [ENSANG:P00000025580]) as shown in Table 1. All identified SNPs are novel and are consistent with being single nucleotide substitutions. Manual inspection of the mass spectra corresponding to the SNPs was carried out and through the clear presence of y ion series (as shown in Figure 7), we attributed the mass difference to amino acid change and eliminated the possibility of post-translational and other modifications. From our manual data analysis, two more SNPs were identified on a peptide derived from the Trio protein (Table 1). The sequence of one of the tryptic peptides that occurs in this protein obtained from translation of the genomic sequence is SLNPASPTGSLGGK. However, we found another peptide, SQNPASPAGSLGGKDVVSK (Figure 7E), which contained two sequence polymorphisms relative to the genomic sequence (the amino acids that are different are in bold). This longer peptide is similar to the former one but contains a tryptic miscleavage and matches Trio protein variant found in NCBI nr database (GenBank Accession # AAL68795) but not in Ensembl. Both of these amino acid substitutions (leucine to glutamine and threonine to alanine), could be explained on the basis of a single base pair changes and hence are likely to be due to cSNPs. Interestingly, Ensembl annotations catalog eight SNPs that occur in Trio protein including three non-synonymous and five synonymous SNPs. These do not include any SNPs that could explain the polymorphisms that we have observed. Thus, it seems that we have identified polymorphisms that arise from cSNPs not yet been obtained by other methods. Thus, mass spectrometry-derived data can be used to complement genomic methods for cSNP identification as well.
Figure 7 MS/MS spectra of five different peptides that identify coding SNPs. (A) The amino acid change Ala→ Ser is shown in the peptide SFASDGTDVTVR that matches the protein [ENSANG:P00000029569]. (B) The sequence of the peptide CNAEAEKVHTSSK that matches the D7-related 3 protein precursor ([ENSANG:P00000025580]) shows the amino acid change Asp→His. (C) The peptide VPYDTKYDTVEGDYPLVVK corresponding to the protein putative 5'-nucleotidase precursor ([ENSANG:P00000012716]) presents the amino acid change Ile→Val. (D) The amino acid change Y→S is identified in the peptide LLPAEYGDGVSVPR that corresponds to the protein peroxidase precursor ([ENSANG:P00000000593]). (E) Two changes L→ Q and T→A occur in the same peptide SQNPASPAGSLGGKDVVSK that corresponds to the TRIO protein ([ENSANG:P00000017522]). The amino acid changes representing the cSNPs for the five proteins are shown in rectangle.
Conclusion
One of the basic components of the annotation of any genome is an accurate representation of protein-coding genes. However, even this seemingly simple task is quite difficult. Here, we demonstrate that mass spectrometry is a powerful tool for annotating protein-coding regions in genomes. Here we report a pilot study to annotate the An. gambiae genome. Using mass spectrometry-derived data, we validated the physical existence of 23 known and 50 predicted transcripts at the protein level and confirmed the N-termini of proteins encoded by two predicted transcripts based on N-terminal acetylation. We also identified two sequence polymorphisms based on peptide evidence that were not annotated as SNPs in the databases. Thus, mass spectrometry is a valuable complementary method for initial discovery of locations of non-synonymous SNPs. Importantly; we also identified a novel gene that was not predicted by automatic annotation pipelines at all. The task of assigning translational start sites is fraught with errors especially in the absence of transcript data. Similarly, UTRs can also be wrongly assigned. Using MS/MS data, we corrected the translational start sites and UTR assignment of proteins, which would otherwise be difficult, or impossible using molecular biology based methods. Our MS/MS derived peptide sequence data has been uploaded onto Ensembl DAS server and can be visualized using the Ensembl genome browser. In summary, we have demonstrated how mass spectrometry-derived data can be used to refine the annotations of a complex eukaryotic genome and share them with the biomedical community.
Methods
Mass spectrometric analysis
Salivary glands from female An. gambiae (G-3 strain) were homogenized and subjected to digestion with trypsin as described previously [19]. The tryptic peptides were subjected to LC-MS/MS and analyzed on a quadrupole time of flight mass spectrometer (QTOF US-API, Micromass, UK) as described [19]. A total of 16 LC-MS/MS runs were carried out and the 3,967 MS/MS spectra acquired were searched against both protein and genome databases, this led to identification of 369 unique peptide sequences. The acquisition and deconvolution of data were performed on a MassLynx Windows NT PC data system (version 4.0).
Data analysis
The An. gambiae genome and proteome database (release 16.2) was downloaded from the Ensembl ftp site . Mass spectrometric data searches were performed using Mascot version 1.9 installed on a Linux cluster [20] against the NCBI non-redundant (nr) database as described earlier [21] The following settings were used: a) trypsin as the specific enzyme (al1ow up to 2 missed cleavages); b) peptide window tolerance (error window on experimental peptide mass values) ± 0.4 Da; and c) fragment mass tolerance of ± 0.3 Da. Moreover, during the searches, N-terminal acetylation, oxidation of methionine and carbamidomethylcysteine modification were the three amino acid modifications allowed. Searches were also carried out against the genome database. For this purpose, the large genome sequence files in FASTA format were trimmed into 100 kb long sequences since Mascot cannot deal with large genomic sequences. Same set of parameters were used for genome search as used for NCBInr search. Only peptides with a Mascot score greater than 30 and containing a sequence tag of at least four consecutive amino acids were considered in this study. The spectra were further investigated and verified by manual interpretation. Any peptide hits that matched transcripts labeled known or novel were investigated further using Ensembl browser. This included validation of existing exons, correction of intron-exon boundaries and mapping of N-termini of mature proteins. All peptide matches to the genome were compared with matches to the non-redundant protein database. Those peptides that did not match any entry in the nr database were analyzed further. This allowed identification of novel genes that are not predicted by gene prediction algorithms or correction of regions annotated as introns or untranslated regions.
In our study, if a peptide maps to more than one transcript, we have assigned such peptides to all of the corresponding transcripts. However, for the purpose of counting the number of transcripts/proteins that we have identified, we have included only the assignments of those peptides that had matches to only one transcript.
In order to identify the presence of potential cSNPs, we utilized the "point mutations" feature in X! Tandem, which allows the user to identify single amino acid changes in peptides. We searched the data against Ensembl Anopheles protein database using the X! Tandem 2 release search algorithm installed on a Linux cluster [22]. The searching parameters were the same as described above for search using Mascot.
Use of Distributed Annotation System (Das)
The Distributed Annotation System provided by Ensembl was used to visualize the peptides in the context of genome and to share our annotations with the community. A genome database search was carried out using the peptides to determine the corresponding regions in the genome for each peptide. The positions of these peptides were obtained by implementing scripts written in Python programming language. These scripts parse the output files obtained by searching genome using TBLASTN algorithm. The genomic coordinates were packaged into a tab delimited format necessary for uploading onto the DAS server at Ensembl. Whenever a user chooses a 'MS data JHU' as a DAS server source, the peptide and its coordinates on the genome are mapped onto the browser and visualized on a separate track.
List of abbreviations
An. gambiae: Anopheles gambiae
NCBI: National Center for Biotechnology Information
EBI: European Bioinformatics Institute
Mbp: Mega base pair
DAS: Distributed Annotation System
UTRs: Annotated untranslated regions
cSNPs: Coding single nucleotide polymorphisms
LC-MS/MS: Liquid chromatography nanoelectrospray tandem mass spectrometry
MS/MS: tandem mass spectrometry
JHU: Johns Hopkins University
Authors' contributions
DK carried out the mass spectrometric analysis, database search and interpretation of the mass spectrometry-data and drafted the manuscript. SP and RR carried out the genome data analysis for inclusion in the Distributed Annotation System (DAS). RR carried out additional genome data analysis to identify potential cSNPs. JZ assisted in the mass spectrometry data analysis. BO dissected the salivary glands from female Anopheles gambiae. NK supervised the mosquito experiments and helped with design of the study and the manuscript. AP conceived the study, coordinated and assisted in drafting the manuscript. All authors read, endorsed and approved the final version of the manuscript.
Supplementary Material
Additional File 1
A list of peptides used to validate annotated exons in known transcripts. A list of peptide sequences obtained by tandem mass spectrometry that were used to validate the presence of protein-coding exons in known transcripts in the Ensembl database.
Click here for file
Additional File 2
A list of peptides used to validate novel transcripts. A list of peptide sequences obtained by tandem mass spectrometry that were used to validate protein-coding exons in novel transcripts in the Ensembl database.
Click here for file
Acknowledgements
AP and NK were supported by a pilot project grant from the Malaria Research Institute at the Johns Hopkins Bloomberg School of Public Health. We thank John Kloss and Jakob Bunkenborg for their help with database searching. We thank Martin Hammond, Ensembl mosquito genome project's coordinator, and Ewan Birney at the EBI for their help with making the peptide data available through the Ensembl DAS server. We also thank members of the Institute of Bioinformatics for their assistance with the genome analysis. We thank Sun Microsystems for providing us a computer cluster under the Academic Equipment Grant mechanism.
==== Refs
Holt RA Subramanian GM Halpern A Sutton GG Charlab R Nusskern DR Wincker P Clark AG Ribeiro JM Wides R Salzberg SL Loftus B Yandell M Majoros WH Rusch DB Lai Z Kraft CL Abril JF Anthouard V Arensburger P Atkinson PW Baden H de Berardinis V Baldwin D Benes V Biedler J Blass C Bolanos R Boscus D Barnstead M Cai S Center A Chaturverdi K Christophides GK Chrystal MA Clamp M Cravchik A Curwen V Dana A Delcher A Dew I Evans CA Flanigan M Grundschober-Freimoser A Friedli L Gu Z Guan P Guigo R Hillenmeyer ME Hladun SL Hogan JR Hong YS Hoover J Jaillon O Ke Z Kodira C Kokoza E Koutsos A Letunic I Levitsky A Liang Y Lin JJ Lobo NF Lopez JR Malek JA McIntosh TC Meister S Miller J Mobarry C Mongin E Murphy SD O'Brochta DA Pfannkoch C Qi R Regier MA Remington K Shao H Sharakhova MV Sitter CD Shetty J Smith TJ Strong R Sun J Thomasova D Ton LQ Topalis P Tu Z Unger MF Walenz B Wang A Wang J Wang M Wang X Woodford KJ Wortman JR Wu M Yao A Zdobnov EM Zhang H Zhao Q Zhao S Zhu SC Zhimulev I Coluzzi M della Torre A Roth CW Louis C Kalush F Mural RJ Myers EW Adams MD Smith HO Broder S Gardner MJ Fraser CM Birney E Bork P Brey PT Venter JC Weissenbach J Kafatos FC Collins FH Hoffman SL The genome sequence of the malaria mosquito Anopheles gambiae Science 2002 298 129 149 12364791 10.1126/science.1076181
Mongin E Louis C Holt RA Birney E Collins FH The Anopheles gambiae genome: an update Trends Parasitol 2004 20 49 52 14747013 10.1016/j.pt.2003.11.003
Mann M Pandey A Use of mass spectrometry-derived data to annotate nucleotide and protein sequence databases Trends Biochem Sci 2001 26 54 61 11165518 10.1016/S0968-0004(00)01726-6
Jaffe JD Berg HC Church GM Proteogenomic mapping as a complementary method to perform genome annotation Proteomics 2004 4 59 77 14730672 10.1002/pmic.200300511
Shevchenko A Jensen ON Podtelejnikov AV Sagliocco F Wilm M Vorm O Mortensen P Boucherie H Mann M Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels Proc Natl Acad Sci U S A 1996 93 14440 14445 8962070 10.1073/pnas.93.25.14440
Kuster B Mortensen P Andersen JS Mann M Mass spectrometry allows direct identification of proteins in large genomes Proteomics 2001 1 641 650 11678034 10.1002/1615-9861(200104)1:5<641::AID-PROT641>3.3.CO;2-I
Desiere F Deutsch EW Nesvizhskii AI Mallick P King NL Eng JK Aderem A Boyle R Brunner E Donohoe S Fausto N Hafen E Hood L Katze MG Kennedy KA Kregenow F Lee H Lin B Martin D Ranish JA Rawlings DJ Samelson LE Shiio Y Watts JD Wollscheid B Wright ME Yan W Yang L Yi EC Zhang H Aebersold R Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry Genome Biol 2005 6 R9 15642101 10.1186/gb-2004-6-1-r9
Dowell RD Jokerst RM Day A Eddy SR Stein L The distributed annotation system BMC Bioinformatics 2001 2 7 11667947 10.1186/1471-2105-2-7
Balakirev ES Ayala FJ Pseudogenes: are they "junk" or functional DNA? Annu Rev Genet 2003 37 123 151 14616058 10.1146/annurev.genet.37.040103.103949
Mighell AJ Smith NR Robinson PA Markham AF Vertebrate pseudogenes FEBS Lett 2000 468 109 114 10692568 10.1016/S0014-5793(00)01199-6
Arca B Lombardo F Lanfrancotti A Spanos L Veneri M Louis C Coluzzi M A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae Insect Mol Biol 2002 11 47 55 11841502 10.1046/j.0962-1075.2001.00309.x
Francischetti IM Valenzuela JG Pham VM Garfield MK Ribeiro JM Toward a catalog for the transcripts and proteins (sialome) from the salivary gland of the malaria vector Anopheles gambiae J Exp Biol 2002 205 2429 2451 12124367
Birney E Clamp M Hubbard T Databases and tools for browsing genomes Annu Rev Genomics Hum Genet 2002 3 293 310 12194990 10.1146/annurev.genom.3.030502.101529
Zhang MQ Computational prediction of eukaryotic protein-coding genes Nat Rev Genet 2002 3 698 709 12209144 10.1038/nrg890
Polevoda B Sherman F Nalpha -terminal acetylation of eukaryotic proteins J Biol Chem 2000 275 36479 36482 11013267 10.1074/jbc.R000023200
Grand RJ Shenolikar S Cohen P The amino acid sequence of the delta subunit (calmodulin) of rabbit skeletal muscle phosphorylase kinase Eur J Biochem 1981 113 359 367 7202416 10.1111/j.1432-1033.1981.tb05074.x
Peri S Pandey A A reassessment of the translation initiation codon in vertebrates Trends Genet 2001 17 685 687 11718907 10.1016/S0168-9525(01)02493-3
Suzuki Y Ishihara D Sasaki M Nakagawa H Hata H Tsunoda T Watanabe M Komatsu T Ota T Isogai T Suyama A Sugano S Statistical analysis of the 5' untranslated region of human mRNA using "Oligo-Capped" cDNA libraries Genomics 2000 64 286 297 10756096 10.1006/geno.2000.6076
Ibarrola N Kalume DE Gronborg M Iwahori A Pandey A A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture Anal Chem 2003 75 6043 6049 14615979 10.1021/ac034931f
Perkins DN Pappin DJ Creasy DM Cottrell JS Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 1999 20 3551 3567 10612281 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2
Kristiansen TZ Bunkenborg J Gronborg M Molina H Thuluvath PJ Argani P Goggins MG Maitra A Pandey A A proteomic analysis of human bile Mol Cell Proteomics 2004 3 715 728 15084671 10.1074/mcp.M400015-MCP200
Craig R Beavis RC TANDEM: matching proteins with tandem mass spectra Bioinformatics 2004 20 1466 1467 14976030 10.1093/bioinformatics/bth092
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1311617429610.1186/1471-2164-6-131Research ArticleEffect of dataset selection on the topological interpretation of protein interaction networks Hakes Luke [email protected] David L [email protected] Stephen G [email protected] Faculty of Life Sciences, The University of Manchester, Manchester, UK2005 20 9 2005 6 131 131 12 7 2005 20 9 2005 Copyright © 2005 Hakes et al; licensee BioMed Central Ltd.2005Hakes et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Studies of the yeast protein interaction network have revealed distinct correlations between the connectivity of individual proteins within the network and the average connectivity of their neighbours. Although a number of biological mechanisms have been proposed to account for these findings, the significance and influence of the specific datasets included in these studies has not been appreciated adequately.
Results
We show how the use of different interaction data sets, such as those resulting from high-throughput or small-scale studies, and different modelling methodologies for the derivation pair-wise protein interactions, can dramatically change the topology of these networks. Furthermore, we show that some of the previously reported features identified in these networks may simply be the result of experimental or methodological errors and biases.
Conclusion
When performing network-based studies, it is essential to define what is meant by the term "interaction" and this must be taken into account when interpreting the topologies of the networks generated. Consideration must be given to the type of data included and appropriate controls that take into account the idiosyncrasies of the data must be selected
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Background
In recent years, there has been an unprecedented growth in both the volume and the type of experimental data available to researchers interested in elucidating the biological networks that underpin the functions of living cells. To date, the majority of available eukaryotic data comes from the yeast Saccharomyces cerevisiae, where a variety of different networks have been subject to investigation, including gene regulatory [1], metabolic [2-4] and protein interaction networks [5]. As the majority of cellular processes are mediated by protein-protein interactions, much attention has been focused on their study in the hope that their investigation on a "global" scale will help us to understand how a dynamically interconnected system manages to perform multiple functionally related tasks while maintaining stability against deleterious perturbations.
The recent deluge of protein interaction data generated from large-scale high-throughput systematic screens [6-9] has presented us with an opportunity to create networks consisting of thousands of interacting proteins. Analysis of the resulting networks has shown that, in common with other naturally occurring and artificial networks, protein-interaction networks display a scale-free topology [10,11] and exhibit "small-world" properties [12]. The scale-free property of these networks is thought to be of particular biological significance as it confers robustness to random node loss, allowing the network to maintain its overall integrity even when a significant number of nodes are removed [13]. The concept of network-mediated robustness appears to be reinforced by the presence of a correlation between the connectivity of neighbouring nodes within the network (a feature not observed in random networks) [14]. In the yeast protein-interaction network, the observed negative correlation between the connectivity of a protein and the average connectivity of its binding partners has been seen as a possible adaptation which allows the network to be resilient to the propagation of deleterious perturbations [14]. Recently, Pereira-Leal and co-workers showed that this correlation is valid only for the yeast protein-interaction network as a whole, and that the network formed by the proteins essential for yeast growth has its own unique topological properties, including a very high degree of connectivity (97% of the proteins form a single distinct sub-network), which they postulate may have some implications for our understanding of the network's evolution [15].
Protein interaction networks are generally described using a graph theoretical approach, in which proteins within the graph (nodes) are connected by undirected links (edges) if they are found to interact. While creating a representation of the network is relatively straight forward, deciding what should be represented is often more difficult. Typically, networks are generated using interactions derived from a plurality of different experimental types, which may include protein interactions identified in both individual small-scale studies and larger systematic genome-scale screens – such as those from yeast two-hybrid (Y2H) and affinity-purification experiments. More often than not, less thought than appropriate is given to how the interactions derived from these different systems have been, or should be, combined and the possible implications that different methodologies for achieving this might have on the outcome of analyses.
The issue of data handling is of particular importance in the study of protein interactions derived from purified protein complexes. For any given purified complex that results from a FLAG or TAP tag-based experiment, it is very unlikely that every "prey" protein identified within the complex interacts directly with the "bait" protein. Other proteins or molecules (such as RNA) present within the mixture may act as scaffolds or bridges between the protein constituents. Consequently, we are unable to determine the true topology of the complex. In order to integrate this type of data with those from other experimental sources, we must first derive a set of hypothetical pair-wise protein interactions using either a "spoke", or "matrix" model [16]. The spoke model assumes that the bait protein physically interacts with each of the prey proteins in the complex but does not acknowledge any type of association between the preys. In contrast, the matrix model assumes that any two proteins within the "complex" are connected.
Here, we investigate the effect that the choice of datasets, and modelling methodology (matrix or spoke), has on the topological properties of the yeast protein interaction network and discuss our results with respect to the notion of a negative correlation between nodes within the network (in some studies, this is referred to as an "anticorrelation"). We go on to investigate the notion of a highly connected essential sub-network and, finally, we discuss the nature of the term "interaction" and how the interpretation of that term might affect research within the field.
Results
Data choice
The topology of the protein interaction network created using data derived from the yeast-subset of the DIP database (15,129 protein interactions involving 4,738 unique proteins) [17] reveals that the nodes within the network obey a power-law degree distribution as previously described (data not shown) [10]. Analysis of node connectivities also reveals the previously reported negative correlation between the connectivity of a central reference node (k0) and the mean connectivity of its neighbouring nodes (<k1>). The previously reported difference between the topologies of the global network and the network of essential yeast proteins is also evident, with the essential network displaying a markedly weaker negative correlation than the global network. For k0 ≤ 60, the global network has a correlation coefficient, rk0:k1 = -0.83 and slope, αk0:k1 = -0.25; for the network of essential proteins, rk0:k1 = -0.39, αk0:k1 = -0.08 between log (<k1>) and log (k0) (Figure 1) [14,15].
Figure 1 DIP-based protein interaction network using the spoke model. Mean connectivity of neighbouring nodes (<k1>) as a function of the connectivity of the central node (k0) displayed on a log.-log. scale for the "global" network (blue) and "essential" sub-network (red), generated from interactions extracted from the yeast subset of the DIP database. Note the difference in topology between the two networks.
The yeast subset of the DIP database consists of interactions derived from a range of different studies that employed a variety of different experimental methods. It is possible that biases within one or more of these individual datasets are having a measurable effect on the outcome of the topological analysis. By performing analyses on portions of the data extracted from the database, we were able to begin to identify some of these biases. Figure 2 shows the topology of the network created from protein interactions identified in protein interaction studies (totalling 3,191 protein interactions involving 1,623 proteins) that may be characterised as "small-scale", defined as an experiment described in a published article listing no more than 100 protein-protein interactions [18]. Again, we observed the previously identified power-law degree distribution for nodes within the network (data not shown). However, in this case, the differences between the topologies of the global and essential gene networks are no longer evident, with both having similar slopes (αk0:k1 -0.10 global v -0.09 essential) and correlation coefficients (rk0:k1 -0.39 global v -0.34 essential) between log(<k1>) and log(k0). This result suggests that the significant negative correlation previously observed within the global network results mainly from data generated using high-throughput methods.
Figure 2 Protein interaction network generated using the results of "small-scale studies" only. Average value of k1 as a function of k0 displayed on a log.-log. scale for the network of non-essential proteins (red) and essential proteins (blue) generated from a dataset including only protein interactions derived from small-scale studies. Both the global and essential networks display similar topological properties with both only exhibiting a very weak negative correlation.
Variation in modelling methodology
Following the dataset-dependent observations described above, the impact of applying different modelling methodologies to experimental data on protein complexes was assessed. The application of the matrix model to the protein complex data contained within the cohort used in Figure 1, and reanalysis of the resulting network, causes a dramatic shift in the topology of the network. Again, the power-law degree-distribution for nodes within the network is found. However, the previously observed negative correlation between log(<k1>) and log(k0) changes to a positive correlation for both the global and essential sub-networks, with both networks showing similar topological characteristics (Figure 3) (for K0 ≤ 195, global network rk0:k1 = 0.68 αk0:k1 = 0.17, essential sub-network; rk0:k1 = 0.88, αk0:k1 = 0.27).
Figure 3 DIP based protein interaction network using matrix model. Average value of k1 as a function of k0 displayed on a log.-log. scale for the global network (blue) and essential network (red); generated by applying the matrix model to determine pair-wise interactions from protein complex data. For both networks, the previously reported negative correlation reverts to a positive correlation, and the previously observed difference between the topologies of the two networks appears to have been lost.
Randomisation strategy
A second prominent finding of earlier work investigating the yeast protein interaction network is that the essential sub-network is very highly connected, with ≈ 97% of all proteins within it being connected in a single giant component [15]. The significance of this result was previously highlighted using a standard randomisation strategy, in which a number of nodes equivalent to that in the essential network were randomly selected from the global network and the connectivity of the resultant sub-network determined. To assess the validity of this finding, a "biased" randomisation strategy was employed that took into account the connectivity of the proteins within the essential sub-network. By mimicking the degree distribution of nodes within the essential network in the generated random networks, the average number of nodes encompassed within the largest connected component increased from 33% (using a standard randomisation strategy) to 88% over 1000 iterations. Although connectivity levels equal to that of the essential sub-network were not observed, levels as high as 92% connectivity were achieved.
Discussion
In this study, we have shown how the choice of dataset and modelling methodology can profoundly affect the outcome of investigations into the topology of the yeast protein interaction network. We show that, while these variables have little effect on the apparent power-law degree distribution of nodes within the network, they can dramatically alter the correlation between the connectivities of neighbouring nodes. These results raise the question of what data should be included in these studies and, in the case of protein complex data, which of the two proposed modelling methods is the most appropriate for its incorporation? In a recent study, Bader and Hogue [16] showed that pairs identified using the spoke model were more likely to be correct (i.e. in agreement with published literature) than interactions derived using the matrix model. However, Cornell and co-workers [19] showed that there is little difference between the two modelling methods when the annotations of protein pairs found using each model were compared. This indicates that pairs derived using the matrix model are equally as meaningful (in terms of their functional annotation) as those derived using the spoke model, suggesting that either method provides a valid approach to modelling interactions. In fact, if we wish to include the "classical" hand-annotated MIPS complexes within our analyses, the matrix model becomes our only viable option, as it is the only method that allows us to define a set of pair-wise interactions for a protein complex whose topology is completely unknown.
Given our results on the topology of the network, it is hard to believe that changes in the strength and polarity of the correlations observed, stem from some underlying biological process [14,15]. Rather, they are presumably the result of biases introduced either through experimental methodologies or the choice of analysis technique. As an example, data from Y2H experiments show a significant asymmetry between the connectivities of baits and preys (i.e., the average connectivity of baits with at least one interaction is almost double the same quantity measured for preys) [14]. This and other factors such as auto-activation and the presence of "sticky-proteins" [20], if not taken into account during network construction and analysis, could create the false impression of a negative correlation. In a similar way, it is obvious (from the volume of published literature) that yeast essential genes have been subject to a greater degree of investigation than non-essential genes, and are therefore liable to have more documented interacting partners. It is possible that this bias is responsible for the appearance of the topologically distinct "essential" sub-network (first identified by Pereira-Leal and co-workers [15], and shown here in Figure 1) because a more complete and "accurate" dataset is available for analysis. This is supported by the fact that this topology is only evident when multiple datasets (which, if taken individually, often show an apparent negative correlation) are combined (Figure 4). Furthermore, we have shown that, by changing the modelling methodology employed to derive pair-wise protein interactions, it is relatively easy to change the overall topology of the observed networks. Although the application of one model seems to indicate the global and essential networks have distinct topological properties (Figure 1), by using the same data and employing another (apparently equally valid [19]) model, we show that the previously observed negative correlation reverts to a positive correlation for both the global and essential networks, and the topologies (in terms of both the correlation between log(k0) and log (<k1>) and the slope of the resulting graphs) of both also appear similar (Figure 3). The difference between the topologies of the spoke and matrix model networks is probably a consequence of the models themselves. In the spoke model, bait proteins tend to have a higher degree than prey proteins; thus typical interactions within the network are between high-degree and low-degree members. Conversely, in the matrix model, all proteins found within a complex are assigned connections with all others; thus typical interactions tend to be between proteins with similar connectivities. The ease with which the overall topology of the networks is flipped, and the disparity between results given above, further highlights the degree of caution that should be exercised when attempting to draw biologically meaningful conclusions from studies of this type.
Figure 4 Effect of combining data on network topology. Average value of k1 as a function of k0 displayed on a log.-log. scale for the network composed of essential yeast genes. In each case, where the dataset includes information derived from protein complex analysis pair-wise interactions are determined using the spoke model. Green: network resulting from the incorporation of all protein interactions not classified as being obtained in "small-scale" studies. Brown: network resulting from the protein complex study performed by Ho and co-workers [7]. Purple: network resulting from Y2H interactions identified by Ito and co-workers [8]. Note that the relatively strong negative correlation present within the two networks generated using data from individual high-throughput studies (brown and purple on the graph) is significantly reduced when all available high throughput data are combined (green).
In addition to the observations made about the correlations between neighbouring nodes, we have also shown the importance of using the correct control when selecting nodes for randomization studies involving network connectivity. We found that, by simply matching the degree distribution of the nodes within the essential network in that of the randomly selected sample (composed entirely of non-essential genes), we were able to achieve very similar levels of network connectivity. This result suggests that the highly connected nature of the sub-network of essential proteins previously reported by Pereira-Leal and co-workers is primarily a consequence of the high-degree bias of its nodes, rather than a manifestation of some specific evolutionary process.
Conclusion
We conclude that, before embarking on these network-based analyses, we must first be clear as to what we mean when we use the term "interaction". Interactions derived from direct physical studies, such as Y2H experiments, are very different from those found in synthetic genetic screens, which (in turn) are different again from "associations" between the proteins found within protein complexes. However, in several recent studies, many of these different interaction types have been lumped together as though they were equivalent and directly comparable. For instance, both Y2H data and synthetic lethal gene pairs count as 'interactions' in the GRID database [18], although the protein products of the latter rarely interact physically [21].
While graph theoretical analysis approaches have been successfully applied to a number of man-made and naturally occurring networks [22], these networks differ from the biological systems investigated in that every link between pairs of nodes within the network is of the same type and is generally independent of other factors. For example, analysis of the HTML pages that make up the content of the World Wide Web is relatively simple. In this network, both the nature of the relationships (hyperlinks) between nodes (pages) and the nodes themselves are usually homogeneous and well-defined. Therefore, meaningful and representative visualizations and quantifications of the structure of the network and its properties are possible. However, the "biological networks" we construct are not representative of the underlying system. Biological systems essentially comprise protein "machines" [23] and biological function is mediated through associations between proteins, either directly through physical contact, or indirectly within protein complexes, or as part of the same biological pathway. Although it is technically possible to create an abstract representation of these associations; in reality, heterogeneity and the spatial and temporal restrictions imposed upon the links mean that the resulting topology and parameters of the network need not convey biologically meaningful information.
Methods
Network analysis was performed by extracting all machine-readable, yeast-derived protein interactions from the DIP database (release 20050605). Node connectivities and network topology were investigated using custom software written in the Perl programming language. Random networks were generated from a pool of non-essential proteins only. Construction of the random network continued until the appropriate number of proteins had been selected, whose degree distribution within the sample was similar to that actually observed in the essential network. This was done using an algorithm that created a sample of nodes that, at each level of connectivity, matched as closely as possible (data-permitting) the observed node numbers in the essential network. In instances where an exact match was not possible, another node with a degree within the same range of the desired node was selected. The essential sub-network is defined by taking into consideration only interactions between essential genes, as defined by the Saccharomyces Gene Deletion Project [24]. Correlations between variables were determined by computing the Pearson's correlation coefficient, r. We also report the slope, α, of a linear fit to the data.
Authors' contributions
LH carried out all the analyses and wrote all the custom algorithms, with advice and help from DR, and involving discussions between all authors. LH wrote the initial draft of the manuscript, which was revised by DR and SGO (who conceived of the study). All authors read and approved the final manuscript.
Acknowledgements
We thank Dr. Simon Lovell and for insights and discussions. LH is supported by a CASE Studentship from the Biotechnology & Biological Sciences Research Council (BBSRC) and AstraZeneca. Work on protein interactions is supported by grants from the BBSRC to SGO and DR, and from the Beacon initiative of the UK Department of Trade & Industry to SGO.
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Amoutzias GD Robertson DL Oliver SG Bornberg-Bauer E Convergent evolution of gene networks by single-gene duplications in higher eukaryotes EMBO Rep 2004 5 274 279 14968135 10.1038/sj.embor.7400096
Jeong H Tombor B Albert R Oltvai ZN Barabasi AL The large-scale organization of metabolic networks Nature 2000 407 651 654 11034217 10.1038/35036627
Ma H Zeng AP Reconstruction of metabolic networks from genome data and analysis of their global structure for various organisms Bioinformatics 2003 19 270 277 12538249 10.1093/bioinformatics/19.2.270
Wagner A Fell DA The small world inside large metabolic networks Proc R Soc Lond B Biol Sci 2001 268 1803 1810 10.1098/rspb.2001.1711
Wuchty S Evolution and topology in the yeast protein interaction network Genome Res 2004 14 1310 1314 15231746 10.1101/gr.2300204
Gavin AC Bosche M Krause R Grandi P Marzioch M Bauer A Schultz J Rick JM Michon AM Cruciat CM Remor M Hofert C Schelder M Brajenovic M Ruffner H Merino A Klein K Hudak M Dickson D Rudi T Gnau V Bauch A Bastuck S Huhse B Leutwein C Heurtier MA Copley RR Edelmann A Querfurth E Rybin V Drewes G Raida M Bouwmeester T Bork P Seraphin B Kuster B Neubauer G Superti-Furga G Functional organization of the yeast proteome by systematic analysis of protein complexes Nature 2002 415 141 147 11805826 10.1038/415141a
Ho Y Gruhler A Heilbut A Bader GD Moore L Adams SL Millar A Taylor P Bennett K Boutilier K Yang L Wolting C Donaldson I Schandorff S Shewnarane J Vo M Taggart J Goudreault M Muskat B Alfarano C Dewar D Lin Z Michalickova K Willems AR Sassi H Nielsen PA Rasmussen KJ Andersen JR Johansen LE Hansen LH Jespersen H Podtelejnikov A Nielsen E Crawford J Poulsen V Sorensen BD Matthiesen J Hendrickson RC Gleeson F Pawson T Moran MF Durocher D Mann M Hogue CW Figeys D Tyers M Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry Nature 2002 415 180 183 11805837 10.1038/415180a
Ito T Chiba T Ozawa R Yoshida M Hattori M Sakaki Y A comprehensive two-hybrid analysis to explore the yeast protein interactome Proc Natl Acad Sci U S A 2001 98 4569 4574 11283351 10.1073/pnas.061034498
Uetz P Giot L Cagney G Mansfield TA Judson RS Knight JR Lockshon D Narayan V Srinivasan M Pochart P Qureshi-Emili A Li Y Godwin B Conover D Kalbfleisch T Vijayadamodar G Yang M Johnston M Fields S Rothberg JM A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae Nature 2000 403 623 627 10688190 10.1038/35001009
Jeong H Mason SP Barabasi AL Oltvai ZN Lethality and centrality in protein networks Nature 2001 411 41 42 11333967 10.1038/35075138
Wagner A The yeast protein interaction network evolves rapidly and contains few redundant duplicate genes Mol Biol Evol 2001 18 1283 1292 11420367
Wuchty S Interaction and domain networks of yeast Proteomics 2002 2 1715 1723 12469341 10.1002/1615-9861(200212)2:12<1715::AID-PROT1715>3.0.CO;2-O
Albert R Jeong H Barabasi AL Error and attack tolerance of complex networks Nature 2000 406 378 382 10935628 10.1038/35019019
Maslov S Sneppen K Specificity and stability in topology of protein networks Science 2002 296 910 913 11988575 10.1126/science.1065103
Pereira-Leal JB Audit B Peregrin-Alvarez JM Ouzounis CA An exponential core in the heart of the yeast protein interaction network Mol Biol Evol 2005 22 421 425 15496552 10.1093/molbev/msi024
Bader GD Hogue CW Analyzing yeast protein-protein interaction data obtained from different sources Nat Biotechnol 2002 20 991 997 12355115 10.1038/nbt1002-991
Salwinski L Miller CS Smith AJ Pettit FK Bowie JU Eisenberg D The Database of Interacting Proteins: 2004 update Nucleic Acids Res 2004 32 D449 51 14681454 10.1093/nar/gkh086
Deane CM Salwinski L Xenarios I Eisenberg D Protein interactions: two methods for assessment of the reliability of high throughput observations Mol Cell Proteomics 2002 1 349 356 12118076 10.1074/mcp.M100037-MCP200
Cornell M Paton NW Oliver SG A critical and integrated view of the yeast interactome Comparative and Functional Genomics 2004 382 402 10.1002/cfg.412
Vidalain PO Boxem M Ge H Li S Vidal M Increasing specificity in high-throughput yeast two-hybrid experiments Methods 2004 32 363 370 15003598 10.1016/j.ymeth.2003.10.001
Tong AH Lesage G Bader GD Ding H Xu H Xin X Young J Berriz GF Brost RL Chang M Chen Y Cheng X Chua G Friesen H Goldberg DS Haynes J Humphries C He G Hussein S Ke L Krogan N Li Z Levinson JN Lu H Menard P Munyana C Parsons AB Ryan O Tonikian R Roberts T Sdicu AM Shapiro J Sheikh B Suter B Wong SL Zhang LV Zhu H Burd CG Munro S Sander C Rine J Greenblatt J Peter M Bretscher A Bell G Roth FP Brown GW Andrews B Bussey H Boone C Global mapping of the yeast genetic interaction network Science 2004 303 808 813 14764870 10.1126/science.1091317
Barabasi AL Albert R Emergence of scaling in random networks Science 1999 286 509 512 10521342 10.1126/science.286.5439.509
Spirin V Mirny LA Protein complexes and functional modules in molecular networks Proc Natl Acad Sci U S A 2003 100 12123 12128 14517352 10.1073/pnas.2032324100
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1331617657510.1186/1471-2164-6-133Research ArticleComparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22 Van Poucke Mario [email protected] David [email protected] François [email protected] Marc [email protected] Zeveren Alex [email protected] Patrick [email protected] Luc J [email protected] Department of Animal Genetics and Breeding, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium2 Department of Organic Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281 S4, B-9000 Ghent, Belgium3 Laboratoire de Radiobiologie et d'Etude du Génome, UMR INRA-CEA, F-78352 Jouy en Josas cedex, France2005 21 9 2005 6 133 133 10 8 2005 21 9 2005 Copyright © 2005 Van Poucke et al; licensee BioMed Central Ltd.2005Van Poucke 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 gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable.
Results
A contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping.
Conclusion
The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence.
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Background
Neonatal diarrhoea, often caused by ETEC F4 bacteria, is a common problem in pig production. These bacteria use their fimbriae to adhere to specific receptors on the brush borders of enterocytes of their host. This adhesion is a prerequisite for infection and promotes bacterial colonization of the small intestine. The colonizing bacteria produce enterotoxins that stimulate the secretion of water and electrolytes into the lumen of the small intestine and lead to diarrhoea and often death in neonatal pigs [1]. ETEC F4 resistance, acquired by receptor phenotype differences of the host, seems to be inherited as an autosomal recessive Mendelian trait [2], whereby the gene(s) encoding the ETEC F4ab/ac receptors have been linked to several loci on Sscr13 [3-7]. Based on the tight linkage of the ETEC F4ab/ac receptor loci to microsatellite markers Swr926 (Locus P) and Swc22 (Locus G) by Peelman [5], a BAC contig covering this region and containing TF was built by chromosome walking. The contig was annotated by comparing BAC sequences with sequences from nucleotide databases and by comparative mapping with the human genome sequence in order to provide a basis for the identification of the ETEC F4ab/ac gene(s) by the candidate gene approach.
Results and discussion
Construction of the BAC contig
The construction of the BAC contig was started at 2 microsatellite marker loci, Swr926 and Swc22, estimated to be 1 cM apart from each other and closely linked to the ETEC F4ab/ac receptor loci, according to the porcine genetic map of Peelman [5]. From those 2 loci, 2 subcontigs were built by chromosome walking in both directions until the gap between the 2 was filled. The resulting BAC contig, comprising 33 BAC clones, is shown in Figure 1C All 66 BAC ends were sequenced and submitted to the GenBank database as GSSs [GenBank:CG993013-CG993078]. On 4 occasions, 2 BAC clones turned out to possess the same end (5'-215D7 with 5'-409C1, 129E6-3' with 225H9-3', 5'-613G8 with 5'-1002E2, and 5'-696F10 with 240G11-3'). From 52 of the 62 unique sequences, primers were designed to construct the contig and to screen for new overlapping clones. By dividing the total number of overlaps between the BAC ends and the BAC clones by the total number of BAC ends an estimated contig depth of 3.3 was calculated. Since the average length of the BAC inserts is 135,000 bp, we have covered a region of approximately (33/3.3) × 135,000 = 1.35 Mb.
Figure 1 Comparative map of the annotated BAC contig with its syntenic region on Hsap3q21-q22. The contig is drawn in part C. Black triangles represent BAC end sequences from which primers were designed to construct the contig. The black circle represents the only internal BAC sequence from which primers were designed to construct the contig. White circles show overlaps of these BAC sequences with other BAC clones. White triangles represent BESs from which it was impossible to design primers. The triangles point towards the 3'-side of the BAC clone. Encircled triangles represent BAC ends that are also present in the Sanger framework map. Black diamonds represent microsatellite positions. Black squares represent genes annotated by PCR, whereas white squares represent genes annotated by hybridization. Annotated sequences (genes are in regular, ESTs in italic) from the BAC contig are represented on a plane map (B) and their homology with the human genome sequence is illustrated with dotted lines (A). The orientation of the human genes and finished HTGs (used to assemble the human genome sequence) are represented in (A) by arrows [10]. Repeat sequences (white rectangle = LINE, black rectangle = SINE, black bow = LTR element, white bow = DNA element) are shown on a plane map in D. The RH mapping results are shown in E.
Annotation of the BAC end sequences
A total of 22,552 bp of the BAC contig (62 unique BAC ends and 1 internal BAC fragment [GenBank:CZ692943]) were sequenced and annotated by NIX [8]. The sequences had an overall GC content of 41.48%, which is less than the 46.17% for Sscr7q found in an analogous study of Barbosa and co-workers [9].
The BESs contained 2 gene fragments (MGC3040 and TF) and 2 ESTs (CA778263 and AA461333) located on the human genome (Figure 1A–C) [10]. In 35 of the 62 BESs, homologous sequences could be found within 12 consecutive finished HTGs used to assemble the Hsap3q21-q22 region of the human genome sequence (Figure 1A–C) [10]. These homologies were studied in detail by BLAST 2 sequence comparisons of the BESs with their orthologs (based on the 35.1 latest human genome build). Repeat sequences were excluded (RepeatMasker) and only single hits were taken into account. Orthologous sequences longer than 50 bp had on average a length of 150 bp, a sequence identity of 80% and an e-value of 1e-20. Smaller fragments were only considered as orthologs if at least 2 of them were located close to each other at their expected orthologous position. An extended conservation of synteny between Sscr13 and Hsap3 was already shown by the comparative map of Van Poucke and co-workers [11], based on chromosome painting results of Goureau and co-workers [12]. But taking into account the orientation of the finished HTGs and the position of the orthologous sequences within these HTGs, a perfect comparative map could be established showing even 100% conserved linkage in this region.
Based on this comparative map the BAC contig covers approximately 1.40 Mb of the human genome (from 134.075 Mb to 135.475 Mb on Hsap3) [10], which is close to the BAC contig length calculated above. The BAC sequences also contained 17 LINEs, 10 SINEs, 3 LTR elements and 1 DNA element (Figure 1D), resulting in an average density of 0.77 LINEs/kb, 0.45 SINEs/kb, 0.14 LTR elements/kb and 0.05 DNA elements/kb. Barbosa and co-workers [9] found an average density of 0.35 LINEs/kb, 0.61 SINEs/kb and 0.17 LTR+DNA elements/kb on Sscr7q.
Comparative mapping with the human syntenic region
Based on this detailed comparative map between Hsap3q21-q22 and the BAC contig, the latter could be annotated by comparative mapping. H41, TOPBP1, TF, SRPRB, RAB6B, SLCO2A1 and RYK could be found in the contig by PCR (Figure 1A–C). Also PICA, a gene not yet described in human but located on the pig EST map of the NCBI human genome map viewer [10] between TOPBP1 and TF, was found in the contig by PCR at the orthologous region (Figure 1A–C). It showed sequence homology with the finished HTG sequence AC083905. MGC3040 and BFSP2 could be found in the contig by BAC colony hybridisation (Figure 1A–C). For MGC3040, TF and SLCO2A1 two regions of the gene were annotated in the contig. Their locations showed that those genes were organised in the same orientation as in human. All the comparative mapping results confirmed the conserved linkage (gene order and orientation) based on the sequence homologies of the BESs (Figure 1A–C).
Fifteen BAC ends are also located on the Sanger porcine framework map [13]. On average, they show 98.5% sequence identity, and are located in the same order (Figure 1C). This was expected because (1) the framework map was constructed by fingerprinting and BES alignment on the human sequence, and (2) this region shows 100% conserved linkage with the human genome. So, for this region, the Sanger map assembly, based on the assumption of conservation between both species, is correct. But because of inter- and intrachromosomal rearrangements between the human and the porcine chromosomes [11,12], the Sanger framework map contains some errors. This underlines the importance of the chromosome walking approach for the development of an exact map.
Based on the characteristics of the genes annotated in this contig, SLCO2A1 could be a candidate gene encoding the ETEC F4ab/ac receptor. It is a single copy gene encoding the prostaglandin transporter, a 12-transmembrane organic anion cell surface transporter that is expressed in the small intestine. The presence of different mRNA transcripts suggests that several functionally distinct mRNAs may arise by alternative splicing and/or alternative promotors [14]. It is also assumed that SLCO2A1 contains several different substrate binding sites, to which binding does not always result in substrate translocation across the membrane [15].
RH mapping
During chromosome walking, 4 loci were mapped with the IMpRH panel (data are submitted to the IMpRH server [16]) in order to detect possible chromosome jumping, to estimate the remaining gap between the 2 subcontigs, and to anchor the contig to the integrated comparative map [11]. Using the IMpRH server, 2-point distances were calculated between BAC ends 409C1-3', 5'-613G8, 5'-991F11 and an internal sequence of BAC 8A9, and microsatellite markers Swr926 and Swc22, that were previously mapped on the IMpRH map (Figure 1E) [17]. Based on these distances, the contig covers a region of approximately 40 cR. Thus, 1 cR equals approximately 33.750 kb in our contig. The distance between Swr926 and Swc22 was measured as 17 cR. Because the same distance was measured as 1 cM on a linkage map of Peelman [5], the cR/cM ratio in our contig is 17. Hawken and co-workers [17] measured values for Sscr13 of 59.9 kb/cR and 30.4 cR/cM with the linkage map of Rohrer and co-workers [18].
Methods
Primer design and amplicon verification
All primers, designed with Primer3 [20], were confirmed to not generate an amplicon of the same length with bacterial DNA as a template. Primers used for RH mapping were also checked not to generate an amplicon of the same length with hamster DNA as a template. The construction of the BAC contig was started with primers amplifying porcine microsatellite markers Swr926 [GenBank:AF235467] and Swc22 [GenBank:AF225193]. During the construction of the BAC contig, new primers were designed based on the BESs [GenBank:CG993013-CG993078]. Information on those primers can be found in the corresponding GenBank files. For annotation by comparative mapping with the human genome, primers were designed based on orthologous human and/or porcine sequences. Information on new primers is presented in Table 1. These PCR products were cloned in pCRII (Invitrogen, Merelbeke, Belgium), sequenced for verification with the Thermo Sequenase Primer Cycle Sequencing Kit (Amersham Biosciences, Uppsala, Sweden) and submitted to GenBank [GenBank:AY5182650-AY5182658, DQ104835, DQ104841]. Because of sequence homology, primers for PICA were confirmed to not amplify TF. Primers for RYK were described earlier [11].
Table 1 Information on new primers for genes annotated by PCR
Gene Forward Primer (5'- 3') Annealing temperature
Porcine Acc.No. Reverse Primer (5'- 3') Amplicon size
H41 GGCAAGAGTGAAGCAAATGG 60°C
AY518265 TCAAAAACATAACCCCAGCAA 395 bp
TOPBP1 CCTGAATCTCTTTATCCACATACTT 57°C
AY518266 CATTTGATGGTGCTGACTCTT 318 bp
PICA TGGACGCGAAGCTCTAT 59°C
U36916 TCCGAGTTACAATTCAAGATG 1.286 bp
TF (exon 2) CCAATAAGTGCTCCAGTTTC 56°C
X12386 CCCTGATGGCTTTGATG 111 bp
SRPRB CGCCTTCCATCCCTACCT 58°C
AY518267 AACCGCCCTTTGACTGCT 756 bp
RAB6B CATTGGGATTGACTTCTTGTC 58°C
AY518268 GATGTAGCTGGGGATCAGG 313 bp
SLCO2A1 (exon 3) GCCGTCCTCATCATCTTTGT 60°C
DQ104835 GAAGTGCGGGAGGGTGA 117 bp
SLCO2A1 (exon 9) CCTTGGGGATGCTGTTTG 60°C
DQ104841 TGGAGATGGTGATGATGGTG 96 bp
BAC screening and contig building by chromosome walking
The INRA porcine BAC library was screened by PCR [21]. Approximately 20 μg BAC DNA was purified from a 100 ml culture of the isolated BAC clones by using the Qiagen Plasmid Midi Kit (Westburg, Leusden, The Netherlands). The primers used to isolate the BAC clones were used to amplify the same amplicon on 20 ng BAC DNA for verification. Both ends of the isolated BAC clones were sequenced with 5 μg BAC DNA as template by using the Thermo Sequenase Primer Cycle Sequencing Kit (Amersham Biosciences, Uppsala, Sweden). Primers based on those BESs were used to construct the contig by defining overlaps with all other BAC clones. Primers at both ends of the growing subcontigs were used to screen the BAC library for new overlapping clones until the gap between Swr926 and Swc22 was filled.
Annotation
Annotation of the contig was performed by analyzing all BAC sequences on the NIX server (allowing integration and display of many gene identification programs, such as BLAST against EMBL, EST, STS and GSS databases [8], but not operational anymore), and by comparative mapping using PCR and BAC colony hybridization. These and similar sequence comparisons such as BLAST 2, can also be performed via the NCBI BLAST server [22]. Gene symbols, names and positions were based on the NCBI Gene Entrez [23] and NCBI Map viewer [10] with the latter also used for the identification of the human HTGs.
BAC colony hybridization
For annotation purposes by comparative mapping with the human genome, 2 IMAGE clones (3163990 [GenBank:BC000568] at the MGC3040 locus and 2472940 [GenBank:AI954686] at the BFSP2 locus), located in the human syntenic region, were ordered (MRC geneservice, Cambridge, UK). Inserts of these clones were used as radiolabeled probes for BAC colony hybridization.
RH mapping
During chromosome walking, 4 loci were mapped on the IMpRH panel [24] in order to detect possible chromosome jumping, to estimate the remaining gap between the 2 subcontigs, and to anchor the contig to the integrated comparative map [11]. Swr926 and Swc22 [17] and TF [25] were already located on the IMpRH map.
Conclusion
A porcine BAC contig containing 33 BAC clones and covering approximately 1.35 Mb of Sscr13q31-q32 was constructed. The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage with Hsap3q21-q22, based on 46 anchor points, providing further evidence for conservation of linkage on a fine scale. This underlines the importance of the comparative mapping strategy between human and pig, not only in the search for genes in pig but also as a basis for the assembly of the porcine genome [13,19]. The contig also contains 15 anchor points with the Sanger porcine framework map [13], 4 anchor points (Swr926, Swc22, TF and RYK) with the integrated map of Sscr13 [11] and 2 (Swr926, Swc22) with the porcine Map Viewer [10].
List of abbreviations
BAC bacterial artificial chromosome
BES BAC end sequence
BFSP2 beaded filament structural protein 2, phakinin
bp basepairs
cM centiMorgan
cR centiRay
EMBL European Molecular Biology Laboratory
EST expressed sequence tag
ETEC enterotoxigenic Escherichia coli
GSS genomic survey sequence
H41 hypothetical protein H41
Hsap Homo sapiens
HTGs high throughput genomic sequences
IMpRH INRA-University of Minnesota porcine Radiation Hybrid
kb kilobasepairs
LINE long interspersed nuclear elements
LTR long terminal repeat
Mb megabasepairs
MGC3040 hypothetical protein MGC3040
PICA porcine inhibitor of carbonic anhydrase
RAB6B RAB6B, member RAS oncogene family
RH radiation hybrid
RYK RYK receptor-like tyrosine kinase
SINE short interspersed nuclear elements
SLCO2A1 solute carrier organic anion transporter family, member 2A1
SRPRB signal recognition particle receptor, B subunit
Sscr Sus scrofa
STS Sequence Tag Site
TF transferrin
TOPBP1 topoisomerase (DNA) II binding protein 1
Authors' contributions
MVP coordinated this work, carried out the contig building and the primer design, and drafted this manuscript. DB carried out the contig annotation. FP assisted the BAC screening. MM carried out the sequencing. AVZ and LJP designed the project. PC supervised the BAC screening. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to thank Dominique Vander Donckt and Linda Impe for excellent technical assistance. This work was supported by the Ministry of Trade and Agriculture Brussels (grant No. 5687A) and co-financed by Gentec and Rattlerow Seghers. We also thank Drs. Martine Yerle and Denis Milan (INRA, Castanet-Tolosan, France) for providing the IMpRH panel.
==== Refs
Nagy B Fekete PZ Enterotoxigenic Escherichia coli (ETEC) in farm animals Vet Res 1999 30 259 284 10367358
Gibbons RA Sellwood R Burrows MR Hunter PA Inheritance of resistance to neonatal E. coli diarrhoea in the pig: examination of the genetic system Theor Appl Genet 1977 51 65 70 10.1007/BF00299479
Guerin G Duval-Iflah Y Bonneau M Bertaud M Guillaume P Ollivier L Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs Anim Genet 1993 24 393 396 7904804
Edfors-Lilja I Gustafsson U Duval-Iflah Y Ellergren H Johansson M Juneja RK Marklund L Andersson L The porcine intestinal receptor for Escherichia coli K88ab, K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen Anim Genet 1995 26 237 242 7661395
Peelman LJ Genetic investigation of the resistance mechanisms of the pig against diarrhea caused by E. coli Verh K Acad Geneeskd Belg 1999 61 489 515 10500474
Python P Jorg H Neuenschwander S Hagger C Stricker C Burgi E Bertschinger HU Stranzinger G Vogeli P Fine-mapping of the intestinal receptor locus for enterotoxigenic Escherichia coli F4ac on porcine chromosome 13 Anim Genet 2002 33 441 447 12464019 10.1046/j.1365-2052.2002.00915.x
Jorgensen CB Cirera S Anderson SI Archibald AL Raudsepp T Chowdhary B Edfors-Lilja I Andersson L Fredholm M Linkage and comparative mapping of the locus controlling susceptibility towards E. COLI F4ab/ac diarrhoea in pigs Cytogenet Genome Res 2003 102 157 162 14970696 10.1159/000075742
NIX
Barbosa A Demeure O Urien C Milan D Chardon P Renard C A physical map of large segments of pig chromosome 7q11-q14: comparative analysis with human chromosome 6p21 Mamm Genome 2004 15 982 995 15599557
NCBI Map viewer
Van Poucke M Yerle M Chardon P Jacobs K Genet C Mattheeuws M Van Zeveren A Peelman LJ A refined comparative map between porcine chromosome 13 and human chromosome 3 Cytogenet Genome Res 2003 102 133 138 14970692 10.1159/000075738
Goureau A Yerle M Schmitz A Riquet J Milan D Pinton P Frelat G Gellin J Human and porcine correspondence of chromosome segments using bidirectional chromosome painting Genomics 1996 36 252 262 8812451 10.1006/geno.1996.0460
Porcine Genome Physical Mapping Project
Lu R Kanai N Bao Y Schuster VL Cloning, in vitro expression, and tissue distribution of a human prostaglandin transporter cDNA(hPGT) J Clin Invest 1996 98 1142 1149 8787677
Pucci ML Bao Y Chan B Itoh S Lu R Copeland NG Gilbert DJ Jenkins NA Schuster VL Cloning of mouse prostaglandin transporter PGT cDNA: species-specific substrate affinities Am J Physiol 1999 277 R734 R741 10484490
Milan D Hawken R Cabau C Leroux S Genet C Lahbib Y Tosser G Robic A Hatey F Alexander L Beattie C Schook L Yerle M Gellin J IMpRH server: an RH mapping server available on the Web Bioinformatics 2000 16 558 559 10980153 10.1093/bioinformatics/16.6.558
Hawken RJ Murtaugh J Flickinger GH Yerle M Robic A Milan D Gellin J Beattie CW Schook LB Alexander LJ A first-generation porcine whole-genome radiation hybrid map Mamm Genome 1999 10 824 830 10430669 10.1007/s003359901097
Rohrer GA Alexander LJ Hu Z Smith TP Keele JW Beattie CW A comprehensive map of the porcine genome Genome Res 1996 6 371 391 8743988
Wernersson R Schierup MH Jorgensen FG Gorodkin J Panitz F Staerfeldt HH Christensen OF Mailund T Hornshoj H Klein A Wang J Liu B Hu S Dong W Li W Wong GK Yu J Wang J Bendixen C Fredholm M Brunak S Yang H Bolund L Pigs in sequence space: a 0.66X coverage pig genome survey based on shotgun sequencing BMC Genomics 2005 6 70 15885146 10.1186/1471-2164-6-70
Rozen S Skaletsky H Primer3 on the WWW for general users and for biologist programmers Methods Mol Biol 2000 132 365 386 10547847
Rogel-Gaillard C Bourgeaux N Billault A Vaiman M Chardon P Construction of a swine BAC library: application to the characterization and mapping of porcine type C endoviral elements Cytogenet Cell Genet 1999 85 205 211 10449899 10.1159/000015294
NCBI BLAST
NCBI Entrez Gene
Yerle M Pinton P Robic A Alfonso A Palvadeau Y Delcros C Hawken R Alexander L Beattie LB Milan D Gellin J Construction of a whole genome radiation hybrid panel for high-resolution gene mapping in pigs Cytogenet Cell Genet 1998 82 182 188 9858812 10.1159/000015095
Van Poucke M Yerle M Tuggle C Piumi F Genet C Van Zeveren A Peelman LJ Integration of porcine chromosome 13 maps Cytogenet Cell Genet 2001 93 297 303 11528129 10.1159/000057001
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-721616806110.1186/1471-2334-5-72Research ArticlePopulation-based laboratory surveillance for Giardia sp. and Cryptosporidium sp. infections in a large Canadian health region Laupland Kevin B [email protected] Deirdre L [email protected] Departments of Critical Care Medicine, Medicine and Pathology & Laboratory Medicine, and Community Health Sciences, University of Calgary, Calgary, Alberta, Canada2 Center for Anti-microbial Resistance, Calgary Health Region, Calgary Laboratory Services and the University of Calgary, Calgary, Canada3 c/o Calgary Laboratory Services, 9-3535 Research Rd. N.W., Calgary, Alberta, T2L 2K8, Canada4 Departments of Pathology & Laboratory Medicine and Medicine, University of Calgary, Calgary, Alberta, Canada5 Calgary Laboratory Services, Calgary, Alberta, Canada2005 16 9 2005 5 72 72 13 12 2004 16 9 2005 Copyright © 2005 Laupland and Church; licensee BioMed Central Ltd.2005Laupland and Church; 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
Giardia lamblia (intestinalis) and Cryptosporidium parvum are the two most important intestinal parasites infecting North Americans but there is a paucity of active population-based surveillance data from Canada. This study determined the incidence of and demographic risk factors for developing Giardia sp. and Cryptosporidium sp. infections in a general Canadian population.
Methods
Population-based laboratory surveillance was conducted among all residents of the Calgary Health Region (CHR; population ≅ 1 million) during May 1, 1999 and April 30, 2002.
Results
Giardia sp. infection occurred at a rate of 19.6 per 100,000 populations per year. Although the yearly incidence was stable, a significant seasonal variation was observed with a peak in late summer to early fall. Males were at higher risk for development of this infection as compared to females (21.2 vs. 17.9 per 100,000/yr; relative risk (RR) 1.19; 95% confidence interval (CI), 1.00–1.40, p = 0.047), and there was a significant decrease in risk associated with an increasing age. Cryptosporidium sp. infection occurred at an overall rate of 6.0 per 100,000 populations per year although a large outbreak of Cryptosporidium sp. infections occurred in the second half of the summer of 2001. During August and September of 2001, the incidence of cryptosporidiosis was 55.1 per 100,000 per year as compared to 3.1 per 100,000 per year for the remainder of the surveillance period (p < 0.0001). Cryptosporidiosis was largely a disease of children with an incidence of 17.8 per 100,000 per year occurring among those aged < 20 years of age compared to 1.25 per 100,000 per year for adults ≥ 20 years of age (RR 14.19; 95% CI, 9.77–21.11; p < 0.0001).
Conclusion
This study provides important information on the occurrence and demographic risk groups for acquisition of giardiasis and cryptosporidiosis in a non-selected Canadian population.
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Background
Giardia lamblia (intestinalis) and Cryptosporidium parvum are the two most important intestinal parasites infecting North Americans [1,2]. Infection with either parasite occurs when cysts are ingested via contaminated hands, food and/or water, or through person to person contact [3,4]. These infections are associated with endemic infection rates in many jurisdictions but they have also caused large outbreaks [5,6]. In Canada, several outbreaks have been reported in recent decades due to Cryptosporidium parvum [7,8] and Giardia lamblia [9]. Population-based surveillance studies conducted in the United States have demonstrated increasing rates for giardiasis between 1992–97 with the highest national rates being amongst children aged 0–5 years, followed closely by persons aged 31–40 years [1,10-12]. In the United States in 1997, giardiasis cases per 100,000 state population ranged from 0.98 to 42.3 with a national average of 9.5 cases per 100,000 population [1]. Similar true population-based rates have not been determined for cryptosporidiosis in North America. Crude estimates from the United States based on monitoring water contamination show an expected Cryptosporidium sp. infection rate of between 1–400 cases per 100,000 population [13].
However, because water-borne transmission is one of the major routes of acquiring infection, it must also be recognized that infection rates for either enteric parasite may vary by geographic location. One study in southern Ontario demonstrated an association between giardiasis and rural areas and this has been corroborated by subsequent GIS spatial scan statistics investigation of clusters of giardiasis in this area [14]. Although a previously study had shown significant associations of giardiasis rates with manure application on agricultural land and livestock density this was not born out using spatial statistics scanning methods [15].
There is a lack of active population-based data on the distribution and determinants of Giardia sp. and Cryptosporidium sp. infections in a Canadian population. The objective of this study was to determine the incidence of and demographic risk factors for acquiring these infections among residents of the Calgary Health Region. Such data is important to establish the burden of disease and assess risk factors for acquiring these infections in a defined geographic locale.
Methods
Patients
The Calgary Health Region (CHR) is a well defined, fully integrated, publicly funded health system that provides virtually all medical and surgical care to the residents of the cities of Calgary and Airdrie and several nearby small towns, villages, and hamlets (2001 population 958,610) [16]. All residents of the CHR who had a positive stool specimen for Giardia sp. or Cryptosporidium sp. during the period from May 1, 1999 and April 30, 2002 were included in this study.
Study design
A laboratory-based surveillance cohort design was utilized since all stool samples for parasitological testing are routinely submitted to a single regional microbiology laboratory [i.e., Calgary Laboratory Services (CLS)] [17]. CHR physicians order stool tests based on the patient's presenting symptoms typically of a diarrhoeal illness and the presence of other risk factors (i.e., recent travel, exposure to contaminated food or water, immune status etc.). Because there is universal coverage for healthcare services including laboratory tests in Canada, lack of health insurance is not a barrier to stool parasitological testing. All stool samples submitted from community-based or hospital collection sites in the CHR during the study period were identified by use of the Cerner PathNet Classic version 306 (Kansas City, MO) database at Calgary Laboratory Services (CLS) [18]. Patients were deemed to have giardiasis or cryptosporidiosis if an approved diagnostic test was positive for one or both of these infections according to the laboratory procedures outlined below. Once these patients were identified basic demographic information on age and gender was obtained at CLS and data were entered manually into an Excel spreadsheet (Microsoft Corp.).
Laboratory procedures
All stool parasitological tests were performed by Calgary Laboratory Services (CLS) a large integrated publicly funded medical laboratory company that provides microbiology services from hospitalized and ambulatory patients 24 hours a day, 7 days a week through a centralized facility located in the community. Physicians can order either a Giardia/Cryptosporidium screen or a full stool ova and parasite (O & P) examination on the CLS requisition. Stool parasitological testing proceeded as outlined below depending on the physician's test request. Only a G/C EIA screen was done unless a clinical history was provided on the requisition. All stool specimens that had a stool O & P procedure were initially screened by the G/C EIA procedure.
a) Giardia/Cryptosporidium assay
A rapid commercially available immunoassay is initially performed when a G/C screen is ordered. A stained slide was also read from all stool samples submitted from children ≤ 14 years of age) in order to screen for the presence of other parasites including Dientamoeba fragilis. Giardia (GSA 65) and Cryptosporidium (CSA) are specific antigens produced by the parasites as they multiply within the host intestinal tract. The ProSpecT® Giardia/Cryptosporidium Microplate (Remel Inc., Lenexa, KS, USA) assay (G/C EIA) is a solid phase immunoassay for the simultaneous detection of both antigens (GSA 65 and CSA). The G/C EIA was performed according to the manufacturer's instructions on unconcentrated formalin-fixed specimens. G/C EIA tests were considered positive if the optical density was 0.050 ≥ at 450 nm. The Meriflor® G/C direct fluorescent antibody (DFA) test (Meridian Bioscience Inc., Cincinnati, OH) was used to confirm the presence of Giardia and/or Cryptosporidium infection. One drop (~10 μl) of the concentrated sediment was thinly spread onto each well of the treated slide and fluorescence light microscopy with a 20X objective was used to read the entire well.
b) Stool ova & parasite (stool O & P) method
Stool O & P testing (i.e. stained slide and concentrate) were only done on samples where the physician had ordered the test and provided one or more clinical indications for the request as follows: 1) recent travel outside North America or Travel Clinic patient, 2) recent immigration, 3) immune-compromised or specific HIV/AIDS, cancer, transplantation clinic locations, 4) request for worms, tapeworms or Ascaris sp., 5) bloody or bloody stool specimen, 6) Gastroenterologist's patient, and 7) other (Microbiologist-on-call is consulted). Repeated standard wash and centrifugation procedures are used to prepare stool sediment that is stained with Iron Hematoxylin/Kinyoun stain. The entire 20 × 50 mm cover-slipped stained smear is scanned with the 10X objective to look for larger parasitic elements (i.e. ova). The stained slide is then examined for 10 min. per slide using the 50X oil immersion lens and confirmation of any parasitic elements is done under the 100X oil immersion lens. A fecal concentrate is also prepared using a standard ethyl acetate concentration method after preparation of the stained smears. A different technologist reads the concentrate of the same specimen using both the 10X and 40X dry objectives.
Analysis
Analyses were performed using Stata version 8.0 (Stata Corp., College Station, TX). Differences in proportions in categorical data were compared using Fisher's exact test. Medians with interquartile ranges (IQR) were used to report non-normally distributed continuous variables and were compared using the Wilcoxon Rank-sum test. Incidence rates were calculated using denominator data from the Alberta Health Registry [19]. Only the first sample positive in a given study year was included in the analysis. Category specific risks were calculated and reported as relative risks (RR) with 95% confidence intervals (CI) as previously described [20]. Patients were classified as CHR residents if they had Alberta Health Care numbers and samples were collected at a CHR site or if they were inpatients in a CHR hospital.
Results
Giardiasis
A total of 562 episodes of Giardia sp. infection were identified among 552 patients for an overall incidence of 19.6 per 100,000 populations per year. There was no difference in the yearly incidence of Giardia sp. infection during the three years of the study (p > 0.2). A significant seasonal variation was observed with a peak in late summer to early fall (Figure 1). The incidence rate in the second quarter of the study (August to October; 31.9 per 100,000/year) was two times higher (relative risk (RR), 2.06; 95 % confidence interval (CI), 1.74–2.45) than in the other three quarters (November to July, 15.5 per 100,000/year; p < 0.0001).
Figure 1 Seasonal distribution of Giardia sp. infections in the Calgary Health Region.
A significant association between age and gender and development of giardiasis was observed (Figure 2). Overall males were at slightly higher risk for development of this infection as compared to females (21.2 vs. 17.9 per 100,000/yr; RR 1.19; 95% CI, 1.00–1.40, p = 0.047). There was a significant decrease in risk associated with a increasing age with children and adolescents less than 20 years of age at highest risk (24.6 per 100,000 per year) followed by adults aged 20 to 64 (19.4 per 100,000 per year), and senior adults aged 65 years and older (6.2 per 100,000 per year; p < 0.01 for each pairwise comparison).
Figure 2 Age and gender specific incidence rates of Giardia sp. infection in the Calgary Health Region.
Cryptosporidiosis
A total of 173 patients were diagnosed with Cryptosporidium sp. infection for an overall incidence of 6.0 per 100,000 populations per year. No patients had repeat episodes of this infection. There was a dramatic difference in the temporal occurrence of Cryptosporidium sp. infection during the three years of the study with a large outbreak occurring in the second half of the summer of 2001 (Figure 3). During August and September of 2001, 88 patients were diagnosed with cryptosporidiosis for an incidence of 55.1 per 100,000 per year as compared to 3.1 per 100,000 per year for the remainder of the surveillance period (p < 0.0001).
Figure 3 Occurrence of infections with Cryptosporidium sp. in the Calgary Health Region.
In contrast to giardiasis where adults remained at notably increased risk, cryptosporidiosis was primarily a disease of children and adolescents (Figure 4). A total of 137 (79%) cases were in individuals < 20 years of age for an incidence of 17.8 per 100,000 per year as compared to 1.25 per 100,000 per year for adults ≥ 20 years of age (RR 14.19; 95% CI, 9.77–21.11; p < 0.0001). However, these findings are influenced by the fact that significant differences in age existed among outbreak related (August and September 2001) as compared to non-outbreak (other study surveillance periods) related patients with Cryptosporidium sp. infections. The median (IQR) age of outbreak related patients were significantly lower than that of non-outbreak patients (7.5 years; IQR; 3.5–11 years vs. 10 years; IQR, 4–23 years, p = 0.02). Ninety-two (81/88) percent of the outbreak patients were < 20 years of age as compared to 66% (56/85) of non-outbreak patients (p < 0.0001).
Figure 4 Age and gender specific incidence rates of Cryptosporidium sp. infection in the Calgary Health Region.
Males were at slightly higher risk overall for development of cryptosporidiosis as compared to females (6.9 vs. 5.1 per 100,000/yr; RR 1.35; 95% CI, 0.99–1.84, p = 0.053) which was similar to the trend found for giardiasis. No significant association was observed between gender and the presence of the outbreak.
Discussion
This is the first study that has investigated the occurrence and demographic risk factors for acquiring Giardia sp. and Cryptosporidium sp. infections using a population-based methodology in Canada. Population-based methodologies are widely viewed as optimal designs for establishing the burden of disease because selection bias is minimized since all patients in a defined geographic area are included in surveillance [21-23]. The American Centers for Disease Control (CDC) that is recognized as a leader in infectious disease epidemiology utilizes a very similar methodology for their Active Bacterial Core Surveillance program .
If adequate denominator data is available then incidence rates may be calculated from population-based datasets. The rates we determined in this study should be viewed as the minimum incidence because multiple stool sampling is not a standard practice in our region, and many patients may have had these diseases without having a stool sample submitted for parasitological testing. In addition, certain groups that seek medical attention less frequently may be underrepresented using a population-based laboratory surveillance approach (i.e., shut-in elderly, intravenous drug users, those with mental illness etc.), but these limitations would also be a reality of other approaches. However, even considering these limitations, Giardia and Cryptosporidium result in a significant disease burden in Calgary. We found that infections with Giardia sp. and/or Cryptosporidium sp. were common in our region.
Epidemiological analysis of regional stool parasitological testing data over a two-year period demonstrated that these two parasites were the most commonly identified pathogenic enteric protozoa in the Calgary Health Region, which is similar to the rates previously reported in the United States [1,15,24]. Population-based studies in the United States have indicated rates of Giardia sp. infections ranging from 0.98 to 42.3 cases per 100,000 per year with an average of 9.5 cases per 100,000 per year [1]. However, true population based rates for Cryptosporidum sp. based on laboratory surveillance data have been difficult to determine in the United States because most diagnostic laboratories do not routinely perform stool parasitological procedures that would allow detection of this parasite. A recent survey indicates that most laboratories only analyze stool parasitological samples for the presence of Cryptosporidium sp., Cyclspora sp. and Microsporidium sp. on specific request of the physician [25]. Based on laboratory surveillance data from clinical microbiology laboratories, there were a total of 443 cases of cryptosporidiosis reported to the CDC in the United States in 2003 [26] which is much lower than that expected from population-based estimates of infection rates. Estimates for the Cryptosporidium sp. infection rates for the general population (i.e., 1 – 400 cases per 100,000) in the United States have been based on US-wide water monitoring data [27]. The population-based rate of giardiasis and cryptosporidiosis cases in Calgary is similar to the national average rate per 100,000 reported from the United States. However, the rate of Cryptosporidium sp. infections found in our region is well above the proposed acceptable annual risk level of 1 case of infection per 10,000 [13]. Substantially more Cryptosporidium sp. infections were found in our region during the study period. To our knowledge studies conducted to date from Canada have also not been adequately designed to report incidence rates to allow comparison. The results of our study demonstrate that Giardia sp. and Cryptosporidium sp. infections are common with the former occurring at a rate of more than 3 times more frequently.
We observed a number of similar and contrasting features among infections due to Giardia and Cryptosporidium sp. Giardia sp. infections occurred more commonly in late summer in our region and may be related to water exposure and the increased use of backcountry recreational areas. On the other hand, there was no apparent yearly seasonal occurrence of sporadic (i.e. non-outbreak related) Cryptosporidium sp. infections in our region (Figure 3). Although few studies of the seasonal incidence of intestinal parasites have been done, prior reports from specific geographic locations in the United States suggest that Cryptosporidium sp. infection is most prevalent in the spring, while no seasonality was found in Giardia sp. infections [28]. Infection with either parasite occurs when cysts are ingested via contaminated hands, food, and/or water or through person-to-person contact. Individual cases as well as outbreaks have therefore been associated with improper food handling, exposure to contaminated water (i.e. municipal sources including swimming pools, surface and groundwater including those found in beaver ponds and springs), travel to less developed countries or close contact with a case (i.e. families, day care centers) [12,29-33]. Specific risk factors that have recently been associated with sporadic cryptosporidiosis among immunocompetent persons in the United States include international travel, contact with cattle, contact with persons >2 to 11 years of age with diarrhea and freshwater swimming [34]. Temporal investigation of fecal shedding of Cryptosporidium parvum oocysts in cow-calf herds has also shown that most fecal shedding of this parasites by infected cattle is limited to calves 1 to 4 months old so that the risk of parasitic watershed contamination is greatest in the spring during calving season [35]. Enteric parasitic infection with either Giardia sp. or Cryptosporidium sp. may also be transmitted through sexual contact and immunocompromised persons (i.e. acquired immunodeficiency syndrome) are particularly at risk of developing severe persistent infection [36]. Differences in the seasonality of intestinal parasite prevalence between disparate geographic locations may therefore be dependent on a variety of factors including the composition of the population and their coming into contact with various contaminated water sources (i.e. urban versus rural areas).
Another contrasting feature among these infections was that the age distribution was different. Giardia sp. infections, although most commonly infecting children, occurred across a broad age range (Figure 2). In contrast, Cryptosporidum sp. infections occurred primarily in young children. The main commonality amongst these two infections was that they both tended to more commonly affect males as compared to females [11,37]. Although this study did not investigate the risk factors associated with disease acquisition in various age groups, the demographics of infection is similar to prior studies [11,13,34,37]. Young children tend to have a higher rate of infection with Giardia sp. and/or Cryptosporidium sp. because of their exposure to contaminated municipal and rural water sources such as well water and swimming pools and contact with cases in communal living situations including day care centers. Epidemiological investigation into the risk factors for enteric infection due to one or both of these parasites is the focus of future studies in our region.
Our observation that an outbreak of Cryptosporidium sp. occurred in the third year of the study demonstrates the importance of their detection by conducting population-based studies over extended periods (years). We have previously raised the theoretical potential for bias in population-based studies designed to assess the burden and risk factors for disease if they are conducted in the setting of an outbreak [38]. If this study had only been conducted over a one-year period in 2001 we would have made erroneous inferences on the occurrence and demographic risk factors for endemic disease as evidenced by the dramatically different rate in that year and that outbreak patients were younger. In our opinion it is important that "baseline" or endemic disease be differentiated from outbreak cases in the description of the epidemiology of an infectious disease. We urge authors of other studies to plot cases over time prior to detect outbreaks prior to description.
Although our study is novel in Canada and provides important information there are some important limitations that merit discussion. First, this was a laboratory based study and as a result we did not have detailed information on patient clinical variables, treatments, and outcome. Such information may be of interest and use to clinicians. Second, we did not have reliable other data on exposures such as food and water sources, travel, and family exposures. These variables are clearly important in these diseases and have been the focus of other important investigations in Canadian populations [2,12,29,32]. Finally, we did not have actual addresses for all patients and used a definition of CHR residency status that may have resulted in at least some non-CHR residents being classified as residents in our study. As a result we likely overestimated the incidence of these infections and may have made biased assessments of demographic risk factors to at least some degree [21].
Conclusion
In conclusion, we present the results of active population-based surveillance for Giardia sp. and Cryptosporidium sp. in a large Canadian region. Giardiasis was endemic, and younger individuals and males were at increased risk. Overall, cryptosporidiosis was much less common than giardiasis, although a large outbreak of cryptosporidiosis occurred during surveillance. These data provide important information on the occurrence and determinants of the two most important intestinal parasitoses in Canada.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DLC performed the laboratory surveillance study at Calgary Laboratory Services and KBL performed the data analysis. Both authors wrote the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The Division of Microbiology, Calgary Laboratory Services performed all of the patient laboratory testing.
==== Refs
Furness BW Beach MJ Roberts JM Giardiasis surveillance – United States, 1992–1997 MMWR CDC Surveill Summ 2000 49 1 13 10955980
Croll NA Gyorkos TW Parasitic disease in humans: the extent in Canada Can Med Assoc J 1979 120 310 2 427669
Odoi A Martin SW Michel P Holt J Middleton D Wilson J Determinants of the geographical distribution of endemic giardiasis in Ontario, Canada: a spatial modelling approach Epidemiol Infect 2004 132 967 76 15473161 10.1017/S0950268804002481
Welch TP Risk of giardiasis from consumption of wilderness water in North America: a systematic review of epidemiologic data Int J Infect Dis 2000 4 100 3 10737847 10.1016/S1201-9712(00)90102-4
Yoder JS Blackburn BG Craun GF Hill V Levy DA Chen N Lee SH Calderon RL Beach MJ Surveillance for waterborne-disease outbreaks associated with recreational water – United States, 2001–2002 MMWR Surveill Summ 2004 53 1 22 15499306
Hayes EB Matte TD O'Brien TR McKinley TW Logsdon GS Rose JB Ungar BL Word DM Pinsky PF Cummings ML Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply N Engl J Med 1989 320 1372 6 2716783
Louie K Gustafson L Fyfe M Gill I MacDougall L Tom L Wong Q Isaac-Renton J An outbreak of Cryptosporidium parvum in a Surrey pool with detection in pool water sampling Can Commun Dis Rep 2004 30 61 6 15109091
Stirling R Aramini J Ellis A Lim G Meyers R Fleury M Werler D Waterborne cryptosporidiosis outbreak, North Battleford, Saskatchewan, spring 2001 Can Commun Dis Rep 2001 27 185 92 11729455
Wallis PM Matson D Jones M Jamieson J Application of monitoring data for Giardia and Cryptosporidium to boil water advisories Risk Anal 2001 21 1077 85 11824683 10.1111/0272-4332.216176
Dietz VJ Roberts JM National surveillance for infection with Cryptosporidium parvum, 1995–1998: what have we learned? Public Health Rep 2000 115 358 63 11059430 10.1093/phr/115.4.358
Addiss DG Davis JP Roberts JM Mast EE Epidemiology of giardiasis in Wisconsin: increasing incidence of reported cases and unexplained seasonal trends Am J Trop Med Hyg 1992 47 13 19 1636878
Isaac-Renton JL Philion JJ Factors associated with acquiring giardiasis in British Columbia residents Can J Public Health 1992 83 155 8 1617559
Makri A Modarres R Parkin R Cryptosporidiosis susceptibility and risk: a case study Risk Anal 2004 24 209 20 15028013 10.1111/j.0272-4332.2004.00424.x
Odoi A Martin SW Michel P Middleton D Holt J Wilson J Investigation of clusters of giardiasis using GIS and a spatial scan statistic Int J Health Geogr 2004 3 11 15176979 10.1186/1476-072X-3-11
Odoi A Martin SW Michel P Holt J Middleton D Wilson J Geographical and temporal distribution of human giardiasis in Ontario, Canada Int J Health Geogr 2003 2 5 12946275 10.1186/1476-072X-2-5
Calgary Health Region Website Accessed October 28, 2004
Pitout JD Hanson ND Church DL Laupland KB Population-based laboratory surveillance for Escherichia coli-producing extended-spectrum beta-lactamases: importance of community isolates with blaCTX-M genes Clin Infect Dis 2004 38 1736 41 15227620 10.1086/421094
Church D Hall P Centralization of a regional clinical microbiology service: The Calgary experience Can J Infect Dis 1999 10 393 402
Calgary Health Region Website Accessed October 28, 2004
Laupland KB Davies HD Low DE Schwartz B Green K McGeer A Invasive group A streptococcal disease in children and association with varicella-zoster virus infection. Ontario Group A Streptococcal Study Group Pediatrics 2000 105 E60 10799624 10.1542/peds.105.5.e60
Laupland KB Population-based epidemiology of intensive care: critical importance of ascertainment of residency status Crit Care 2004 8 R431 R436 15566588 10.1186/cc2947
Davies HD McGeer A Schwartz B Green K Cann D Simor AE Invasive group A streptococcal infections in Ontario, Canada. Ontario Group A Streptococcal Study Group N Engl J Med 1996 335 547 54 8684408 10.1056/NEJM199608223350803
Laupland KB Gregson DB Zygun DA Doig CJ Mortis G Church DL Severe bloodstream infections: a population-based assessment Crit Care Med 2004 32 992 7 15071391 10.1097/01.CCM.0000119424.31648.1E
Crompton DW Savioli L Intestinal parasitic infections and urbanization Bull World Health Organ 1993 71 1 7 8440028
Jones JL Lopez A Wahlquist SP Nadle J Wilson M Survey of clinical laboratory practices for parasitic diseases Clin Infect Dis 2004 38 S198 202 15095190 10.1086/381587
Centers for Disease Control (CDC) Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food – Selected sites, United States, 2003 MMWR 2004 53 338 343 15123986
U.S. Environmental Protection Agency. Office of Water Human Health Criteria Document for Cryptosporidium (Publication No. EPA-822-K-94-001) 2001 Washington, D.C.:U.S. Government Printing Office
Amin OM Seasonal prevalence of intestinal parasites in the United States during 2000 Am J Trop Med Hyg 2002 66 799 803 12224595
Isaac-Renton J Blatherwick J Bowie WR Fyfe M Khan M Li A King A McLean N Medd L Moorehead W Ong CS Robertson W Epidemic and endemic seroprevalence of antibodies to Cryptosporidium and Giardia in residents of three communities with different drinking water supplies Am J Trop Med Hyg 1999 60 578 83 10348231
Gradus M Cryptosporidium and public health: From watershed to water glass Clin Microbiol News 2000 22 25 32 10.1016/S0196-4399(00)87495-4
Okhuysen PC Traveler's diarrhea due to intestinal protozoa Clin Infect Dis 2001 33 110 4 11389503 10.1086/320894
Keystone JS Krajden S Warren MR Person-to-person transmission of Giardia lamblia in day-care nurseries Can Med Assoc J 1978 119 241 2 247-8 679128
Thielman NM Guerrant RL Persistent diarrhea in the returned traveler Infect Dis Clin North Am 1998 12 489 501 9658255 10.1016/S0891-5520(05)70015-5
Roy SL DeLong SM Stenzel SA Shiferaw B Roberts JM Khalakdina A Marcus R Segler SD Shah DD Thomas S Vugia DJ Zansky SM Dietz V Beach MJ Risk factors for sporadic cryptosporidiosis among immunocompetent persons in the United States from 1999 to 2001 J Clin Microbiol 2004 42 2944 51 15243043 10.1128/JCM.42.7.2944-2951.2004
Atwill ER Johnson E Klingborg DJ Verserat GM Markegard G Jensen WA Pratt DW Delmas RE George HA Forero LC Philips RL Barry SJ McDougald NK Gildsleeve RR Frost WE Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow-calf herds Am J Vet Res 1999 60 420 5 10211683
Griffiths JK Treatment for AIDS-associated cryptosporidiosis J Infect Dis 1998 178 915 6 9728573
Clavel A Olivares JL Fleta J Castillo J Varea M Ramos FJ Arnal AC Quilez J Seasonality of cryptosporidiosis in children Eur J Clin Microbiol Infect Dis 1996 15 77 9 8641309 10.1007/BF01586190
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-771618802610.1186/1471-2334-5-77Research ArticlePrevalence of human papillomavirus cervical infection in an Italian asymptomatic population Centurioni Maria G [email protected] Andrea [email protected] Domenico F [email protected] Gennaro [email protected] Enzo R [email protected] Rodolfo [email protected] Claudio A [email protected] Department of Surgical Therapies, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16100 Genova, Italy2 Department of Epidemiology and Prevention, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16100 Genova, Italy3 Department of Obstetrics and Gynaecology, Ospedale Evangelico Internazionale, Corso Solferino 1, 16100 Genova, Italy2005 27 9 2005 5 77 77 15 7 2004 27 9 2005 Copyright © 2005 Centurioni et al; licensee BioMed Central Ltd.2005Centurioni 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 the last decade many studies have definitely shown that human papillomaviruses (HPVs) are the major cause of cervical carcinogenesis and, in the last few years, HPV testing has been proposed as a new and more powerful tool for cervical cancer screening. This issue is now receiving considerable attention in scientific and non scientific press and HPV testing could be considered the most important change in this field since the introduction of cervical cytology. This paper reports our prevalence data of HPV infection collected in the '90s, while a follow up of these patients is ongoing.
Methods
For this study we used polymerase chain reaction (PCR) to search HPV DNA sequences in cervical cell scrapings obtained from 503 asymptomatic women attending regular cervical cancer screening program in the city of Genova, Italy. All patients were also submitted to a self-administered, standardized, questionnaire regarding their life style and sexual activity. On the basis of the presence of HPV DNA sequences women were separated into two groups: "infected" and "non infected" and a statistical analysis of the factors potentially associated with the infection group membership was carried out.
Results
The infection rate was 15.9% and the most frequent viral type was HPV 16.
Conclusion
Our HPV positivity rate (15.9%) was consistent to that reported by other studies on European populations.
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Background
Although the Pap-test has given a great contribution to early diagnosis of cervical cancer, this is still a very common, worldwide distributed, female neoplasm and a major cause of death in developing countries. Moreover, in developed countries, the treatment of preneoplastic cervical lesions is a considerable public health problem. There is now strong evidence that infection with carcinogenic types of HPV represents a nearly universal event in cervical carcinoma development and sexual transmission is the predominant mode of HPV infection [1-3]. The relative risk of cervical cancer associated with high-risk types of HPV is even higher than the risk of lung cancer associated with smoking. More than 50 HPV types infecting the genital tract have been identified and sequenced and they can be associated with distinct clinical manifestation. Some HPV types, like 6 and 11, are cause of benign condylomas (low risk group) while a wider number of subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) has been proved to be involved in cervical carcinogenesis (high risk group). Several oncogenes have been identified in the oncogenic types and the biologic mechanism of malignant transformation has been increasingly well characterized [4].
HPV testing for cancer-associated HPV DNA is now accepted as a viable and validated option in the management of women with equivocal cytologic findings [5-7] and, in the last few years, there has been an increasing interest in using the HPV testing also in cervical samples from asymptomatic women without cytological abnormalities [8-10]. This strategy seems to allow an early identification of populations at different risk level for this neoplasia because the absence of infection makes the risk of cancer negligible. Large-scale screening studies demonstrated that HPV testing is more sensitive for the detection of high-grade cervical lesions than cytology.
Nevertheless, the lower specificity and the high cost of HPV testing make its use in primary cervical cancer screening still questionable. At the same time very promising trials are ongoing to evaluate the efficacy of prophylactic HPV 16 vaccines [11,12]. These results suggest that the increasing knowledge about the importance of HPV infection is probably leading to new prevention strategies for this disease. Polymerase Chain Reaction (PCR) is a sensitive technique for the detection of very small amounts of HPV's nucleic acids in clinical specimens and it has been used in most epidemiological studies that have evaluated the role of these viruses in cervical cancer causation. In the '90s we performed a study of prevalence of this infection in cervical samples obtained from women attending their annual screening. This paper presents our historical data and, in our knowledge, this is the first report regarding an Italian population.
Methods
This study included 514 women attending the Istituto Nazionale per la Ricerca sul Cancro in Genova (Italy) for their routine annual Pap smear from June 1992 to June 1993. At the time of their visit all the participants were informed of the research and its purpose and gave their informed consent. Then, they were invited to fill in a self administered questionnaire including items about lifetime number of male sexual partners, age at first intercourse, history of pregnancy and sexually transmitted diseases, contraceptives methods, smoking, prior abnormal Pap smears and other variables. Pap smears were all reviewed by the same pathologist according to the Bethesda System 1991 (figure 1) [13], without knowledge of clinical or laboratory data.
Figure 1 Bethesda System 1991.
Sample preparation
Cervical specimens were collected with a cotton swab pre-wet with PBS and suspended in a 50 ml conical tube containing 10 ml PBS. The tube was vortexed to remove all the material from the swab, which was discarded. The tube was then centrifuged at 2000 g for 10 min, the supernatant was removed and discarded, the pellet resuspended in 500 μl PBS, transferred into a 1.5 ml Eppendorf tube and frozen at -80°C until DNA extraction was performed with the following procedure: phenol/chlorophorm:isoamyl alcohol 24:1, precipitation with 0.3 M sodium acetate in 2.5 V/V pure ethanol, centrifugation at 14,000 rpm for 20 min, washing with 70% ethanol, resuspension in double distilled water.
PCR
The consensus primers MY09 and MY11 were used for the amplification in the following 100 μl reaction mixture: 10 μl of 10 × PCR buffer, 0.2 μM of each primer, 200 μM of each dNTP, 2.5 units of Taq polymerase (Perkin Elmer/Cetus), 200 ng of sample DNA, double distilled water. The apmplification was performed in a DNA Thermal Cycler (Perkin Elmer/Cetus Instruments), using the following program set: 95°C for 30 sec., 55°C for 30 sec., 72°C for 1 min. for a total number of 30 cycles plus an additional 9 min. at 72°C. Separate rooms were used for: preparation of DNA template, preparation and storage of reagents, setting up the amplification reaction. All reagents used in PCR were prepared, aliquotted and stored in an area that was free of PCR- amplified products. Amplification of a single copy human gene (β-globin), as control of DNA suitability for amplification, was performed in each reaction tube; we also used He-La cells as positive control and K562 cells as negative control in each reation.
Analysis of the amplification products
10 μl of the amplification mixture were analysed by electrophoresis in 1.5 agarose gel and visualized by UV light after ethidium bromide staining. Dot-blot analysis was then performed using 2 and 5 μl of the PCR mixture after alkaline denaturation treatment (NaOH 1.5 M and NaCl 0.5 M). DNA was transferred onto nylon membranes Hybond N+ (Amersham, UK) using the Dot-blot apparatus (Millipore, USA) under constant vacuum conditions. Replicate membranes were used for Hybridization with each of the type specific probes (MY12, MY13, MY14, WD74 and MY16) and the generic probe mix (GP1 and GP2). For detection we used the ECL chemoilluminescence kit (Amersham, UK).
Statistical analysis
The associations between HPV infection (defined as a positive HPV-PCR) and individual covariates, including age, age at first sexual intercourse, marital status, smoking habits, education, number of lifetime partners and use of oral contraceptives was investigated using univariate and multiple logistic regression (MLR) analyses. The analyses were done with the SPSS Statistical Software (SPSS Inc. Chicago Illinois). Odds ratios point estimates (OR) and their 95% confidence intervals (95% CIs) were computed by MLR procedure for binary data to estimate the association between each covariate levels and HPV infections while adjusting for the effect of other variables retained in the model. Statistical tests were considered to be significant if the p value was 0.05 or less.
Results
Amongst 514 women enrolled in this study, 503 samples were adequate for PCR. The most important patient characteristics are shown in table 1. The mean age was 51 years (SD = 12.3). Our group was heterogeneous for schooling and other socio-economic variables. Three out of 503 women had cytological abnormal findings (SIL) and no one had genital warts on examination at enrollment. As shown in table 2, 80/503 (15.9%) samples were found positive for HPV infection. The distribution of different viral types by age group is shown in table 2. HPV 16 was the most frequently found subtype (46/80, 57.5%). 19/80 (23.7%) samples were positive for more than one type. Patients carrying a SIL (3/503) were all found positive for HPV (2 for type 16; 1 for type 18+33). The results of the univariate and multiple logistic regression analyses are shown in table 3 and 4, respectively. The number of male lifetime partners was the only covariate significantly associated with HPV infection in univariate analysis (p = 0.03). In multiple regression analysis increased ORs were found to be associated with higher levels of education (p trend = 0.03) while none of the other covariates, including the number of male lifetime partners, was associated with HPV infection.
Table 1 Distribution of selected characteristics observed in the 503 patients included in the study.
N° 503
Age Mean (s.d.) 51 (12.3)
Range 20–81
Age at first intercourse Mean (s.d.) 20.8 (6.3)
Range 5*-38
Marital status Single 123 (24.45%)
Married 369 (73.36%)
Unknown 11 (2.19%)
Education High school or University 207 (41.1%)
Middle School 167 (33.2%)
Primary School 120 (23.8%)
Unknown 9 (1.9%)
History of pregnancy 0 90 (17.8%)
1–2 263 (52.2%)
≥ 3 137 (27.2%)
n.a. 13 (2.5%)
N° of lifetime partners 1 294 (58.45%)
2–4 74 (14.71%)
5–9 33 (6.56%)
≥ 10 47 (9.34%)
Unknown 55 (10.93%)
History of sexually transmitted diseases No 479 (95.2%)
Yes 24 (4.8%)
Smoking No 348 (69.2%)
Yes 155 (30.8%)
Oral contraceptives No 337 (67%)
Yes/ex 166 (33%)
History of abnormal Pap-smear 50/503 (9.94%)
SIL in the present Pap smear 3/503 (0.6%)
* sexual abuse
Table 2 Viral type distribution by age group assessed by PCR and dot-blot analyses. The percentage of PCR and dot-blot positive samples (among HPV-PCR +) are shown in parenthesis.
PCR analysis Dot-blot analysis
Age HPV (all) Generic probe 6/11 16 18 33 Others
≤ 44 18/119 (15.1%) 16 (88.9%) 1 (5.6%) 8 (44.4%) 4 (22.2%) 2 (11.1%) 2 (11.1%)
45–52 21/120 (17.5%) 19 (90.5%) 2 (9.5%) 10 (47.6%) 11 (52.4%) 3 (14.3%) 2 (9.5%)
53–59 14/122 (11.5%) 13 (92.9%) 0 11 (78.6%) 4 (28.6%) 0 1 (7.1%)
≤ 60 27/142 (19.0%) 26 (96.3%) 1 (3.7%) 17 (63.0%) 10 (37.0%) 2 (7.4%) 2 (7.4%)
All 80/503 (15.9%) 74 (92.5%) 4 (5.0%) 46 (57.5) 29 (36.3) 7 (8.8%) 7 (8.8%)
Table 3 Findings from the univariate analysis: HPV positive patients by selected covariates
Covariates All Women HPV Positive P value χ2 test
No. No. %
Age ≤ 44 119 18 15.1 0.37
45–52 120 21 17.5
53–59 122 14 11.5
≥ 60 142 27 19.0
Education Primary School 120 12 10.0 0.09
Secondary School 167 27 16.2
Higher 207 40 19.3
Unknown 9 - -
Smoking habits Current + previous 155 26 16.8 0.79
Never smokers 348 54 15.5
N° of lifetime partners 1 293 35 11.9 0.03
> 1 ≤ 5 74 13 17.6
> 5 78 19 24.4
Unknown 58 12 20.0
Age at 1° sexual intercourse ≤ 18 128 22 17.2 0.63
19 – 21 138 24 17.4
22 – 24 109 13 11.9
≥ 24 125 19 15.2
Marital status Single 123 27 21.95 0.06
Married 369 52 14.1
Unknown 11 - -
Use of Oral Contraceptives Current + previous 166 27 16.3 0.88
Never 337 53 15.7
Table 4 Findings from multiple logistic regression: Odds Ratios (OR) point estimates and their 95% Confidence Intervals (95% C.I.)
Covariate No. OR (a) 95% C.I. P (b) P trend(c)
Age
≤ 44 106 1 (ref.) 0.21 0.13
45 – 52 113 1.88 0.80 – 4.41
53 – 59 103 1.20 0.45 – 3.18
≥ 60 110 2.29 0.89 – 5.89
No. of lifetime partners
1 283 1 (ref.) 0.29 0.11
1 – 5 71 1.30 0.61 – 2.78
> 5 78 1.82 0.86 – 3.85
Use of Oral Contraceptives.
No 284 1 (ref.) 0.64
Yes 148 1.16 0.61 – 2.21
Education
Primary 93 1 (ref.) 0.44 0.03
Secondary 153 1.39 0.61 – 3.16
Higher 186 1.72 0.73 – 4.03
Smoking
No 290 1 (ref.) 0.92
Yes 142 0.97 0.54 – 1.74
Age 1° intercourse
≤ 18 94 1 (ref.) 0.74 0.63
19 – 21 124 1.04 0.48 – 2.24
22 – 24 100 0.70 0.28 – 1.70
> 24 114 0.77 0.32 – 1.86
Marital status
Single 101 1 (ref.) 0.31
Married 331 0.71 0.37 – 1.36
The analysis is based on 432 women with complete data; all variables included in the model. (a) (ref.) = reference level; (b) Wald test for the contribution of the covariate to the logistic model; (c) Test for linear trend within individual categorical covariates.
Discussion
In the nineteenth century some investigators noticed that cervical cancer was almost exclusively detected in married women, being rare in the unmarried ones and exceptional in nuns [20,21]. Since then, a large number of studies have investigated the importance of sexual activity as the main risk factor for cervical carcinoma development (increased risk related to early onset and number of lifetime partners), suggesting a role for a sexually transmitted infectious agent. More recently, many epidemiological studies have identified HPV as the main factor in cervical cancer causation [14,22]. At the time of this study we wanted to investigate whether there was any association between genital HPV infection and known risk factors for cervical cancer. The sample we studied was a non selected population attending our hospital from the whole Liguria Region, in Italy. Our HPV positivity rate (15.9%) was low compared to that detected by the first studies conducted using PCR [14,23], but it matches well with prevalence data more recently detected in other European countries [8,15,24,25]. A significant relationship was observed in our population between age and HPV infection. Moreover, in the group aged ≤ 30 (33 women) the HPV prevalence was higher (27.3% vs 15.1%, data not shown) and similar to that reported by other investigators [14-19]. The different HPV frequency between age groups could be due to their different sexual behaviour, although this cannot completely explain the findings of the intermediate age group (>30 y.o., <50 y.o.). In fact the sexual habits of this population, as confirmed by the questionnaires, are similar to those of younger subjects (<30 y.o.), but HPV is detectable in a lower rate (16%), close to the value found for the older group. A better explanation of these results, also reported by other studies [18,26], is that most HPV genital infections are transient because they are cleared by host immune response. In fact serum IgG antibodies develop in response to infection in 55–92% of women [12,27]. These data are very helpful now that the introduction of HPV testing in cervical cancer screening seems to be incoming, and new guidelines are needed. Our data support the evidence that this kind of screening should not start before age 30 (later in comparison to the cytology – based screening), because in young women HPV infection is too frequent [8,19,24].
In our population the "higher education" was the most represented group (205/436 subjects), and this may have affected the observed association with HPV infection, due to a an easier access to health system services.
According to other studies, the most frequent viral type was HPV 16 [15], an important finding when planning new prevention strategies (e.g. prophylactic vaccines). Smoking and use of oral contraceptives, previously reported to be risk factors for cervical carcinoma development, were not related to HPV infection in our data. A major confounding factor of the present investigation is the lack of objective and anamnestic data regarding male partners. Moreover other factors (such as the immune response to the HPV) still need to be better evaluated.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MGC conceived of the study, carried out part of the molecular analysis and drafted the manuscript, AP helped to draft the manuscript, DFM performed the statistical analysis, GP carried out most of the molecular assays, ERC and RS enrolled the patients, CAG participated in the design and coordination of the study and helped to draft the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This research was supported by the Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. The authors would like to thank Dr. Cristina Belpassi for her help in the writing of the manuscript.
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Mc Cance DJ Campion MJ Clakson PK Chesters PM Jenkins D Singer A Prevalence of human papillomavirus type 16 DNA in cervical intraepithelial neoplasia and invasive carcinoma of the cervix Br J Obstet Gynaecol 1985 92 1101 1105 2998438
Franco E Bergeron J Villa L Arella M Richardson L Arseneau J Stanimir G Human Papillomavirus DNA in Invasive Cervical Carcinomas and Its Association with Patient Survival: a Nested Case-Control Study Cancer Epidemiol Biomarkers Prev 1996 5 271 275 8722218
Wolf JK Franco EL Arbeit JM Shroyer KR Wu TC Runowicz CD Tortolero Luna G Herrero R Crum CP Innovation in Understanding the Biology of Cervical Cancer Cancer Suppl 2003 98 2064 2069
Matlashewski G Schneider J Banks L Jones N Murray A Crawford L Human papillomavirus type 16 DNA co-operates with activated ras in transforming primary cells EMBO J 1987 6 1741 1746 3038534
Arbyn M Buntinx F Van Ranst M Paraskevaidis E Martin-Hirsch P Dillner J Virologic Versus Cytologic Triage of Women Eith Equivocal Pap Smears; a Meta-analysis of the Accuracy To Detect High-Grade Intraepithelial Neoplasia J Natl Cancer Inst 2004 96 280 293 14970277
Arnold K Study Results Help Define HPV's Role as Diagnostic Tool J Natl Cancer Inst 2001 93 259 260 11181767 10.1093/jnci/93.4.259
Solomon D Schiffman M Tarone R Comparison of Three Management Strategies for Patients With Atypical Squamous Cells of Undetermined Significance: Baseline Results From a Randomized Trial J Natl Cancer Inst 2001 93 293 299 11181776 10.1093/jnci/93.4.293
Petry KU Menton S Menton M van Loenen-Frosch F de Carvalho Gomes H Holz B Schopp B Garbrecht-Buettner S Davies P Boehmer G van den Akker E Iftner T Inclusion of HPV testing in routine cervical cancer screening for women above 29 years in Germany: results for 8466 patients Br J Cancer 2003 88 1570 1577 12771924 10.1038/sj.bjc.6600918
Wright TC JrSchiffman M Adding a Test for Human Papillomavirus DNA to Cervical-Cancer Screening N Engl J Med 2003 348 489 490 12571255 10.1056/NEJMp020178
Cuzick J Szarewski A Cubie H Hulman G Kitchener H Luesley D McGoogan E Menon U Terry G Edwards R Brooks C Desai M Gie C Ho L Jacobs I Pickles C Sasieni P Management of women who test positive for high-risk types of human papillomavirus:the HART study Lancet 2003 362 1871 1876 14667741 10.1016/S0140-6736(03)14955-0
Koutsky LA Ault KA Wheeler CM Brown DR Barr E Alvarez FB Chiacchierini LM Jansen KU A controlled trial of a human papillomavirus type 16 vaccine N Engl J Med 2002 347 1645 1651 12444178 10.1056/NEJMoa020586
Galloway DA Papillomavirus vaccines in clinical trials Lancet Infectious Diseases 2003 3 469 475 12901889 10.1016/S1473-3099(03)00720-5
The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses (1992) Report of the 1991 Bethesda Workshop Am J Surg Pathol 1992 16 914 916 1415910
Ley C Bauer HM Reingold A Schiffman MH Chambers JC Tashiro CJ Manos MM Determinants of genital human papillomavirus infection in young women J Natl Cancer Inst 1991 83 997 1003 1649312
Jacobs MW Walboomers JM Snijders PJ Voorhorst FJ Verheijen RH Fransen-Daalmeijer N Meijer JL Distribution of 37 mucosotropic HPV types in women with cytologically normal cervical smears: the age-related patterns for high-risk and low-risk types Int J Cancer 2000 87 221 227 10861478 10.1002/1097-0215(20000715)87:2<221::AID-IJC11>3.3.CO;2-U
Cuschieri KS Cubie HA Whitley MW Seagar AL Arends MJ Moore C Gilkisson G McGoogan E Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population J Clin Pathol 2004 57 68 72 14693839 10.1136/jcp.57.1.68
Dalstein V Riethmuller D Prétet J-L Le Bail Carval K Sautière J-L Carbillet J-P Kantelip B Schaal JP Mougin C Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal french cohort study Int J Cancer 2003 106 396 403 12845680 10.1002/ijc.11222
Evander M Edlund K Gustafsson A Jonsson M Karlsson R Rylander E Wadell G Human papillomavirus infection is transient in young women: a population based cohort study J Infect Dis 1995 171 1026 1030 7706782
Peto J Gilham C Deacon J Taylor C Evans C Binns W Haywood M Elanko N Coleman D Yule R Desai M Cervical HPV infection and neoplasia in a large population-based prospective study: the Manchester cohort Br J Cancer 2004 91 942 953 15292939
Rigoni Stern D Fatti statistici relativi alle malattie cancerose Giorn, Prog Patol Terap 1842 2 507 517
Skrabanek P Cervical cancer in nuns and prostitutes: a plea for scientific continence J Clin Epidemiol 1988 41 577 582 3290397 10.1016/0895-4356(88)90062-5
Daling JR Madeleine MM McKnight B Carter JJ Wipf GC Ashley R Schwartz SM Beckmann AM Hagensee ME Mandelson MT Galloway DA The Relationship of Human Papillomavirus-related Cervical Tumors to Cigarette Smoking, Oral Contraceptive Use, and Prior Herpes Simplex Virus Type 2 Infection Cancer Epidemiol Biomarkers Prev 1996 5 541 548 8827359
Bauer HM Ting Y Greer CE Genital human papillomavirus infection in female university students as determined by a PCR-based method JAMA 1991 265 472 477 1845912 10.1001/jama.265.4.472
Baay MF Tjalma WA Weyler J Goovaerts G Buytaert P Van Marck EA Lardon F Vermorken JB Human papillomavirus infection in the female population of Antwerp, Belgium: prevalence in healthy women, women with premalignant lesions and cervical cancer Eur J Gynaecol Oncol 2001 22 204 208 11501772
Forslund O Antonnson A Edlund K van den Brule AJ Hansson B-G Meijer CJ Ryd W Rylander E Strand A Wadell G Dillner J Johansson B Population-Based Type-Specific Prevalence of High-Risk Human Papillomavirus Infection in Middle-Aged Swedish Women J Med Virol 2002 66 535 541 11857534 10.1002/jmv.2178
Kang m Lagakos SW Evaluating the role of human papillomavirus vaccine in cervical cancer prevention Stat Methods Med Res 2004 13 139 55 15068258
Wikstrom A van Doornum GJ Quint WG Schiller JT Dillner J Identification of human papillomavirus seroconversions J Gen Virol 1995 76 529 539 7897345
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BMC ImmunolBMC Immunology1471-2172BioMed Central London 1471-2172-6-231617909110.1186/1471-2172-6-23Research ArticleFollicular dendritic-like cells derived from human monocytes Heinemann Dagmar EH [email protected] J Hinrich [email protected] Max-Planck-Institut für Biophysikalische Chemie, D-37077 Goettingen, Germany2 Abteilung für Immunologie, D-37075 Göttingen, Germany2005 22 9 2005 6 23 23 19 11 2004 22 9 2005 Copyright © 2005 Heinemann and Peters; licensee BioMed Central Ltd.2005Heinemann and Peters; 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
Follicular dendritic cells (FDCs) play a central role in controlling B-cell response maturation, isotype switching and the maintenance of B-cell memory. These functions are based on prolonged preservation of antigen and its presentation in its native form by FDCs. However, when entrapping entire pathogens, FDCs can turn into dangerous long-term reservoirs that may preserve viruses or prions in highly infectious form.
Despite various efforts, the ontogeny of FDCs has remained elusive. They have been proposed to derive either from bone marrow stromal cells, myeloid cells or local mesenchymal precursors. Still, differentiating FDCs from their precursors in vitro may allow addressing many unsolved issues associated with the (patho-) biology of these important antigen-presenting cells. The aim of our study was to demonstrate that FDC-like cells can be deduced from monocytes, and to develop a protocol in order to quantitatively generate them in vitro.
Results
Employing highly purified human monocytes as a starter population, low concentrations of Il-4 (25 U/ml) and GM-CSF (3 U/ml) in combination with Dexamethasone (Dex) (0.5 μM) in serum-free medium trigger the differentiation into FDC-like cells. After transient de-novo membrane expression of alkaline phosphatase (AP), such cells highly up-regulate surface expression of complement receptor I (CD35). Co-expression of CD68 confirms the monocytic origin of both, APpos and CD35pos cells. The common leukocyte antigen CD45 is strongly down-regulated. Successive stimulation with TNF-α up-regulates adhesion molecules ICAM-1 (CD54) and VCAM (CD106). Importantly, both, APpos as well as APneg FDC-like cells, heterotypically cluster with and emperipolese B cells and exhibit the FDC characteristic ability to entrap functionally preserved antigen for prolonged times. Identical characteristics are found in monocytes which were highly expanded in vitro by higher doses of GM-CSF (25 U/ml) in the absence of Dex and Il-4 before employing the above differentiation cocktail.
Conclusion
In this work we provide evidence that FDC-like cells can be derived from monocytes in vitro. Monocyte-derived FDC-like cells quantitatively produced offer a broad utility covering basic research as well as clinical application.
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Background
Until today, the ontogeny of follicular dendritic cells (FDCs) has remained unresolved [1,2]. Even transplantation experiments have led to contradictory results [3-11]. Researchers contemplating a bone marrow origin have suggested that these professional antigen-presenting cells derive either from the lymphoid [5] or the myeloid lineages [10]; from monocytes in particular [12,13]; or even from stromal cells of the bone marrow [6,11,14,15]. Alternatively, FDCs have been claimed to originate within lymphoid organs from local mesenchymal precursors [6,7,11,14,16-18]. So far, however, no protocol is available to generate FDCs in vitro from their putative progenitors [2].
The monocyte is increasingly acknowledged as the cornerstone of a plastic differentiation system. Accordingly, the findings of a monocytic origin of both macrophages [19] and dendritic cells (DCs) [20]; (for review see [21]) have subsequently been complemented by evidence proving that microglia [22] and osteoclasts [23] can be derived from this cell type as well. More recent results have revealed that even osteoblasts [24], neural cells, hepatocytes [25] and endothelial cells [26] can be generated from certain monocyte subsets. Therefore, its remarkable developmental capacity strongly indicates that the monocyte actually represents a somatic stem cell.
Our previous findings demonstrated that the bone/liver/kidney isozyme of Alkaline Phosphatase (AP) is likewise expressed in cells obtained from ex vivo-explanted foreign-body granuloma as well as in monocyte-derived in-vitro granuloma. Co-expression of CD68 assigned such APpos cells to the myeloid lineage. Intriguingly, cultured osteoblasts were found to be phagocytic and co-expressed AP and CD68. Taken together, these results strongly implied osteoblasts and macrophages to derive from a common source [27,28]. AP of the bone/liver/kidney isozyme is also expressed by osteoblasts, activated endothelial cells, stromal reticulum cells [29]) as well as by FDCs [30]. Consequently, we hypothesized that AP expression may constitute a link between monocytes and mesenchymal cells [27]. Indeed, when attempting to identify signals provoking AP expression in monocytes, we found it transiently up-regulated in GM-CSF-induced proliferating monocyte cultures. AP expression was further enhanced and prolonged by IL-4. In the absence of IL-4, AP was down-regulated, while typical osteoclast markers such as tartrate-resistant acid phosphatase and vitronectin receptor, were expressed [27].
Our present study aimed at determining whether FDCs can be derived from the monocytic lineage. As a result, we present evidence that under defined serum-free conditions highly purified human monocytes differentiate into functionally competent FDC-like cells. Low concentrations of Il-4 and GM-CSF in combination with Dexamethasone (Dex) were used to induce AP expression as a striking feature of these cultures accompanied by some of the most important features of FDCs, expression of CD35 [31,32], B cell rosetting and emperipolesis [33], as well as antigen trapping and retaining it for prolonged time [34].
Results
Generation and phenotyping of monocyte-derived primary FDC-like cells
Highly enriched human monocytes highly depleted of lymphocytes and cultured for 12 or 15 days in the presence of IL-4 (25 U/ml), GM-CSF (3 U/ml) and Dex (0,5 μM), tightly adhered to the substratum, exhibited multiple shapes as well as numerous projections and thrived closely adjacent to one another. Typically, these cell variants strongly expressed AP (Fig. 1), with a peak on day 12. These cells were referred to as primary FDC-like cells. For a maximal expression of AP the three inducers had to be present to act synergistically, whereas GM-CSF plus IL-4 in the absence of Dex induced expression of AP to a lesser extent only (Fig. 2a–d). IL-4 could be replaced by IL-13 (100 U/ml) (not demonstrated).
Figure 1 AP expression in FDC-like cells. After 15 days of culture the presence of Il-4 (25 U/ml), GM-CSF (3 U/ml) and Dex (0, 5 μM), monocyte-derived cells were fixed and stained for AP (blue).
Figure 2 Synergistic action of AP-inducing factors. Monocytes were cultured in the presence of a, 25 U/ml Il-4 and 3 U/ml GM-CSF; b, 0, 5 μM Dex; c, 3 U/ml GM-CSF; d, 25 U/ml Il-4, 3 U/ml GM-CSF and 0, 5 μM Dex (full mix). On day 15, the cultures were fixed and stained for expression of AP.
By triple-staining we found APpos and APneg cells expressing membrane-bound CD35 (complement receptor I) (Fig. 3a). Both, APpos as well as CD35pos cells, could further be co-stained for the monocyte/macrophage marker CD68, thus revealing the myeloid origin of the cells (Fig. 3a). For control, AP expression was virtually absent, and CD35 was only occasionally seen in parallel cultures where monocytes had been induced to differentiate into macrophages (Fig. 3b).
Figure 3 APpos cells express the FDC-marker CD35 and the myeloid marker CD68. Monocytes were cultured under conditions described in Fig. 1 for differentiating into FDC-like cells (a) or for macrophages (b) and triple-stained for AP, CD 35 and CD68. a, FDC-like cells co-expressing CD68 (red; filled arrow), AP (blue; open arrow), and CD35 (brown; arrowhead) at different degrees; b, control cells cultured in parallel in the presence of macrophage differentiation conditions stained for AP (negative), CD35 (negative), CD68 (positive, red).
The common leukocyte marker CD45 was strongly down-regulated in FDC-like cells, obviously correlating with AP expression (Fig. 4a) as compared with constitutively CD45pos macrophages (Fig. 4b).
Figure 4 Down-regulation of CD45 in FDC-like cells. Co-staining for AP and CD45 in FDC-like cells or macrophages. a, CD45 (brown) is down-regulated in FDC-like AP-positive (blue) cells compared with b, macrophages as controls where CD45 is fully expressed, and AP is negative.
B-cell rosetting and emperipolesis
When cultured for 14 days or longer, spontaneous homotypic clustering, B cell rosetting and emperipolesis, as well as antigen retaining in its native form (see below) were verifiable as characteristic functional features of FDCs. For these experiments, initially autologous B cells (not shown) and thereafter the Raji B-cell line was employed (cf. Fig. 5a, and 5b for B-cell rosetting and emperipolesis by FDC-like cells).
Figure 5 Heterotypic B-cell rosetting and emperipolesis. Primary FDC-like cells at day 14 were co-cultured with B-cells (Raji) at a ratio of 1:1 for 4 hours, fixed and double-stained for AP and CD22. a, APpos FDC-like cells (blue) with filamentous dendrites capturing CD22+ Raji cells (brown); b, survey of rosettes and various stages of emperipolesis.
Antigen trapping and retention
Antigen retention for a longer period of time may be regarded as the most defining and distinctive function of FDCs. In order to investigate whether cultured monocyte derivatives express this property, we employed horseradish peroxidase (HRPO) as a model antigen which offers the advantage to be easily detectable by a standard HRPO substrate reaction. The enzyme was offered in the form of immune complexes of human Ig/HRPO-anti-human IgG.
Complexes were likewise trapped by monocyte-derived FDC-like cells and macrophages. In FDC-like cells HRPO remained fully enzymatically active up to 16-days tested (Fig. 6a, c). In contrast, control macrophages had almost completely degraded the enzyme at 4 and 16 days, respectively (Fig. 6b, d), already observable after 4 h (not shown). Immune-complex loaded cells strongly co-expressed CD35 as a typical FDC marker (Fig. 6c), whereas control macrophages only marginally expressed CD35. Only minimal numbers of cells revealed HRPO activity (Fig. 6d).
Figure 6 Antigen trapping and long-term retention. FDC-like cells (a, c) or macrophages (b, d), were pulsed for 30 min with HRPO/human IgG complexes. a, b, Cells were fixed after 4 days and double stained by a standard HRPO substrate reaction (brown) and for CD35 (red). a, In FDC-like cells antigen was present in its enzymatic active form, and CD35 was expressed. b, In macrophages only traces of antigen was detectable and CD35 is weakly positive. c, d, Cells were cultured for 16 days. In FDC-like cells (c) enzyme activity is still detectable, whereas in macrophages enzyme activity is absent.
Secondary stimulation
Based on these findings, an even more mature FDC phenotype was differentiable from the APpos phenotype under secondary stimulation. When day12 APpos FDC-like cells were pulsed for a further 3 to 5 days with 10 ng/ml TNF-α, they were induced for increased expression of ICAM-1 (CD54) (Fig. 7a) and VCAM (CD106) was slightly up-regulated (Fig. 7c; see also Table 1). In control macrophage cultures these markers were seen only occasionally (Fig. 7b,d). Both adhesion molecules as well as CD35 are hallmarks of functionally competent FDCs in vivo [15,35]. Supplementing the cultures with TNF-β (LT-α) [36,37] did not reveal similar effects and IFN-γ up-regulated CD54 only, but down-regulated CD35 (not shown). Functional markers such as B-cell rosetting and antigen retention were not further up-regulated by secondary stimulation (not shown).
Figure 7 Up-regulation of adhesion molecules after secondary stimulation. TNF-α added to FDC-like cells on day 13 for further three days expressed ICAM-1 (CD54) (a) and VCAM (CD106) (c) on day 16; b, d, macrophages as controls.
Table 1 Phenotype and function of primary and secondary FDC-like cells as compared with macrophages generated in vitro and with freshly prepared monocytes
Monocytes [day 0] Macrophages [day 15] Primary FDC-like cells [day 15] Secondary FDC-like cells [day 18]
HLA-DR + ++ + ++
CD22 - - ± ±
CD35 (CR1) + + +++ +++
CD45 +++ +++ ± N.D.
CD54 (ICAM-1) + - - ++
CD68 ++ +++ +± +±
CD106 (VCAM-1) - - - +
AP - - +++ +++
Clustering. - - ++ +++
B cell Rosetting - - +++ +++
Emperipolesis - - ++ ++
Antigen Retention - - +++ +++
Markers and functions are expressed as absent (-), faintly or few positive cells (+), positive with different intensities (+, ++, +++), variable with respect to donor variations (±). The term, clustering, refers to the ability to establish homotypic and heterotypic aggregates. N.D. not determined.
Monocyte proliferation
Throughout the entire period of culture, the differentiating monocyte derivatives were tested in serial two day-intervals for 5-bromodeoxyuridine (BrdU) incorporation. The maximum of 1% positive cells was measured within days 6 to 10 (not shown). This result clearly excluded the outgrowth of a certain cell subset(s) while underscoring a direct derivation of APpos cells from monocytes.
As an alternative source, we used monocytes derived from 8- to 10-day cultures in which – as a first step – proliferation had been induced with GM-CSF at 25 U/ml. Cells transferred from this condition, and immediately introduced to FDC-inducing conditions specified above, ceased to proliferate and acquired the complete phenotype of primary FDC-like cells (not shown).
Discussion
In the present study, we demonstrate for the first time the in-vitro generation of FDC-like cells from monocytes as their putative precursors. Human monocytes were prepared by selective adherence combined with reverse sedimentation as a novel procedure in order to eliminate undesired non-adherent cell populations. This protocol allowed for a virtual enrichment of monocytes while avoiding any interference with antibodies posing the potential hazard to evoke adverse cellular trigger events.
Our data clearly demonstrate that peripheral blood monocytes can be induced to differentiatiate into FDC-like cells. Similar to the earlier conundrum on the ontogeny of T cell-tropic dendritic cells (DCs) [21], the search for the origin of FDCs has always been complicated by their unstable marker profile, showing that FDCs isolated from lymphoid organs readily lose typical antigens such as DRC-1, CD21, CD23, and KiM4 in culture [35,38,39]. For practical reasons, we therefore decided to take advantage of an AP isoenzyme that has been described to be expressed by FDCs [30], but not by monocytes, macrophages or monocyte-derived DCs (cf. Tab. 1). Actually, our earlier experiments had revealed that this anchor marker is inducible in monocytes under defined conditions, suggesting AP expression as indicative for the developmental plasticity of these cells [27]. As a logical step, when attempting to generate FDCs in vitro, we first converted monocytes into APpos cells, here referred to as primary FDC-like cells.
Typically, the common leukocyte antigen CD45 was strongly down-regulated in APpos cells as well as in cells with similar morphology suggesting a transdifferentiation into non-leukocytic cells. Absence of CD45 has been described for FDCs by Schriever et al. [40]. The co-expression of CD68, however, indicates the monocytic origin of the APpos cells.
Labelling of CD35 (complement receptor I) is routinely used in histology to discriminate FDCs from other types of dendritic cells as well as from histiocytes [31,32]. In our work CD35 was stably expressed in monocyte-derived FDC-like cells. Again, we found the monocyte/macrophage marker CD68 in CD35pos cells which was also obvious in CD35/AP co-expressing cells. Control macrophages were stained only marginally or negatively for CD35.
When establishing conditions for the generation of APpos cells, sera were found to give variable results. Therefore, we developed a completely serum-free differentiation protocol. Low concentrations of IL-4 and GM-CSF plus Dex were found to act as basic inducers. Within 12 to 15 days of culture, these conditions elicited a cell showing markers and functions of FDCs. Conversely, this combination of factors prevented the differentiation of macrophages or T-cell tropic dendritic cells from monocytes.
IL-4 at concentrations one order of magnitude lower than those established for the differentiation of T-cell tropic DCs had previously been shown to generate large flattened and adherent cells with fine dendritic protrusions. Such cells had shown to express both, AP as well as the myeloid marker CD68 [27]. Our forthcoming experiments revealed that IL-4 can successfully be replaced by IL-13, which will be a subject of further research.
Importantly, corticosteroids such as Dex synergistically enhanced AP expression. Dex, previously employed to induce osteoblasts from bone-marrow derived mesenchymal stem cells [41], has been shown to inhibit macrophage and DC differentiation [42]. When differentiating FDCs from monocytes, we now have found that IL-4 and Dex synergize in inducing the APpos phenotype.
GM-CSF which was applied routinely at low concentrations appeared merely to serve as a survival factor in addition to its contribution to differentiation. Its actual requirement was largely donor-dependent, indicating that developmental properties of starter monocytes may vary according to the donor's immunological status. Moreover, we have experienced that occasionally exogenous GM-CSF can even be omitted without affecting the differentiation towards APpos cells, which might be explained by an autocrine production of GM-CSF by monocytes in culture [43].
After 12–15 days of culture, the majority of monocyte-derived cells displayed the APpos phenotype, while not proliferating throughout this period of time. This high percentage of APpos cells therefore reflects quantitative monocytic differentiation and clearly argues against the possibility that FDC-like cells might have expanded from a small contaminating cell population.
Alternatively, we used monocytes in which – as a first step – proliferation had been induced by adding GM-CSF at higher concentrations. Cells transferred from this condition and introduced to FDC-inducing conditions acquired the APpos phenotype as well. As a consequence, combining sequential proliferation and differentiation might emerge as the method of choice when considering large-scale production of FDC-like cells from monocytes, for example when faced with the need to avail such cells for therapeutic application.
Membrane expression of ICAM-I and VCAM on FDCs has been described as a prerequisite for the interaction of FDCs and B cells within lymphoid tissues. We found a strong up-regulation of ICAM-I after secondary stimulation with TNF-α. However, we already obtained functionally competent FDC-like cells capable of B-cell rosetting and antigen retention which strongly expressed CD35 under the TNF-α-free basic condition.
Development of functional germinal centres as well as the maintenance of FDC function has been claimed to strongly depend on the engagement of TNF family members (TNF-α and LT-α/β) [36,37]. In-vitro generation of FDC-like cells, as described herein, appears to be widely independent of LT-α/β, whereas TNF-α may cause a further maturation of the FDC phenotype. The expression of HLA-DR which is controversial, but positively found by some authors [35,39], was up-regulated by TNF-α as well (Table 1).
Other factors were, however, inhibitory. Lymphocytes had to be eliminated as far as possible – which, especially when activated, inhibited the observed transdifferentiation. Specifically, IFN-γ may be the main factor: when added to monocytes as early as at the onset of culture, it strictly inhibited APpos phenotype, as we have shown before [27]. When added in a secondary step after 12 days of culture, IFN-γ up-regulated expression of CD54, but down-regulated CD35.
Of note, emperipolesis specifying the prolonged engulfment of B cells is a unique FDC property which, within germinal centres, is implicated in the protection of transiently internalised B cells from apoptosis [33]. It is envisioned that this property may be useful for preserving immunogenic or tolerogenic B cells triggered in vitro for their subsequent clinical application.
Identifying these newly differentiated cells as FDC-like cells culminates in the demonstration of storing antigen for long periods of time in its native form. This ability has been described as a unique feature of FDCs [34] but, to our knowledge, neither for other types of dendritic cells nor for macrophages. Specifically, in FDC-like cells the antigen, provided as HRPO-IgG complexes, was retained in an enzymatic active form up to 16 days tested. Enzyme activity was localized perinuclearly. In contrast, macrophages eliminated the material within a few hours. Further studies will reveal the precise kinetics of uptake, sub-cellular distribution, storage and possible re-expression on the FDC surface by ultrastructural investigations.
Monocyte-derived FDC-like cells can now be generated in vitro. Obviously, one pertinent employment of this system is to spur on the further elucidation of the underpinnings of immunological B-cell memory. Furthermore, cultures of FDCs may greatly facilitate research on the mechanisms underlying the capture and functional preservation of antigen. Next, because antigen retention is known to be exploited by pathogenic entities such as the human immunodeficiency virus-1 (HIV-1) [44] as well as prions [45], close investigation of these processes in vitro provides hope for important progress in these areas. For example, Smith et al. nicely demonstrated on isolated human FDCs, and in murine models alike, that FDC-entrapped HIV-1 remains highly infective for prolonged times, which appears to be due to protection of this virus from degradation [46]. Last but not least, this novel protocol might even foreshadow later clinical applications of autologous FDCs or FDC-preserved B cells akin to the clinical studies currently conducted with T cell-tropic DCs. In conclusion, the present findings may open up new horizons for unraveling the secrets of antigen and pathogen storage by FDCs, with self-evident immunological and clinical implications.
Other groups have shown other cell types to transdifferentiate from monocytic precursors. However, all of these studies suffered from the lack of defined conditions (such as by using serum, conditioned media or undefined lymphocyte-derived signals) and/or employed a monocyte subfraction as the starter population. In contrast, we now introduce a common pathway, and define the signals required, for allowing monocytes to quantitatively convert into a state of high developmental plasticity from which FDC-like cells can be deduced. It needs to be stressed that our results do not per se give a definitive clue as to the normal pathway of FDC differentiation. In fact, contemplating the actual existence of such a principal differentiation path may either unravel the main pathway of FDC generation or a salvage route, respectively. Normal FDCs in situ express some myeloid markers such as CD14 and CD11b but not CD68. However, FDC tumors have been described to also express CD68 [47-49]. In any case, the myeloid pathway desribed here obviously complements our previous findings on the ontogeny of T cell-directed DCs in that monocytes [20] and their myeloid precursors [50] can both be induced to quantitatively develop into T cell-tropic DCs, as is now commonly acknowledged.
Interestingly, AP and CD35 expression as well as antigen capture were occasionally observed to spontaneously emerge in single cells within control macrophage cultures. It thus becomes increasingly obvious that monocytes, macrophages, T-cell tropic DCs and FDCs belong to a continuum of a developmentally plastic system of cells which are highly interconvertible. Further data expand this view to osteoblasts, which were recently differentiated from a monocyte subset [24], thus supporting our results on overlapping phenotypes between osteoclasts, osteoblasts, dendritic cells and macrophages [27].
Moreover, as mentioned before, even cell classes generally considered to be developmentally distant from the myeloid lineage can be readily obtained from monocytes when the appropriate signals are provided. Hence, when acknowledging the monocyte as a previously ignored species of somatic stem cell, it appears unlikely that this fascinating cell type has already disclosed its entire potential. It will indeed be exciting to witness which ontogenetic surprises and therapeutic promises the future may hold.
Conclusion
Here we provide evidence that monocytes can be transdifferentiated in vitro into cells that resemble FDCs by several markers and functions. Immunocytochemical co-stainings suggest thatthese cells had a myeloid origin. An attractive feature of our findings isthat theyprecisely describe stimuli enabling a transdifferentiation of monocytes into FDC-like cells at defined serum-free conditions. As a result, FDC-like cells can now be quantitatively produced in vitro for the first time, thereby offering a broad application spectrum ranging from basic research to clinical employment.
Methods
Monocyte isolation and culture
Leukocytes were obtained from leukapheresis of healthy blood donors, in addition with serum supplied by the blood transfusion service, University Hospital, Goettingen. Mononuclear cells were prepared by standard Ficoll-Hypaque (Nycomed, Oslo, Norway) gradient sedimentation, diluted with PBS to a final density of 1.068, resulting in an enrichment of monocytes of about 70%, and washed free of platelets. In some experiments, monocytes were obtained from counterflow elutriation (Elutra, Gambro, Martinsried, Germany). Cells were seeded into microwells at 30.000 monocytes/well, in the presence of 10% of 0.45 μm filtered human pooled serum and allowed to attach for a one hour. In order to obtain highly purified monocytes non-attached cells were separated from monocytes by a hanging-drop technique here referred to as reverse sedimentation: Flat-bottom wells were filled up with culture medium to form a convex meniscus, the microplates were then gently turned upside-down and cultured for 2 h in the inverted position. The medium containing non-attached cells was snicked off, and the adherent cells of about 95% purity (FACS analysis of CD14 positive cells, not demonstrated) were further cultured in 100 μl of differentiation medium: CellGro serum free medium (CellGenix, Freiburg, Germany) supplemented with N-acetyl-L-alanyl-L-glutamine (Biochrom, Berlin, Germany), penicillin/streptomycin (Biochrom), 10-6 M dexamethasone dinatrium phosphate (Ratiopharm, Ulm, Germany), 25 U/ml recombinant human Il-4, 3 U/ml recombinant human GM-CSF (R&D Systems, Wiesbaden, Germany). Lymphocytes, if still present, were additionally washed away on the second day. Once a week, medium plus additives were completely exchanged. During the entire cell preparation and culture care was taken to minimize evaporation and pH shift to alkaline. At day 15 the cultures were terminated or, alternatively, additional inducers (recombinant TNF-α, LT-α/β, IFN-γ (R&D Systems)) were added at day 13 for another three days. For control, macrophages were differentiated in medium consisting of each 50% "Medium 199" and RPMI (Biochrom) supplemented with penicillin/streptomycin and 10% heat-inactivated human serum (pooled from 30 healthy donors).
Immunocytochemistry
For staining procedures cells were fixed in ethanol/acetic acid (95%/5%) for 30 min or alternatively with cold methanol for 5 min and rehydrated by 3× washing in distilled water. Antibodies (from Dako, Glowstrup, Denmark, if not stated otherwise) were diluted in medium plus 10% human sera: anti-CD68 clone EMB11 1:100, anti-human CD35 (FDC) 1:30, anti-human CD54 1:800, anti-human CD106 1:30, anti-human HLA-DR 1:200, anti-human CD45 1:100, anti-human CD22 1:50, AP-anti-human IgM 1:50, goat-AP-anti-mouse IgG 1:30, anti-BrdU, (Amersham, Braunschweig, Germany) undiluted, sheep-PO-anti mouse IgG, (Amersham) 1:300. The primary antibodies were incubated over night at 4°C, the secondary antibodies at RT for 45 min. After each incubation step the preparations were washed with PBS. The PO-staining was developed with H2O2 (1 μl/5 ml) and diaminobenzidine (50 μg/ml) in PBS/distilled water 1:1. The AP-conjugated secondary antibodies were detected with Naphthol/Fast Red, Sigma Deisenhofen, Germany, including levamisole to inhibit edogenous AP activity. Staining controls were done by omitting the primary antibodies. The substrate reactions were observed microscopically and stopped with PBS. The preparations were stored in PBS/glycerine 1:1 at 20°C. For immunodoublestaining the preparation after the first staining procedure was washed extensively with PBS, the next immunostaining was performed using a differently conjugated secondary antibody.
Enzyme cytochemistry for AP
The enzymecytochemical detection of AP was performed using a test kit from Sigma according to the manufactures instructions with the following modification: The cultures were fixed as described for immunocytochemistry. The enzyme reaction was carried out at RT for 20 min.
Enzyme cytochemical/immunocytochemical double or triple staining
Enzymecytochemical staining was performed prior to immunostaining. After enzymestaining the preparation was washed 3× in distilled water, the subsequent immunostaining was performed as described above. For further immunostaining the preparation was washed again extensively before the next immunostaining was performed using a differently conjugated secondary antibody.
Cell proliferation assay
Cell proliferation was determined by incorporation of BrdU for 24 h into the nuclei follwed by immunostaining with PO using a test kit from Amersham according to the manufacturer's instructions.
Immunoglobulin aggregation
In order obtain aggregated immunoglobulines a purified immunoglobulin preparation (Polyglobin, Behringwerke, 10% Ig in physiologic saline) was used at 10% in PBS and incubated for 60 min at 60°C.
B-cell rosetting and emperipolesis
Raji cells (human B cell line) were used as a source of B lymphocytes. For demonstrating rosetting or emperipolesis, the Raji cells were cocultured at 3 × 104 cells/microwell with the monocyte-derived cells. After 4 hours the cultures were fixed with ethanol/acetic acid stained, as above and evaluated microscopically.
Antigen trapping
Horseradish peroxidase (Calbiochem, Bad Soden/Ts, Germany) was employed as a model antigen. The enzyme was offered in the form of immune complexes of human Ig (Behringwerke Marburg, Germany), HRPO-anti-human IgG (goat) plus HRPO-anti-goat IgG for enhancing the reaction. Immune complexes were added at 10% on day 12 to 15 for 30 min, thereafter the medium was changed. After further incubation up to 16 days the cultures were fixed with methanol for 5 min. and stained for peroxidase as described for immunocytochemistry and reacted for about 10–15 min. The reaction was stopped by washing with distilled water.
Authors' contributions
DH has proposed the APpos phenotype to be the link to FDCs. She has established the main protocol for generation of APpos cells from monocytes as well as most of the experiments. JHP has developed the novel described monocyte purification, introduced the use of Il-13 as equivalent to Il-4. He contributed to the theoretical framework.
Acknowledgements
This work was generously supported by a grant of the Gesellschaft für Biologische Krebsabwehr. The DC Society for Dendritic Cell Therapy, Goettingen, kindly supplied support. We are indebted to Anja Bornholdt, Dorothea Ostermeier, Christina Schipper and Andrea Struck for technical help. We cordially thank Robert Gieseler (LTBH Medical Research Institute) for critically reading the manuscript.
==== Refs
Heinen E Bosseloir A Bouzahzah F Follicular dendritic cells: origin and function Curr Top Microbiol Immunol 1995 201 15 47 7587349
van Nierop K de Groot C Human follicular dendritic cells: function, origin and development Semin Immunol 2002 14 251 257 12163300 10.1016/S1044-5323(02)00057-X
Imazeki N Senoo A Fuse Y Is the follicular dendritic cell a primarily stationary cell? Immunology 1992 76 508 510 1526656
Yoshida K van den Berg TK Dijkstra CD The functional state of follicular dendritic cells in severe combined immunodeficient (SCID) mice: role of the lymphocytes Eur J Immunol 1994 24 464 468 8299696
Yoshida K Kaji M Takahashi T van den Berg TK Dijkstra CD Host origin of follicular dendritic cells induced in the spleen of SCID mice after transfer of allogeneic lymphocytes Immunology 1995 84 117 126 7890295
Kapasi ZF Qin D Kerr WG Kosco-Vilbois MH Shultz LD Tew JG Szakal AK Follicular dendritic cell (FDC) precursors in primary lymphoid tissues J Immunol 1998 160 1078 1084 9570519
Yamakawa M Imai Y Dobashi M Kasajima T Development of follicular dendritic cells: a study using short-term bone marrow cell grafting in SCID mice Histol Histopathol 1999 14 135 142 9987658
Schriever F Freeman G Nadler LM Follicular dendritic cells contain a unique gene repertoire demonstrated by single-cell polymerase chain reaction Blood 1991 77 787 791 1825182
Humphrey JH Grennan D Sundaram V The origin of follicular dendritic cells in the mouse and the mechanism of trapping of immune complexes on them Eur J Immunol 1984 14 859 864 6479210
Fliedner A Parwaresch MR Feller AC Induction of antigen expression of follicular dendritic cells in a monoblastic cell line. A contribution to its cellular origin J Pathol 1990 161 71 77 2370601 10.1002/path.1711610112
Krenacs T Rosendaal M Immunohistological detection of gap junctions in human lymphoid tissue: connexin43 in follicular dendritic and lymphoendothelial cells J Histochem Cytochem 1995 43 1125 1137 7560895
Parwaresch MR Radzun HJ Feller AC Peters KP Hansmann ML Peroxidase-positive mononuclear leukocytes as possible precursors of human dendritic reticulum cells J Immunol 1983 131 2719 2725 6358354
Bosseloir A Heinen E Defrance T Bouzhazha F Antoine N Simar LJ Moabs MAS516 and 5B5, two fibroblast markers, recognize human follicular dendritic cells Immunol Lett 1994 42 49 54 7829129 10.1016/0165-2478(94)90034-5
Muretto P Immunohistochemical study of tonsils from newborn infants with emphasis on follicular dendritic reticulum cells Eur J Histochem 1998 42 189 195 9857244
Liu YJ Grouard G de Bouteiller O Banchereau J Follicular dendritic cells and germinal centers Int Rev Cytol 1996 166 139 179 8881775
Lee IY Choe J Human follicular dendritic cells and fibroblasts share the 3C8 antigen Biochem Biophys Res Commun 2003 304 701 707 12727211 10.1016/S0006-291X(03)00649-1
Muller-Hermelink HK von Gaudecker B Drenckhahn D Jaworsky K Feldmann C Fibroblastic and dendritic reticulum cells of lymphoid tissue. Ultrastructural, histochemical, and 3H-thymidine labeling studies J Cancer Res Clin Oncol 1981 101 149 164 7276068 10.1007/BF00405075
Imal Y Yamakawa M Morphology, function and pathology of follicular dendritic cells Pathol Int 1996 46 807 833 8970191
Takahashi K [Development and differentiation of macrophages and their related cells] Hum Cell 1994 7 109 115 7873492
Peters JH Ruhl S Friedrichs D Veiled accessory cells deduced from monocytes Immunobiology 1987 176 154 166 3502337
Peters JH Gieseler R Thiele B Steinbach F Dendritic cells: from ontogenetic orphans to myelomonocytic descendants Immunol Today 1996 17 273 278 8962630 10.1016/0167-5699(96)80544-5
Sievers J Parwaresch R Wottge HU Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: morphology Glia 1994 12 245 258 7890329 10.1002/glia.440120402
Udagawa N Takahashi N Akatsu T Tanaka H Sasaki T Nishihara T Koga T Martin TJ Suda T Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells Proc Natl Acad Sci U S A 1990 87 7260 7264 2169622
Kuwana M Okazaki Y Kodama H Izumi K Yasuoka H Ogawa Y Kawakami Y Ikeda Y Human circulating CD14+ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation J Leukoc Biol 2003 74 833 845 12960274 10.1189/jlb.0403170
Zhao Y Glesne D Huberman E A human peripheral blood monocyte-derived subset acts as pluripotent stem cells Proc Natl Acad Sci U S A 2003 100 2426 2431 12606720 10.1073/pnas.0536882100
Fernandez Pujol B Lucibello FC Gehling UM Lindemann K Weidner N Zuzarte ML Adamkiewicz J Elsasser HP Muller R Havemann K Endothelial-like cells derived from human CD14 positive monocytes Differentiation 2000 65 287 300 10929208 10.1046/j.1432-0436.2000.6550287.x
Heinemann DE Siggelkow H Ponce LM Viereck V Wiese KG Peters JH Alkaline phosphatase expression during monocyte differentiation. Overlapping markers as a link between monocytic cells, dendritic cells, osteoclasts and osteoblasts Immunobiology 2000 202 68 81 10879691
Heinemann DE Lohmann C Siggelkow H Alves F Engel I Koster G Human osteoblast-like cells phagocytose metal particles and express the macrophage marker CD68 in vitro J Bone Joint Surg Br 2000 82 283 289 10755442 10.1302/0301-620X.82B2 .9730
Beresford JN Bennett JH Devlin C Leboy PS Owen ME Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures J Cell Sci 1992 102 ( Pt 2) 341 351 1400636
Rademakers LH De Weger RA Roholl PJ Identification of alkaline phosphatase positive cells in human germinal centres as follicular dendritic cells Adv Exp Med Biol 1988 237 165 169 3254050
Pileri SA Grogan TM Harris NL Banks P Campo E Chan JK Favera RD Delsol G De Wolf-Peeters C Falini B Gascoyne RD Gaulard P Gatter KC Isaacson PG Jaffe ES Kluin P Knowles DM Mason DY Mori S Muller-Hermelink HK Piris MA Ralfkiaer E Stein H Su IJ Warnke RA Weiss LM Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases Histopathology 2002 41 1 29 12121233 10.1046/j.1365-2559.2002.01418.x
Maeda K Matsuda M Suzuki H Saitoh HA Immunohistochemical recognition of human follicular dendritic cells (FDCs) in routinely processed paraffin sections J Histochem Cytochem 2002 50 1475 1486 12417613
Tsunoda R Nakayama M Heinen E Miyake K Suzuki K Sugai N Kojima M Emperipolesis of lymphoid cells by human follicular dendritic cells in vitro Virchows Arch B Cell Pathol Incl Mol Pathol 1992 62 69 78 1355323
Tew JG Mandel TE Phipps RP Szakal AK Tissue localization and retention of antigen in relation to the immune response Am J Anat 1984 170 407 420 6475810 10.1002/aja.1001700314
Tsunoda R Nakayama M Onozaki K Heinen E Cormann N Kinet-Denoel C Kojima M Isolation and long-term cultivation of human tonsil follicular dendritic cells Virchows Arch B Cell Pathol Incl Mol Pathol 1990 59 95 105 1977238
Matsumoto M Mariathasan S Nahm MH Baranyay F Peschon JJ Chaplin DD Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers Science 1996 271 1289 1291 8638112
Wang Y Wang J Sun Y Wu Q Fu YX Complementary effects of TNF and lymphotoxin on the formation of germinal center and follicular dendritic cells J Immunol 2001 166 330 337 11123309
Kim HS Zhang X Choi YS Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes J Immunol 1994 153 2951 2961 7522246
Parmentier HK van der Linden JA Krijnen J van Wichen DF Rademakers LH Bloem AC Schuurman HJ Human follicular dendritic cells: isolation and characteristics in situ and in suspension Scand J Immunol 1991 33 441 452 1826796
Schriever F Nadler LM The central role of follicular dendritic cells in lymphoid tissues Adv Immunol 1992 51 243 284 1502976
Jaiswal N Haynesworth SE Caplan AI Bruder SP Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro J Cell Biochem 1997 64 295 312 9027589 10.1002/(SICI)1097-4644(199702)64:2<295::AID-JCB12>3.0.CO;2-I
Matasic R Dietz AB Vuk-Pavlovic S Dexamethasone inhibits dendritic cell maturation by redirecting differentiation of a subset of cells J Leukoc Biol 1999 66 909 914 10614771
Ujihara M Nomura K Yamada O Shibata N Kobayashi M Takano K Granulocyte-macrophage colony-stimulating factor ensures macrophage survival and generation of the superoxide anion: a study using a monocytic-differentiated HL60 subline Free Radic Biol Med 2001 31 1396 1404 11728811 10.1016/S0891-5849(01)00711-0
Spiegel H Herbst H Niedobitek G Foss HD Stein H Follicular dendritic cells are a major reservoir for human immunodeficiency virus type 1 in lymphoid tissues facilitating infection of CD4+ T-helper cells Am J Pathol 1992 140 15 22 1530997
Jeffrey M McGovern G Goodsir CM Brown KL Bruce ME Sites of prion protein accumulation in scrapie-infected mouse spleen revealed by immuno-electron microscopy J Pathol 2000 191 323 332 10878556 10.1002/1096-9896(200007)191:3<323::AID-PATH629>3.0.CO;2-Z
Smith BA Gartner S Liu Y Perelson AS Stilianakis NI Keele BF Kerkering TM Ferreira-Gonzalez A Szakal AK Tew JG Burton GF Persistence of infectious HIV on follicular dendritic cells J Immunol 2001 166 690 696 11123354
Masunaga A Nakamura H Katata T Furubayashi T Kanayama Y Yamada A Shiroko Y Itoyama S Follicular dendritic cell tumor with histiocytic characteristics and fibroblastic antigen Pathol Int 1997 47 707 712 9361106
Riedel F Back W Gotte K Hormann K [Follicular dendritic reticulum cell sarcoma in a cervical lymph node] Hno 2001 49 837 841 11699145 10.1007/s001060170033
Andriko JW Kaldjian EP Tsokos M Abbondanzo SL Jaffe ES Reticulum cell neoplasms of lymph nodes: a clinicopathologic study of 11 cases with recognition of a new subtype derived from fibroblastic reticular cells Am J Surg Pathol 1998 22 1048 1058 9737236 10.1097/00000478-199809000-00002
Gieseler RK Rober RA Kuhn R Weber K Osborn M Peters JH Dendritic accessory cells derived from rat bone marrow precursors under chemically defined conditions in vitro belong to the myeloid lineage Eur J Cell Biol 1991 54 171 181 1851701
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-511616474410.1186/1471-2180-5-51Research ArticleMycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves Manteca Angel [email protected] Marisol [email protected] Jesus [email protected] Universidad de Oviedo, Facultad de Medicina, Area de Microbiologia, Departamento de Biologia Funcional, 33006, Julian Claveria s/n, Oviedo, Spain2 Laboratorio de Proteomica, Centro Nacional de Biotecnologia, Cantoblanco, 28049 Madrid, Spain2005 15 9 2005 5 51 51 31 5 2005 15 9 2005 Copyright © 2005 Manteca et al; licensee BioMed Central Ltd.2005Manteca 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 current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.
Results
The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.
Conclusion
An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.
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Background
Streptomyces is a naturally occurring bacterium in soil and is likely to be present in aquatic habitats as well [1]. Since the early discovery of this microorganism's ability to to produce clinically useful antibiotics [2,3], the bacterium has received tremendous scientific attention [4]. Furthermore, other noteworthy characteristics, such as its remarkably complex developmental features, make this microorganism an interesting subject of study. Early on, Streptomyces was seen to form two distinct structures when grown on culture surfaces [5]: a substrate (vegetative) mycelium and an aerial (reproductive) mycelium. Substrate mycelium, which is assumed to grow into the medium, has a mean diameter of 0.7 μm and is bound by a 0.01–0.02 μm thick mucopeptide cell wall (reviewed in 6). This mycelium is assumed to be present in different stages of cellular degeneration during all growth phases. Early reports stated that aerial hyphae were the result of simple branching of substrate hyphae [7] and were preceded by a short period of decreased macromolecular synthesis [8]. One important feature of the aerial hyphae is that their outer surface is covered with a superficial fibrous sheath [7,9-11]. All these reports described the Streptomyces life cycle as a bottom-up (substrate-aerial) process. Consequently, it was assumed that development was uniform throughout the entire plate surface.
Our previous works have presented a detailed analysis of S. antibioticus development [12-14]. To obtain a reliable picture of the cell death phenomena that accompany this process, we have used a technique to analyse bacterial viability that involves staining the nucleic acids of the damaged (leaky) cells with propidium iodide (PI) [12]. This dye only enters cells with damaged membranes and substantially enhances fluorescence by binding to nucleic acids with little or no sequence preference (references in 15). PI staining, alone or in combination with fluorescein derivatives, has been widely used for cell death analysis in bacteria [16-19] and also in eukaryotic cells [20]. The reliability of this method has been also assessed in Streptomyces in submerged [13,21] and surface [12,14] conditions. Here we have extended our studies to a third dimension: the longitudinal axes on the plate surface. As illustrated below, this perspective is fundamental to understanding the developmental cycle of this bacterium.
Results
Confocal laser-scanning fluorescence microscopy (CLSM) analysis of development-linked cell death processes of Streptomyces antibioticus ATCC11891 in confluent surface cultures
Figure 1 presents a global perspective of some of the most relevant features of the different developmental steps analysed in Streptomyces antibioticus ATCC11891 on surface GAE cultures. To facilitate a sequential view of the process, we have divided it into several phases (A-H; Figure 1).
Figure 1 Confocal laser-scanning fluorescence microscopy analysis of the development-linked cell death processes of Streptomyces antibioticus ATCC11891 in confluent surface cultures. Developmental phases (A-H) and culture times (hours) are indicated. Picture D2 was obtained under the phase contrast microscope. The other images correspond to culture sections stained with SYTO 9 and propidium iodide. E2 is a cross section view; the other images correspond to longitudinal sections (see methods). Arrows in E2 indicate the eccentric circles of live mycelium developing from the bottom upwards, forming distinct layers with well-defined boundaries. Arrows in the rest of the images indicate circle edges. For details see text.
Phase A (0–7 hours) consists of germination and early hyphae development. When the spores are spread out on the surface (normally with a bent glass stick), all of them are viable (stained green) and remain in relatively large groups, probably owing to their hydrophobic properties (not shown). Germination begins at 4 hours by the asynchronous emission of a single (or less frequently, double) germ tube (Figure 1A, 5 and 6 hours). Most of these young hyphae undergo a very early death process (Figure 1A, 5 and 6 hours), which is remarkably symmetrical in a large proportion of the cases: live and dead segments alternate in a highly regular fashion within the same hyphae (Figure 1A, 6 hours); we have named them variegated hyphae [14]. This death process affects most of the young hyphae, although a small proportion of spores emit a germ tube that, while it is initially totally viable (Figure 1A, 6 hours), will eventually die. This is not unexpected, given that the spores are not distributed homogeneously throughout the plate and the microenvironment encountered by each of them may differ.
In Phase B (7–10 hours), the plate is completely covered with a thin layer of variegated mycelium (Figure 1B1, 8 hours). At this stage, the appearance of thin, green rings (Figure 1B1, 8 hours) measuring approximately 0.5 cm in diameter is particularly noteworthy. These rings are formed by the hyphae of late-germinating spores that remain live (high magnification in figure 1B2, 8 hours), and contrast with the mycelium originating from the previously germinated spores; hence, the variegated appearance. The center of the circle delimitated by the ring, as well as the mycelium located between the rings, is made up of variegated hyphae.
During Phase C (10–14 hours), the young, non-variegated hyphae referred to in Phase B that comprise the border of the circles undergo rapid, profuse growth (Figure 1C, 11 hours).
Phase D (14–16 hours) shows an overall decline in the grow rate (see below, Figure 3A). The borders of the circles cease to grow and live mycelium begins to slowly develop in the center (Figure 1D, 15 hours). This mycelium differs from the live mycelium in the borders of the circles in that it originates from the extension of the viable segments within the variegated hyphae (Figure 1A, 6 hours) and not from late-germinating spores, as occurs on the outer surface of the circles (see above and Discussion). The mycelium develops in the form of islands (Figure 1D, 15 hours), which appear near the edge of the circles and spread in radial waves towards the center (Figures 1D1-D3, 15 hours). This radial pattern of growth from the edge of the circles inwards leaves clear areas near the center, unoccupied by islands (Figure 1D3, 15 hours). Hence, at this point the plate is uniformly covered with mycelium arranged in dense circles separated by less dense areas. Figures 1D1 and 1D2 (15 hours) show a mycelial circle in Phase D; arrows indicate the edge of the circle as observed under CLSM (Figure 1D1) and under non-fluorescence, phase-contrast microscopy (Figure 1D2). The mycelium outside the circle is evident in the latter. This mycelium does not reveal fluorescence in Figure 1D1 at the magnification used, due to its lower density. The circles are distributed quite symmetrically (approximately one per 2.5 cm2), as deduced from the microscopic analyses (not shown in the pictures).
Figure 3 Growth curves (total protein and fresh weight per plate) of Streptomyces antibioticus ATCC11891 on surface GAE cultures developed from normal (A, 1.5 × 106 spores per plate) and diluted (B, 6 × 103 spores per plate) inocula. Arrows point to the exponential growth stages. Developmental phases (A-H and A'-H') are reflected in the curve. Error bars indicate ± SD.
During Phase E (16–20 hours), there is profuse growth of the live mycelial islands located in the center of the circles described above. Occasionally, several eccentric circles can be seen in the center of the largest circle (Figure 1E1, 18 hours). Figure 1E2 shows a cross section of the mycelial layer during Phase E: the eccentric circles of live mycelium develop from the bottom up, forming separate layers with well defined boundaries (arrows in Figure 1E2). Figure 1E3 shows the edge of a circle during this phase. No fluorescence is observed outside the circle, owing to the lower density of mycelium located there (see also Figures 1D1 and 1D2).
Phase F (20–30 hours) is characterized by the mycelial growth between the circles described in the earlier phases (Figure 1F, 23 hours). These areas remain less dense than the circles and are made up of live hyphae.
Phase G (30–45 hours) represents the culmination of the second death round in the substrate mycelium, as well as the pre-sporulating areas of aerial mycelium [6,22-25]; Figure 1G, 38 hours]. The majority of the hyphae located in the center of the circles are dead and present segmented DNA in the nucleoids (Figure 1G2, 38 hours).
Phase H (45–96 hours) is the sporulation phase. Live hyphae grow and form spores inside and outside the circles (Figure 1H1, 80 hours). The mycelium between the circles has also suffered a second death round prior to sporulation, although it is less extensive than the one that takes place inside the circles and is not visible at the magnification shown. The change in colour (arrow in Figure 1H1, 80 hours) indicates the border of the original circle, which continues to reveal greater density than the areas between circles. Figure 1H2 (80 hours) shows a detail of the viable chains of spores.
The result of this entire process is a variability in the developmental phases across the plane of a solid agar surface. In this work will refer to this phenomenon as "longitudinal heterogeneity".
Effect of the inoculum: density determines the presence of circles during development
The experiments described above were performed with a sufficient density of spore inocula as to produce rapid, confluent growth on the plates and represent the conditions often encountered in the laboratory during any given morphological and physiological analysis of Streptomyces [26], see Methods]. When a highly diluted inoculum was used (6 × 103 spores per plate), a significant delay was observed; sporulation was less efficient, and circles did not form (Figure 2). The phases indicated with apostrophes to distinguish them from their equivalents with the undiluted inoculum) are described below.
Figure 2 Longitudinal sections of Streptomyces antibioticus ATCC11891 surface cultures obtained using a diluted inoculum (6 × 103 spores per plate). The developmental phases (A'-H') and culture times are indicated. Samples were stained with SYTO 9 and propidium iodide. Arrow in picture A' indicates a group of hyphae in transition from presenting uniform green fluorescence to a variegated appearance, in which live (green) and dead (red) segments alternate in the same hypha. See text for details.
Phase A' (0–15 hours) corresponds to the germination and first death round similar to the corresponding cycle phase obtained with a normal inoculum. The main difference encountered with respect to the normal inoculum is the higher proportion of spores that present a viable, non-variegated germ tube (Phase A; not shown). However, when density increased and the hyphae touch one another, all of them die giving rise to the variegated appearance. Figure 2A' (10 hours) shows a group of hyphae that are beginning to lose their uniform green fluorescence (arrow), developing the variegated appearance which results from the alternating live and dead segments within the same hypha (on the right of the picture). This is indicative of the change in membrane permeability revealed by the propidium iodide that stains the dying segments red (see Methods).
Phase B'-E' (15–24 hours). No late-germinating spores remain at these time points (see Discussion). The live segments of the initial mycelium begin to develop in relatively regular, fast-growing islands. In this case, a unique, exponential growth phase is observed, unlike the various growth phases seen with normal inoculum. Figure 2B'–E' (20 hours) is a superficial view of an island formed by the emerging live mycelium.
Phase F' (24–40 hours) corresponds to the growth phase of the mycelium located between the islands. It is comparable to the mycelial growth between the circles described in Phase F of the developmental cycle in the presence of normal inoculum (Figures 2F'1 and 2F'2, 24 hours)
Phase G'-H' (40–96 hours) is the sporulation phase. The mycelial distribution in islands that began in Phases B'-E' is maintained. Like the circles, the islands also delimit the areas with a higher spore density (Figure 2G'–H', 80 hours). Figure 2G'–H' (80 hours) presents a detail of the surface revealing the spore chains.
In conclusion, the main difference observed with the diluted inoculum is the absence of circles originating from the delayed spore germination. The relevance of this will be discussed later.
Longitudinal heterogeneity determines multiphase growth curves
The methodological approach applied in this study eneabled us to obtain a high degree of synchronization in the plate cultures, thereby making it possible for us to analyze the relationships between growth rates and successive developmental steps. Figure 3 shows the growth curves (total protein and fresh weight per plate) obtained using normal or diluted inocula. The different phases described (see above) are indicated on the graph. Note the existence of three distinct stages of exponential growth in the normal cycle (indicated with arrows on the normal inoculum graph, Figure 3A), separated by two phases of temporary growth arrest (see the protein curve in Figure 3A). The second temporary growth arrest is not seen in the weight curve, probably because this parameter is less sensitive. The three waves of exponential growth correspond to Phase C (growth of the hyphae forming the edge of the circles), Phase E (rapid growth of the live hyphae from the center of the circles outwards) and Phase F (growth of the mycelium between the circles). The growth arrest periods correspond to Phase B (slow formation of the mycelial rings from late-germinating spores), Phase D (slow growth induction in the live segments of the dead hyphae located in the center of the circles) and the latter stage of Phase E (slow growth induction of the live segments of the dead hyphae located between the larger circles). Total protein per plate declines during the second death round, whereas weight continues to increase at a slower rate. This may be due to the accumulation of reserve compounds (glycogen and trehalose) within the cells [27]. Figure 3B shows the growth curves (fresh weight and total protein) for diluted inoculum (6 × 103 spores per plate). In these conditions, only two periods of exponential growth are observed, coinciding with mycelial development in the form of islands and the development of mycelium located between the islands, respectively (Figure 3B, arrows). Hence, there is a close correlation between morphological phases and growth curves. These developmental dynamics lead to the appearance of a layer of mycelium arranged in circles measuring approximately 0.5 cm or in islands measuring 0.9 mm separated by areas with a lower density of mycelium.
The first death round and longitudinal heterogeneity are general phenomena in surface cultures of the Streptomyces genus
The Streptomyces strains were grown in media in which the complete life cycle takes place with abundant sporulation (see Methods). All the Streptomyces species/strains analysed in our laboratory (with the exception of S. coelicolor A3(2); see below), present the pattern reported for S. antibioticus ATCC11891: circles when dense inocula are used and islands in the presence of highly diluted inocula. The only differences between the developmental cycles studied are the times required to reach the different phases and the diameter of the circles. Figures 4b and 4c show Streptomyces glaucescens ETH22794 and Streptomyces. antibioticus ETH7451 circles (compare with Figures 1C and 1E). As already mentioned, S. coelicolor A3(2) is the only exception to the described pattern: circles are not formed in any inoculum condition (concentrated or diluted) and development always occurs in the form of islands (Figure 4a).
Figure 4 Longitudinal sections of different Streptomyces strains growing in confluent surface cultures. (a) S. coelicolor A3(2). (b) S. glaucescens ETH22794. (c) S. antibioticus ETH7451. Culture times are indicated. Samples were stained with SYTO 9 and propidium iodide.
In media in which S. antibioticus ATCC11891 do not sporulate (GYM, GAE plus 2% casamino acids and R5; see Methods), the first death round and the variegated hyphae are also present (Figure 5) and longitudinal heterogeneity is observed (not shown). However, the second death round and sporulation are absent.
Figure 5 Cross sections of S. antibioticus ATCC11891 cultures in different solid media. Developmental time points and culture media are indicated. Casαα is GAE plus 2% casamino acids. Samples were stained with SYTO 9 and propidium iodide.
Discussion
All studies to date describing the differentiation and developmental cycle of Streptomyces refer to a completely live, viable substrate mycelium that grows inside the culture medium from which a reproductive (aerial) mycelium emerges after a massive death round. The most superficial part of the aerial mycelium forms spores by septating into chains of uninucleate compartments, whereas the non-sporulating aerial mycelium and the substrate mycelium eventually die [6,22-24]. In accordance with this model, the substrate mycelium in Streptomyces antibioticus ATCC11891 is the mycelium formed from spores and developed until approximately 35 hours of cultivation at 28–30°C. The formation of aerial mycelium commences at this point and at approximately 60 hours of cultivation, the sporulation process begins [22,28]. The three mycelial types (substrate, aerial and sporulated aerial) exist simultaneously at certain times. Again, according to this model, this cycle is present in all Streptomyces species, albeit with certain temporal variations.
In this work we have analysed the developmental cycle of Streptomyces in three spatial dimensions, the transverse and longitudinal axes, a perspective we believe is essential to understanding the developmental cycle. The developmental features of S. antibioticus ATCC11891 on confluent surface cultures are summarized in Figure 6. An early death round takes place affecting the young substrate hyphae, which to date, has not been described. This death process occurs in a remarkably symmetrical form: live and dead segments alternate within the same hyphae in a very regular pattern (Figure 1A, 6 hours). Subsequently, the remaining mycelium grow in successive waves, creating longitudinal heterogeneity of the bacterium on the plate surface. In all prior studies of the Streptomyces developmental cycle in solid media, it is implicitly assumed that the cultures are homogeneous all over the plate surfaces; hence, the point at which the plate is analysed would not be critical. The only heterogeneity present would be in the transverse plane; that is from the bottom up. Our data clearly demonstrate that substrate and aerial mycelial growth is heterogeneous and orderly, forming a remarkable pattern of circles and islands. This finding suggests that diffusible signals are involved in their induction.
Figure 6 Model for the developmental cycle of S. antibioticus ATCC11891 on surface GAE cultures (detailed in text). Times are indicated in hours.
As occurs with other bacteria, Streptomyces cultures are niches where quorum-sensing phenomena take place [29], particularly in the relatively crowded conditions normally employed in the laboratory for growing surface cultures. Many signals of this type have been reported in the Streptomyces genus [30]. The absence of circles in diluted inoculum conditions might also be related to the lack of appropriate signals. The circles are formed by spores that germinate at a later time, under the influence of signals that determine their growth as viable mycelium that does not die at the earlier time points (see above). Bearing in mind that the circles initially form at very early time points (Figures 1 and 6), it can be speculated that at low inoculum densities, the hypothetical signals do not accumulate enough to induce these structures. Another simple alternative explanation concerns the origin of the circles. Given that they are initially comprised of late-germinating spores that are logically less common in highly diluted inocula, it can be hypothesized that when the mycelial layer is dense enough to produce the signals, no late-germinating spores remain and hence, the edge of the circles is not formed. The phenomena described here are clearly different from other surface phenotypes, such as the previously described Streptomyces "pocks" [31]. The pocks are caused by the presence of conjugative plasmids and their appearance is due to the slower rate of mycelial growth at these points [26,31]. The opposite happens with the circles described in the present work, which are made up of a high mycelial density (see above). Some authors have described the existence of circular areas of unknown origin, which were "not genuine pocks", yet were present in many Streptomyces cultures [26]. These areas probably correspond to the circles described in this paper.
Our data clearly show that longitudinal heterogeneity is at least as important as transverse heterogeneity in the development of Streptomyces surface cultures. Very different developmental phases may therefore be taking place simultaneously at two different points of the surface cultures (Figures 1 and 6). For example, in Phase E, the live mycelium inside the circles is fully developed and in the process of dying, whereas the mycelium between the circles is just beginning to grow. This is fundamental in order to properly understand and interpret the distinct stages that occur during the differentiation cycle of Streptomyces. With the exception of S. coelicolor A3(2), all the Streptomyces species analysed in our laboratory present the pattern reported for S. antibioticus ATCC11891. S. coelicolor do not form circles in any inoculum condition and instead, only present surface development in the form of islands (Figure 4). Knowing whether one specific species or strain of Streptomyces grows in "circles" or "islands" is key for the correct interpretation of the morphological/biochemical data obtained in solid cultures, as illustrated by the growth curves (Figure 3). In media in which there is no sporulation, the first death round takes place and longitudinal heterogeneity is present; however, the second death round does not occur. Consequently the first death round and the appearance of longitudinal heterogeneity are general events inherent to the development of Streptomyces on surface cultures. We are currently analysing the peculiarities of several Streptomyces species in different culture media, in order to integrate these into a consensus and create a reliable model of Streptomyces development on surface cultures.
A vast collection of mutant Streptomyces [32,33] will be generated in the future thanks to the new, powerful techniques currently available. Factoring the features described here into the analysis of these and other previously reported differentiation mutants of Streptomyces will greatly expand the number of potential phenotypes to be considered and hence, their corresponding genetic determinants. This in turn, will hopefully facilitate the discovery of new signal cascades in this important bacterium. Furthermore, as the differentiation of hyphae and antibiotic production share common genetic control elements [34-36], the aspects considered above will also aid in better understanding antibiotic production in solid-state fermentation [37-39], as well as in submerged conditions [40], A. Manteca and J. Sanchez, unpublished data].
Methods
Strains and media
Streptomyces antibioticus ATCC11891, Streptomyces. antibioticus ETH7451, Streptomyces glaucescens ETH22794 and Streptomyces coelicolor A3(2) were the species used in this research. The microorganisms were grown in solid media in which they present a complete life cycle with abundant sporulation. S. antibioticus ATCC11891 and S. glaucescens ETH22794 were grown on GAE medium [22]; S. coelicolor A3(2) was grown on GYM medium (glucose, yeast extract, malt extract; 41). S. antibioticus was also grown on media in which it does not sporulate: GAE plus 2% casamino acids, GYM [40] and R5 [25]. The cultures were prepared in Petri dishes (8.5 cm diameter) as lawns on solid medium (30 ml/plate). When indicated, sterile cellophane disks were placed on the surface prior to inoculation. Under the conditions used, the differentiation processes follow a pattern similar to that of mycelium incubated directly on the culture medium [22]. Plates (with or without cellophane) were inoculated directly with 100 μl of a spore suspension (1.5 × 107 viable spores/ml), followed by incubation at 30°C. In some cases, plates were inoculated with a highly diluted spore suspension (6 × 103 spores per plate).
Microscopy
Culture samples were obtained and processed for microscopy at different incubation times, as described previously for submerged and surface-grown Streptomyces cultures [12,13]. Petri dishes prepared with Difco agar and inoculated as described above were used to obtain solid blocks of the agar cultures with a scalpel. These blocks were further trimmed to squares of approximately 10 mm in size and introduced into a hand microtome (11 mm hole diameter) previously cooled to 4°C, with the surface of growth facing sideways. Sections of about 0.3 mm were obtained (cross sections). To analyse longitudinal sections, we used solid cultures covered with cellophane disks: the disks were removed from the culture, cut into squares measuring approximately 1.5 cm × 1.5 cm and placed on slides before staining.
The permeability assay previously described for Streptomyces was used to stain all samples [12]. This technique involves staining the cells with cell-impermeant nucleic acid stain (propidium iodide, PI) in order to detect the dead cell population of S. antibioticus and with SYTO 9 green fluorescent nucleic acid stain (LIVE/DEAD Bac-Light Bacterial Viability Kit, Molecular Probes, L-13152) to detect viable cells. The SYTO 9 green fluorescent stain labels all the cells, i.e. those with intact membranes, as well as those with damaged ones. In contrast, PI penetrates only bacteria with damaged membranes, decreasing SYTO 9 stain fluorescence when both dyes are present. Thus, in the presence of both stains, bacteria with intact cell membranes appear in fluorescent green, whereas bacteria with damaged membranes appear in red [15]. The stain mixture was prepared as per the manufacturer's instructions and was added directly on the samples on the slide. The coverslide was placed on top and after staining for at least 10 minutes in the dark, the samples were then examined under a Leica TCS-SP2-AOBS confocal laser-scanning microscope at a wavelength of 488 nm and 568 nm excitation and 530 nm (green) or 630 nm (red) emission. Images were mixed using the Leica Confocal Software. In some cases, the samples were also examined in differential interference contrast mode, available with the same equipment.
Streptomyces cell sampling and processing
Cells from S. antibioticus grown on the surface of cellophane disks were scraped off with a plain spatula at different time points. Growth curves were obtained by means of weight determinations and total protein analysis. Fresh weight data (obtained without subjecting the mycelium to any drying treatment) were used, given that these data are reproducible and afford reliable determination of growth values in the early stages of the cultures when the small amount of mycelium per plate at these time points makes the use of other alternative approaches difficult. Determinations were repeated a minimum of three times. Total protein was analysed in the collected mycelium as follows: the weighted mycelium was resuspended in buffer A (Tris-HCl 20 mM pH 8, EDTA 1 mM, β-mercaptoetanol 7 mM and PMSF 0.5 mM), maintaining a constant ratio between fresh weight of mycelium and volume of buffer A (65 mg mycelium/ml buffer A). The suspension was ruptured in an MSE soniprep 150, in 6 cycles of 10 seconds, on ice, after which the samples were centrifuged at 10000 r.p.m. in an Eppendorf microcentrifuge for 30 min at 4°C and the protein was determined by the Lowry assay [42].
List of abbreviations
CLSM, confocal laser-scanning fluorescence microscopy; PI, propidium iodide.
Authors' contributions
AM performed all microscopic and biochemical analyses. MF participated in some of the experiments and in the initial study design. JS conceived the study, participated in its design, and coordinated and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We wish to thank Angel Martinez Nistal, Image Processing and Analysis Service of the University of Oviedo for his indispensable assistance with the confocal microscope and Priscilla A. Chase for revising the text. This research was funded by grant BIO2000-0577 from the DGI, Subdirección General de Proyectos de Investigación, MCYT, Spain.
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Moran MA Rutherford LT Hodson RE Evidence for indigenous Streptomyces populations in a marine environment determined with a 16S rRNA probe Appl Environ Microbiol 1995 61 3695 3700 7487005
Schatz A Bugie E Waksman SA Streptomycin, a substance exhibiting antibiotic activity against gram-negative and gram-positive bacteria Proc Soc ExptlBiol Med 1944 55 66 69
Wasksman SA Woodruff HB The soil as a source of microorganisms antagonistic to disease-producing bacteria J Bacteriol 1940 40 581 600 16560371
Paradkar A Trefzer A Chakraburtty R Stassi D Streptomyces genetics: a genomic perspective Crit Rev Biotechnol 2003 23 1 27 12693442
Waksman SA The Genus Streptomyces The Actinomycetes A Summary of Current Knowledge 1967 Chapter 9 The Ronald Press Co. NY
Hodgson DA Mohan S, Dow C, Cole JA Differentiation in Actinomycetes Prokaryotic Structure and Function: A New Perspective Society for General Microbiology Symposium 1992 47 Cambridge University Press, Cambridge 407 440
Hopwood DA Glauert AM Electron microscope observations on the surface structures of Streptomyces violaceoruber J Gen Microbiol 1961 26 325 30 14448835
Granozzi C Billeta R Passantino R Sollazzo M Puglia AM A breakdown in macromolecular synthesis preceding differentiation in Streptomyces coelicolor A3(2) J Gen Microbiol 1990 136 713 716 1697885
Claessen D Rink R de Jong W Siebring J de Vreugd P Boersma FG Dijkhuizen L Wosten HA A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils Genes Dev 2003 17 1714 1726 12832396 10.1101/gad.264303
Elliot MA Karoonuthaisiri N Huang J Bibb MJ Cohen SM Kao CM Buttner MJ The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor Genes Dev 2003 17 1727 1740 12832397 10.1101/gad.264403
Wildermuth H Wehrli E Horne RW The surface structure of spores and aerial mycelium in Streptomyces coelicolor J Ultrastruct Res 1971 35 168 80 4102961 10.1016/S0022-5320(71)80149-1
Fernandez M Sanchez J Nuclease activities and cell death processes associated with the development on surface cultures of Streptomyces antibioticus ETH7451 Microbiology 2002 148 405 412 11832504
Fernandez M Sanchez J Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451 J Microbiol Methods 2001 47 293 298 11714519 10.1016/S0167-7012(01)00332-3
Manteca A Fernandez M Sanchez J Cytological and Biochemical Analysis of two Lytic programmed Cellular Dismantling Rounds accompanying the development of Streptomyces antibioticus in Surface Cultures Res Microbiol 16171979
Haugland RP Gregory J Nucleic acid detection and genomics technology Handbook of Fluorescent Probes and Research Chemicals 2002 Chapter 8 Ninth Molecular Probes, Inc Eugene, OR
Bunthof CJ van Schalkwijk S Meijer W Abee T Hugenholtz J Fluorescent method for monitoring cheese starter permeabilization and lysis Appl Environ Microbiol 2001 67 4264 71 11526032 10.1128/AEM.67.9.4264-4271.2001
Bunthof CJ Sabina van den Braak S Breeuwer P Rombouts FM Abee T Rapid Fluorescence Assessment of the Viability of Stressed Lactococcus lactis Appl Envir Microbiol 1999 65 3681 3689
Lloyd D Hayes AJ Vigour, vitality and viability of microorganisms FEMS Microbiol Lett 1995 133 1 7 10.1016/0378-1097(95)00322-V
Miller JS Quarles JM Flow cytometric identification of microorganisms by dual staining with FITC and PI Cytometry 1990 11 667 675 1696535 10.1002/cyto.990110603
Darzynkiewicz Z Bruno S Del Bino G Gorczyca W Hotz MA Lassota P Traganos F Features of apoptotic cells measured by flow cytometry Cytometry 1992 13 795 808 1333943 10.1002/cyto.990130802
Sebastine IM Stocks PW Cox PW Thomas CR Characterization of percentage viability of Streptomyces clavuligerus using image analysis Biotechnol Tech 1999 13 419 423 10.1023/A:1008902330043
Mendez C Braña AF Manzanal MB Hardisson C Role of substrate mycelium in colony development in Streptomyces Can J Microbiol 1985 31 446 450 3891055
Kalakoutskii LV and Agre NS Comparative aspects of development and differentiation in actinomycetes Bacteriol Rev 1976 40 469 524 786257
Wildermuth H Development and organization of the aerial mycelium in Streptomyces coelicolor J Gen Microbiol 1970 60 43 50 5488465
Chater KF Hershberger CL, Queener SW, Hegeman G Aspects of multicellular differentiation in Streptomyces coelicolor A3(2) Genetics and Molecular Biology of Industrial Microorganisms 1989 American Society for Microbiology, Washington, DC 99 107
Kieser T Bibb MJ Buttner MJ Chater KF Hopwood DA Practical Streptomyces Genetics 2000 The John Innes Foundation, Norwich, England
Braña AF Mendez C Diaz LA Manzanal MB Hardisson C Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus J Gen Microbiol 1986 132 1319 1326 3534138
Miguelez EM Hardisson C Manzanal MB Hyphal death during colony development in Streptomyces antibioticus : morphological evidence for the existence of a process of cell deletion equivalent to apoptosis in a multicellular prokaryote J Cell Biol 1999 145 515 525 10225953 10.1083/jcb.145.3.515
Bayles KW Are the molecular strategies that control apoptosis conserved in bacteria? Trends Microbiol 2003 11 306 311 12875813 10.1016/S0966-842X(03)00144-6
Bibb MJ Regulation of secondary metabolism in Streptomycetes Curr Opin Microbiol 2005 8 208 215 15802254 10.1016/j.mib.2005.02.016
Pettis GS Ward N Schully KL Expression characteristics of the transfer-related kilB gene product of Streptomyces plasmid pIJ101: implications for the plasmid spread function J Bacteriol 2001 183 1339 1345 11157947 10.1128/JB.183.4.1339-1345.2001
Gehring AM Nodwell JR Beverley SM Losick R Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation Proc Natl Acad Sci U S A 2000 97 9642 9647 10931952 10.1073/pnas.170059797
Sprusansky O Zhou L Jordan S White J Westpheling J Identification of three new genes involved in morphogenesis and antibiotic production in Streptomyces coelicolor J Bacteriol 2003 185 6147 6157 14526027 10.1128/JB.185.20.6147-6157.2003
Champness WC Chater KF Piggot PJ, Morgan CP, Youngman P Regulation and integration of antibiotic production and morphological differentiation in Streptomyces sp Regulation of Bacterial Development 1994 American Society for Microbiology, Washington, DC 61 93
Chater KF Bibb MJ Kleinkauf H, v Dören H Regulation of bacterial antibiotic production Biotechnology Products of Secondary Metabolism 1997 7 VCH Press, Weinheim, Germany 57 105
Horinouchi S Ohnishi Y Kang DK The A-factor regulatory cascade and cAMP in the regulation of physiological and morphological development in Streptomyces griseus J Ind Microbiol Biotechnol 2001 27 177 182 11780789 10.1038/sj.jim.7000068
Ellaiah P Srinivasulu B Adinarayana K Optimisation studies on neomycin production by a mutant strain of Streptomyces marinensis in solid state fermentation Process Biochem 2004 39 1331 1339 10.1016/S0032-9592(03)00263-2
Jermini MF Demain AL Solid state fermentation for cephalosporin production by Streptomyces clavuligerus and Cephalosporium acremonium Experientia 1989 45 1061 1065 2599054 10.1007/BF01950159
Kota KP Sridhar P Solid state cultivation of Streptomyces clavuligerus for cephamycin C production Process Biochem 1999 34 325 328 10.1016/S0032-9592(98)00078-8
Stocks SM Thomas CR Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation Biotechnol Bioeng 2001 75 702 709 11745148 10.1002/bit.10017
Novella IS Barbes C Sanchez J Sporulation of Streptomyces antibioticus ETH 7451 in submerged culture Can J Microbiol 1992 38 769 773 1458369
Lowry OH Rosenbrough NJ Farr AL Randall RJ Protein measurement with the Folin phenol reagent J Biol Chem 1951 193 265 275 14907713
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-511616474410.1186/1471-2180-5-51Research ArticleMycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves Manteca Angel [email protected] Marisol [email protected] Jesus [email protected] Universidad de Oviedo, Facultad de Medicina, Area de Microbiologia, Departamento de Biologia Funcional, 33006, Julian Claveria s/n, Oviedo, Spain2 Laboratorio de Proteomica, Centro Nacional de Biotecnologia, Cantoblanco, 28049 Madrid, Spain2005 15 9 2005 5 51 51 31 5 2005 15 9 2005 Copyright © 2005 Manteca et al; licensee BioMed Central Ltd.2005Manteca 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 current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.
Results
The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.
Conclusion
An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.
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Background
Streptomyces is a naturally occurring bacterium in soil and is likely to be present in aquatic habitats as well [1]. Since the early discovery of this microorganism's ability to to produce clinically useful antibiotics [2,3], the bacterium has received tremendous scientific attention [4]. Furthermore, other noteworthy characteristics, such as its remarkably complex developmental features, make this microorganism an interesting subject of study. Early on, Streptomyces was seen to form two distinct structures when grown on culture surfaces [5]: a substrate (vegetative) mycelium and an aerial (reproductive) mycelium. Substrate mycelium, which is assumed to grow into the medium, has a mean diameter of 0.7 μm and is bound by a 0.01–0.02 μm thick mucopeptide cell wall (reviewed in 6). This mycelium is assumed to be present in different stages of cellular degeneration during all growth phases. Early reports stated that aerial hyphae were the result of simple branching of substrate hyphae [7] and were preceded by a short period of decreased macromolecular synthesis [8]. One important feature of the aerial hyphae is that their outer surface is covered with a superficial fibrous sheath [7,9-11]. All these reports described the Streptomyces life cycle as a bottom-up (substrate-aerial) process. Consequently, it was assumed that development was uniform throughout the entire plate surface.
Our previous works have presented a detailed analysis of S. antibioticus development [12-14]. To obtain a reliable picture of the cell death phenomena that accompany this process, we have used a technique to analyse bacterial viability that involves staining the nucleic acids of the damaged (leaky) cells with propidium iodide (PI) [12]. This dye only enters cells with damaged membranes and substantially enhances fluorescence by binding to nucleic acids with little or no sequence preference (references in 15). PI staining, alone or in combination with fluorescein derivatives, has been widely used for cell death analysis in bacteria [16-19] and also in eukaryotic cells [20]. The reliability of this method has been also assessed in Streptomyces in submerged [13,21] and surface [12,14] conditions. Here we have extended our studies to a third dimension: the longitudinal axes on the plate surface. As illustrated below, this perspective is fundamental to understanding the developmental cycle of this bacterium.
Results
Confocal laser-scanning fluorescence microscopy (CLSM) analysis of development-linked cell death processes of Streptomyces antibioticus ATCC11891 in confluent surface cultures
Figure 1 presents a global perspective of some of the most relevant features of the different developmental steps analysed in Streptomyces antibioticus ATCC11891 on surface GAE cultures. To facilitate a sequential view of the process, we have divided it into several phases (A-H; Figure 1).
Figure 1 Confocal laser-scanning fluorescence microscopy analysis of the development-linked cell death processes of Streptomyces antibioticus ATCC11891 in confluent surface cultures. Developmental phases (A-H) and culture times (hours) are indicated. Picture D2 was obtained under the phase contrast microscope. The other images correspond to culture sections stained with SYTO 9 and propidium iodide. E2 is a cross section view; the other images correspond to longitudinal sections (see methods). Arrows in E2 indicate the eccentric circles of live mycelium developing from the bottom upwards, forming distinct layers with well-defined boundaries. Arrows in the rest of the images indicate circle edges. For details see text.
Phase A (0–7 hours) consists of germination and early hyphae development. When the spores are spread out on the surface (normally with a bent glass stick), all of them are viable (stained green) and remain in relatively large groups, probably owing to their hydrophobic properties (not shown). Germination begins at 4 hours by the asynchronous emission of a single (or less frequently, double) germ tube (Figure 1A, 5 and 6 hours). Most of these young hyphae undergo a very early death process (Figure 1A, 5 and 6 hours), which is remarkably symmetrical in a large proportion of the cases: live and dead segments alternate in a highly regular fashion within the same hyphae (Figure 1A, 6 hours); we have named them variegated hyphae [14]. This death process affects most of the young hyphae, although a small proportion of spores emit a germ tube that, while it is initially totally viable (Figure 1A, 6 hours), will eventually die. This is not unexpected, given that the spores are not distributed homogeneously throughout the plate and the microenvironment encountered by each of them may differ.
In Phase B (7–10 hours), the plate is completely covered with a thin layer of variegated mycelium (Figure 1B1, 8 hours). At this stage, the appearance of thin, green rings (Figure 1B1, 8 hours) measuring approximately 0.5 cm in diameter is particularly noteworthy. These rings are formed by the hyphae of late-germinating spores that remain live (high magnification in figure 1B2, 8 hours), and contrast with the mycelium originating from the previously germinated spores; hence, the variegated appearance. The center of the circle delimitated by the ring, as well as the mycelium located between the rings, is made up of variegated hyphae.
During Phase C (10–14 hours), the young, non-variegated hyphae referred to in Phase B that comprise the border of the circles undergo rapid, profuse growth (Figure 1C, 11 hours).
Phase D (14–16 hours) shows an overall decline in the grow rate (see below, Figure 3A). The borders of the circles cease to grow and live mycelium begins to slowly develop in the center (Figure 1D, 15 hours). This mycelium differs from the live mycelium in the borders of the circles in that it originates from the extension of the viable segments within the variegated hyphae (Figure 1A, 6 hours) and not from late-germinating spores, as occurs on the outer surface of the circles (see above and Discussion). The mycelium develops in the form of islands (Figure 1D, 15 hours), which appear near the edge of the circles and spread in radial waves towards the center (Figures 1D1-D3, 15 hours). This radial pattern of growth from the edge of the circles inwards leaves clear areas near the center, unoccupied by islands (Figure 1D3, 15 hours). Hence, at this point the plate is uniformly covered with mycelium arranged in dense circles separated by less dense areas. Figures 1D1 and 1D2 (15 hours) show a mycelial circle in Phase D; arrows indicate the edge of the circle as observed under CLSM (Figure 1D1) and under non-fluorescence, phase-contrast microscopy (Figure 1D2). The mycelium outside the circle is evident in the latter. This mycelium does not reveal fluorescence in Figure 1D1 at the magnification used, due to its lower density. The circles are distributed quite symmetrically (approximately one per 2.5 cm2), as deduced from the microscopic analyses (not shown in the pictures).
Figure 3 Growth curves (total protein and fresh weight per plate) of Streptomyces antibioticus ATCC11891 on surface GAE cultures developed from normal (A, 1.5 × 106 spores per plate) and diluted (B, 6 × 103 spores per plate) inocula. Arrows point to the exponential growth stages. Developmental phases (A-H and A'-H') are reflected in the curve. Error bars indicate ± SD.
During Phase E (16–20 hours), there is profuse growth of the live mycelial islands located in the center of the circles described above. Occasionally, several eccentric circles can be seen in the center of the largest circle (Figure 1E1, 18 hours). Figure 1E2 shows a cross section of the mycelial layer during Phase E: the eccentric circles of live mycelium develop from the bottom up, forming separate layers with well defined boundaries (arrows in Figure 1E2). Figure 1E3 shows the edge of a circle during this phase. No fluorescence is observed outside the circle, owing to the lower density of mycelium located there (see also Figures 1D1 and 1D2).
Phase F (20–30 hours) is characterized by the mycelial growth between the circles described in the earlier phases (Figure 1F, 23 hours). These areas remain less dense than the circles and are made up of live hyphae.
Phase G (30–45 hours) represents the culmination of the second death round in the substrate mycelium, as well as the pre-sporulating areas of aerial mycelium [6,22-25]; Figure 1G, 38 hours]. The majority of the hyphae located in the center of the circles are dead and present segmented DNA in the nucleoids (Figure 1G2, 38 hours).
Phase H (45–96 hours) is the sporulation phase. Live hyphae grow and form spores inside and outside the circles (Figure 1H1, 80 hours). The mycelium between the circles has also suffered a second death round prior to sporulation, although it is less extensive than the one that takes place inside the circles and is not visible at the magnification shown. The change in colour (arrow in Figure 1H1, 80 hours) indicates the border of the original circle, which continues to reveal greater density than the areas between circles. Figure 1H2 (80 hours) shows a detail of the viable chains of spores.
The result of this entire process is a variability in the developmental phases across the plane of a solid agar surface. In this work will refer to this phenomenon as "longitudinal heterogeneity".
Effect of the inoculum: density determines the presence of circles during development
The experiments described above were performed with a sufficient density of spore inocula as to produce rapid, confluent growth on the plates and represent the conditions often encountered in the laboratory during any given morphological and physiological analysis of Streptomyces [26], see Methods]. When a highly diluted inoculum was used (6 × 103 spores per plate), a significant delay was observed; sporulation was less efficient, and circles did not form (Figure 2). The phases indicated with apostrophes to distinguish them from their equivalents with the undiluted inoculum) are described below.
Figure 2 Longitudinal sections of Streptomyces antibioticus ATCC11891 surface cultures obtained using a diluted inoculum (6 × 103 spores per plate). The developmental phases (A'-H') and culture times are indicated. Samples were stained with SYTO 9 and propidium iodide. Arrow in picture A' indicates a group of hyphae in transition from presenting uniform green fluorescence to a variegated appearance, in which live (green) and dead (red) segments alternate in the same hypha. See text for details.
Phase A' (0–15 hours) corresponds to the germination and first death round similar to the corresponding cycle phase obtained with a normal inoculum. The main difference encountered with respect to the normal inoculum is the higher proportion of spores that present a viable, non-variegated germ tube (Phase A; not shown). However, when density increased and the hyphae touch one another, all of them die giving rise to the variegated appearance. Figure 2A' (10 hours) shows a group of hyphae that are beginning to lose their uniform green fluorescence (arrow), developing the variegated appearance which results from the alternating live and dead segments within the same hypha (on the right of the picture). This is indicative of the change in membrane permeability revealed by the propidium iodide that stains the dying segments red (see Methods).
Phase B'-E' (15–24 hours). No late-germinating spores remain at these time points (see Discussion). The live segments of the initial mycelium begin to develop in relatively regular, fast-growing islands. In this case, a unique, exponential growth phase is observed, unlike the various growth phases seen with normal inoculum. Figure 2B'–E' (20 hours) is a superficial view of an island formed by the emerging live mycelium.
Phase F' (24–40 hours) corresponds to the growth phase of the mycelium located between the islands. It is comparable to the mycelial growth between the circles described in Phase F of the developmental cycle in the presence of normal inoculum (Figures 2F'1 and 2F'2, 24 hours)
Phase G'-H' (40–96 hours) is the sporulation phase. The mycelial distribution in islands that began in Phases B'-E' is maintained. Like the circles, the islands also delimit the areas with a higher spore density (Figure 2G'–H', 80 hours). Figure 2G'–H' (80 hours) presents a detail of the surface revealing the spore chains.
In conclusion, the main difference observed with the diluted inoculum is the absence of circles originating from the delayed spore germination. The relevance of this will be discussed later.
Longitudinal heterogeneity determines multiphase growth curves
The methodological approach applied in this study eneabled us to obtain a high degree of synchronization in the plate cultures, thereby making it possible for us to analyze the relationships between growth rates and successive developmental steps. Figure 3 shows the growth curves (total protein and fresh weight per plate) obtained using normal or diluted inocula. The different phases described (see above) are indicated on the graph. Note the existence of three distinct stages of exponential growth in the normal cycle (indicated with arrows on the normal inoculum graph, Figure 3A), separated by two phases of temporary growth arrest (see the protein curve in Figure 3A). The second temporary growth arrest is not seen in the weight curve, probably because this parameter is less sensitive. The three waves of exponential growth correspond to Phase C (growth of the hyphae forming the edge of the circles), Phase E (rapid growth of the live hyphae from the center of the circles outwards) and Phase F (growth of the mycelium between the circles). The growth arrest periods correspond to Phase B (slow formation of the mycelial rings from late-germinating spores), Phase D (slow growth induction in the live segments of the dead hyphae located in the center of the circles) and the latter stage of Phase E (slow growth induction of the live segments of the dead hyphae located between the larger circles). Total protein per plate declines during the second death round, whereas weight continues to increase at a slower rate. This may be due to the accumulation of reserve compounds (glycogen and trehalose) within the cells [27]. Figure 3B shows the growth curves (fresh weight and total protein) for diluted inoculum (6 × 103 spores per plate). In these conditions, only two periods of exponential growth are observed, coinciding with mycelial development in the form of islands and the development of mycelium located between the islands, respectively (Figure 3B, arrows). Hence, there is a close correlation between morphological phases and growth curves. These developmental dynamics lead to the appearance of a layer of mycelium arranged in circles measuring approximately 0.5 cm or in islands measuring 0.9 mm separated by areas with a lower density of mycelium.
The first death round and longitudinal heterogeneity are general phenomena in surface cultures of the Streptomyces genus
The Streptomyces strains were grown in media in which the complete life cycle takes place with abundant sporulation (see Methods). All the Streptomyces species/strains analysed in our laboratory (with the exception of S. coelicolor A3(2); see below), present the pattern reported for S. antibioticus ATCC11891: circles when dense inocula are used and islands in the presence of highly diluted inocula. The only differences between the developmental cycles studied are the times required to reach the different phases and the diameter of the circles. Figures 4b and 4c show Streptomyces glaucescens ETH22794 and Streptomyces. antibioticus ETH7451 circles (compare with Figures 1C and 1E). As already mentioned, S. coelicolor A3(2) is the only exception to the described pattern: circles are not formed in any inoculum condition (concentrated or diluted) and development always occurs in the form of islands (Figure 4a).
Figure 4 Longitudinal sections of different Streptomyces strains growing in confluent surface cultures. (a) S. coelicolor A3(2). (b) S. glaucescens ETH22794. (c) S. antibioticus ETH7451. Culture times are indicated. Samples were stained with SYTO 9 and propidium iodide.
In media in which S. antibioticus ATCC11891 do not sporulate (GYM, GAE plus 2% casamino acids and R5; see Methods), the first death round and the variegated hyphae are also present (Figure 5) and longitudinal heterogeneity is observed (not shown). However, the second death round and sporulation are absent.
Figure 5 Cross sections of S. antibioticus ATCC11891 cultures in different solid media. Developmental time points and culture media are indicated. Casαα is GAE plus 2% casamino acids. Samples were stained with SYTO 9 and propidium iodide.
Discussion
All studies to date describing the differentiation and developmental cycle of Streptomyces refer to a completely live, viable substrate mycelium that grows inside the culture medium from which a reproductive (aerial) mycelium emerges after a massive death round. The most superficial part of the aerial mycelium forms spores by septating into chains of uninucleate compartments, whereas the non-sporulating aerial mycelium and the substrate mycelium eventually die [6,22-24]. In accordance with this model, the substrate mycelium in Streptomyces antibioticus ATCC11891 is the mycelium formed from spores and developed until approximately 35 hours of cultivation at 28–30°C. The formation of aerial mycelium commences at this point and at approximately 60 hours of cultivation, the sporulation process begins [22,28]. The three mycelial types (substrate, aerial and sporulated aerial) exist simultaneously at certain times. Again, according to this model, this cycle is present in all Streptomyces species, albeit with certain temporal variations.
In this work we have analysed the developmental cycle of Streptomyces in three spatial dimensions, the transverse and longitudinal axes, a perspective we believe is essential to understanding the developmental cycle. The developmental features of S. antibioticus ATCC11891 on confluent surface cultures are summarized in Figure 6. An early death round takes place affecting the young substrate hyphae, which to date, has not been described. This death process occurs in a remarkably symmetrical form: live and dead segments alternate within the same hyphae in a very regular pattern (Figure 1A, 6 hours). Subsequently, the remaining mycelium grow in successive waves, creating longitudinal heterogeneity of the bacterium on the plate surface. In all prior studies of the Streptomyces developmental cycle in solid media, it is implicitly assumed that the cultures are homogeneous all over the plate surfaces; hence, the point at which the plate is analysed would not be critical. The only heterogeneity present would be in the transverse plane; that is from the bottom up. Our data clearly demonstrate that substrate and aerial mycelial growth is heterogeneous and orderly, forming a remarkable pattern of circles and islands. This finding suggests that diffusible signals are involved in their induction.
Figure 6 Model for the developmental cycle of S. antibioticus ATCC11891 on surface GAE cultures (detailed in text). Times are indicated in hours.
As occurs with other bacteria, Streptomyces cultures are niches where quorum-sensing phenomena take place [29], particularly in the relatively crowded conditions normally employed in the laboratory for growing surface cultures. Many signals of this type have been reported in the Streptomyces genus [30]. The absence of circles in diluted inoculum conditions might also be related to the lack of appropriate signals. The circles are formed by spores that germinate at a later time, under the influence of signals that determine their growth as viable mycelium that does not die at the earlier time points (see above). Bearing in mind that the circles initially form at very early time points (Figures 1 and 6), it can be speculated that at low inoculum densities, the hypothetical signals do not accumulate enough to induce these structures. Another simple alternative explanation concerns the origin of the circles. Given that they are initially comprised of late-germinating spores that are logically less common in highly diluted inocula, it can be hypothesized that when the mycelial layer is dense enough to produce the signals, no late-germinating spores remain and hence, the edge of the circles is not formed. The phenomena described here are clearly different from other surface phenotypes, such as the previously described Streptomyces "pocks" [31]. The pocks are caused by the presence of conjugative plasmids and their appearance is due to the slower rate of mycelial growth at these points [26,31]. The opposite happens with the circles described in the present work, which are made up of a high mycelial density (see above). Some authors have described the existence of circular areas of unknown origin, which were "not genuine pocks", yet were present in many Streptomyces cultures [26]. These areas probably correspond to the circles described in this paper.
Our data clearly show that longitudinal heterogeneity is at least as important as transverse heterogeneity in the development of Streptomyces surface cultures. Very different developmental phases may therefore be taking place simultaneously at two different points of the surface cultures (Figures 1 and 6). For example, in Phase E, the live mycelium inside the circles is fully developed and in the process of dying, whereas the mycelium between the circles is just beginning to grow. This is fundamental in order to properly understand and interpret the distinct stages that occur during the differentiation cycle of Streptomyces. With the exception of S. coelicolor A3(2), all the Streptomyces species analysed in our laboratory present the pattern reported for S. antibioticus ATCC11891. S. coelicolor do not form circles in any inoculum condition and instead, only present surface development in the form of islands (Figure 4). Knowing whether one specific species or strain of Streptomyces grows in "circles" or "islands" is key for the correct interpretation of the morphological/biochemical data obtained in solid cultures, as illustrated by the growth curves (Figure 3). In media in which there is no sporulation, the first death round takes place and longitudinal heterogeneity is present; however, the second death round does not occur. Consequently the first death round and the appearance of longitudinal heterogeneity are general events inherent to the development of Streptomyces on surface cultures. We are currently analysing the peculiarities of several Streptomyces species in different culture media, in order to integrate these into a consensus and create a reliable model of Streptomyces development on surface cultures.
A vast collection of mutant Streptomyces [32,33] will be generated in the future thanks to the new, powerful techniques currently available. Factoring the features described here into the analysis of these and other previously reported differentiation mutants of Streptomyces will greatly expand the number of potential phenotypes to be considered and hence, their corresponding genetic determinants. This in turn, will hopefully facilitate the discovery of new signal cascades in this important bacterium. Furthermore, as the differentiation of hyphae and antibiotic production share common genetic control elements [34-36], the aspects considered above will also aid in better understanding antibiotic production in solid-state fermentation [37-39], as well as in submerged conditions [40], A. Manteca and J. Sanchez, unpublished data].
Methods
Strains and media
Streptomyces antibioticus ATCC11891, Streptomyces. antibioticus ETH7451, Streptomyces glaucescens ETH22794 and Streptomyces coelicolor A3(2) were the species used in this research. The microorganisms were grown in solid media in which they present a complete life cycle with abundant sporulation. S. antibioticus ATCC11891 and S. glaucescens ETH22794 were grown on GAE medium [22]; S. coelicolor A3(2) was grown on GYM medium (glucose, yeast extract, malt extract; 41). S. antibioticus was also grown on media in which it does not sporulate: GAE plus 2% casamino acids, GYM [40] and R5 [25]. The cultures were prepared in Petri dishes (8.5 cm diameter) as lawns on solid medium (30 ml/plate). When indicated, sterile cellophane disks were placed on the surface prior to inoculation. Under the conditions used, the differentiation processes follow a pattern similar to that of mycelium incubated directly on the culture medium [22]. Plates (with or without cellophane) were inoculated directly with 100 μl of a spore suspension (1.5 × 107 viable spores/ml), followed by incubation at 30°C. In some cases, plates were inoculated with a highly diluted spore suspension (6 × 103 spores per plate).
Microscopy
Culture samples were obtained and processed for microscopy at different incubation times, as described previously for submerged and surface-grown Streptomyces cultures [12,13]. Petri dishes prepared with Difco agar and inoculated as described above were used to obtain solid blocks of the agar cultures with a scalpel. These blocks were further trimmed to squares of approximately 10 mm in size and introduced into a hand microtome (11 mm hole diameter) previously cooled to 4°C, with the surface of growth facing sideways. Sections of about 0.3 mm were obtained (cross sections). To analyse longitudinal sections, we used solid cultures covered with cellophane disks: the disks were removed from the culture, cut into squares measuring approximately 1.5 cm × 1.5 cm and placed on slides before staining.
The permeability assay previously described for Streptomyces was used to stain all samples [12]. This technique involves staining the cells with cell-impermeant nucleic acid stain (propidium iodide, PI) in order to detect the dead cell population of S. antibioticus and with SYTO 9 green fluorescent nucleic acid stain (LIVE/DEAD Bac-Light Bacterial Viability Kit, Molecular Probes, L-13152) to detect viable cells. The SYTO 9 green fluorescent stain labels all the cells, i.e. those with intact membranes, as well as those with damaged ones. In contrast, PI penetrates only bacteria with damaged membranes, decreasing SYTO 9 stain fluorescence when both dyes are present. Thus, in the presence of both stains, bacteria with intact cell membranes appear in fluorescent green, whereas bacteria with damaged membranes appear in red [15]. The stain mixture was prepared as per the manufacturer's instructions and was added directly on the samples on the slide. The coverslide was placed on top and after staining for at least 10 minutes in the dark, the samples were then examined under a Leica TCS-SP2-AOBS confocal laser-scanning microscope at a wavelength of 488 nm and 568 nm excitation and 530 nm (green) or 630 nm (red) emission. Images were mixed using the Leica Confocal Software. In some cases, the samples were also examined in differential interference contrast mode, available with the same equipment.
Streptomyces cell sampling and processing
Cells from S. antibioticus grown on the surface of cellophane disks were scraped off with a plain spatula at different time points. Growth curves were obtained by means of weight determinations and total protein analysis. Fresh weight data (obtained without subjecting the mycelium to any drying treatment) were used, given that these data are reproducible and afford reliable determination of growth values in the early stages of the cultures when the small amount of mycelium per plate at these time points makes the use of other alternative approaches difficult. Determinations were repeated a minimum of three times. Total protein was analysed in the collected mycelium as follows: the weighted mycelium was resuspended in buffer A (Tris-HCl 20 mM pH 8, EDTA 1 mM, β-mercaptoetanol 7 mM and PMSF 0.5 mM), maintaining a constant ratio between fresh weight of mycelium and volume of buffer A (65 mg mycelium/ml buffer A). The suspension was ruptured in an MSE soniprep 150, in 6 cycles of 10 seconds, on ice, after which the samples were centrifuged at 10000 r.p.m. in an Eppendorf microcentrifuge for 30 min at 4°C and the protein was determined by the Lowry assay [42].
List of abbreviations
CLSM, confocal laser-scanning fluorescence microscopy; PI, propidium iodide.
Authors' contributions
AM performed all microscopic and biochemical analyses. MF participated in some of the experiments and in the initial study design. JS conceived the study, participated in its design, and coordinated and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We wish to thank Angel Martinez Nistal, Image Processing and Analysis Service of the University of Oviedo for his indispensable assistance with the confocal microscope and Priscilla A. Chase for revising the text. This research was funded by grant BIO2000-0577 from the DGI, Subdirección General de Proyectos de Investigación, MCYT, Spain.
==== Refs
Moran MA Rutherford LT Hodson RE Evidence for indigenous Streptomyces populations in a marine environment determined with a 16S rRNA probe Appl Environ Microbiol 1995 61 3695 3700 7487005
Schatz A Bugie E Waksman SA Streptomycin, a substance exhibiting antibiotic activity against gram-negative and gram-positive bacteria Proc Soc ExptlBiol Med 1944 55 66 69
Wasksman SA Woodruff HB The soil as a source of microorganisms antagonistic to disease-producing bacteria J Bacteriol 1940 40 581 600 16560371
Paradkar A Trefzer A Chakraburtty R Stassi D Streptomyces genetics: a genomic perspective Crit Rev Biotechnol 2003 23 1 27 12693442
Waksman SA The Genus Streptomyces The Actinomycetes A Summary of Current Knowledge 1967 Chapter 9 The Ronald Press Co. NY
Hodgson DA Mohan S, Dow C, Cole JA Differentiation in Actinomycetes Prokaryotic Structure and Function: A New Perspective Society for General Microbiology Symposium 1992 47 Cambridge University Press, Cambridge 407 440
Hopwood DA Glauert AM Electron microscope observations on the surface structures of Streptomyces violaceoruber J Gen Microbiol 1961 26 325 30 14448835
Granozzi C Billeta R Passantino R Sollazzo M Puglia AM A breakdown in macromolecular synthesis preceding differentiation in Streptomyces coelicolor A3(2) J Gen Microbiol 1990 136 713 716 1697885
Claessen D Rink R de Jong W Siebring J de Vreugd P Boersma FG Dijkhuizen L Wosten HA A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils Genes Dev 2003 17 1714 1726 12832396 10.1101/gad.264303
Elliot MA Karoonuthaisiri N Huang J Bibb MJ Cohen SM Kao CM Buttner MJ The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor Genes Dev 2003 17 1727 1740 12832397 10.1101/gad.264403
Wildermuth H Wehrli E Horne RW The surface structure of spores and aerial mycelium in Streptomyces coelicolor J Ultrastruct Res 1971 35 168 80 4102961 10.1016/S0022-5320(71)80149-1
Fernandez M Sanchez J Nuclease activities and cell death processes associated with the development on surface cultures of Streptomyces antibioticus ETH7451 Microbiology 2002 148 405 412 11832504
Fernandez M Sanchez J Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451 J Microbiol Methods 2001 47 293 298 11714519 10.1016/S0167-7012(01)00332-3
Manteca A Fernandez M Sanchez J Cytological and Biochemical Analysis of two Lytic programmed Cellular Dismantling Rounds accompanying the development of Streptomyces antibioticus in Surface Cultures Res Microbiol 16171979
Haugland RP Gregory J Nucleic acid detection and genomics technology Handbook of Fluorescent Probes and Research Chemicals 2002 Chapter 8 Ninth Molecular Probes, Inc Eugene, OR
Bunthof CJ van Schalkwijk S Meijer W Abee T Hugenholtz J Fluorescent method for monitoring cheese starter permeabilization and lysis Appl Environ Microbiol 2001 67 4264 71 11526032 10.1128/AEM.67.9.4264-4271.2001
Bunthof CJ Sabina van den Braak S Breeuwer P Rombouts FM Abee T Rapid Fluorescence Assessment of the Viability of Stressed Lactococcus lactis Appl Envir Microbiol 1999 65 3681 3689
Lloyd D Hayes AJ Vigour, vitality and viability of microorganisms FEMS Microbiol Lett 1995 133 1 7 10.1016/0378-1097(95)00322-V
Miller JS Quarles JM Flow cytometric identification of microorganisms by dual staining with FITC and PI Cytometry 1990 11 667 675 1696535 10.1002/cyto.990110603
Darzynkiewicz Z Bruno S Del Bino G Gorczyca W Hotz MA Lassota P Traganos F Features of apoptotic cells measured by flow cytometry Cytometry 1992 13 795 808 1333943 10.1002/cyto.990130802
Sebastine IM Stocks PW Cox PW Thomas CR Characterization of percentage viability of Streptomyces clavuligerus using image analysis Biotechnol Tech 1999 13 419 423 10.1023/A:1008902330043
Mendez C Braña AF Manzanal MB Hardisson C Role of substrate mycelium in colony development in Streptomyces Can J Microbiol 1985 31 446 450 3891055
Kalakoutskii LV and Agre NS Comparative aspects of development and differentiation in actinomycetes Bacteriol Rev 1976 40 469 524 786257
Wildermuth H Development and organization of the aerial mycelium in Streptomyces coelicolor J Gen Microbiol 1970 60 43 50 5488465
Chater KF Hershberger CL, Queener SW, Hegeman G Aspects of multicellular differentiation in Streptomyces coelicolor A3(2) Genetics and Molecular Biology of Industrial Microorganisms 1989 American Society for Microbiology, Washington, DC 99 107
Kieser T Bibb MJ Buttner MJ Chater KF Hopwood DA Practical Streptomyces Genetics 2000 The John Innes Foundation, Norwich, England
Braña AF Mendez C Diaz LA Manzanal MB Hardisson C Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus J Gen Microbiol 1986 132 1319 1326 3534138
Miguelez EM Hardisson C Manzanal MB Hyphal death during colony development in Streptomyces antibioticus : morphological evidence for the existence of a process of cell deletion equivalent to apoptosis in a multicellular prokaryote J Cell Biol 1999 145 515 525 10225953 10.1083/jcb.145.3.515
Bayles KW Are the molecular strategies that control apoptosis conserved in bacteria? Trends Microbiol 2003 11 306 311 12875813 10.1016/S0966-842X(03)00144-6
Bibb MJ Regulation of secondary metabolism in Streptomycetes Curr Opin Microbiol 2005 8 208 215 15802254 10.1016/j.mib.2005.02.016
Pettis GS Ward N Schully KL Expression characteristics of the transfer-related kilB gene product of Streptomyces plasmid pIJ101: implications for the plasmid spread function J Bacteriol 2001 183 1339 1345 11157947 10.1128/JB.183.4.1339-1345.2001
Gehring AM Nodwell JR Beverley SM Losick R Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation Proc Natl Acad Sci U S A 2000 97 9642 9647 10931952 10.1073/pnas.170059797
Sprusansky O Zhou L Jordan S White J Westpheling J Identification of three new genes involved in morphogenesis and antibiotic production in Streptomyces coelicolor J Bacteriol 2003 185 6147 6157 14526027 10.1128/JB.185.20.6147-6157.2003
Champness WC Chater KF Piggot PJ, Morgan CP, Youngman P Regulation and integration of antibiotic production and morphological differentiation in Streptomyces sp Regulation of Bacterial Development 1994 American Society for Microbiology, Washington, DC 61 93
Chater KF Bibb MJ Kleinkauf H, v Dören H Regulation of bacterial antibiotic production Biotechnology Products of Secondary Metabolism 1997 7 VCH Press, Weinheim, Germany 57 105
Horinouchi S Ohnishi Y Kang DK The A-factor regulatory cascade and cAMP in the regulation of physiological and morphological development in Streptomyces griseus J Ind Microbiol Biotechnol 2001 27 177 182 11780789 10.1038/sj.jim.7000068
Ellaiah P Srinivasulu B Adinarayana K Optimisation studies on neomycin production by a mutant strain of Streptomyces marinensis in solid state fermentation Process Biochem 2004 39 1331 1339 10.1016/S0032-9592(03)00263-2
Jermini MF Demain AL Solid state fermentation for cephalosporin production by Streptomyces clavuligerus and Cephalosporium acremonium Experientia 1989 45 1061 1065 2599054 10.1007/BF01950159
Kota KP Sridhar P Solid state cultivation of Streptomyces clavuligerus for cephamycin C production Process Biochem 1999 34 325 328 10.1016/S0032-9592(98)00078-8
Stocks SM Thomas CR Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation Biotechnol Bioeng 2001 75 702 709 11745148 10.1002/bit.10017
Novella IS Barbes C Sanchez J Sporulation of Streptomyces antibioticus ETH 7451 in submerged culture Can J Microbiol 1992 38 769 773 1458369
Lowry OH Rosenbrough NJ Farr AL Randall RJ Protein measurement with the Folin phenol reagent J Biol Chem 1951 193 265 275 14907713
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-6-591615938810.1186/1471-2202-6-59Research ArticleEthanol induces cell-cycle activity and reduces stem cell diversity to alter both regenerative capacity and differentiation potential of cerebral cortical neuroepithelial precursors Santillano Daniel R [email protected] Leena S [email protected] Terasa L [email protected] Cynthia [email protected] Joseph D [email protected] Rajesh C [email protected] Department of Human Anatomy & Medical Neurobiology, Texas A&M University System Health Science Center, College of Medicine, College Station, TX, USA2 Centre for Environmental and Rural Health, Texas A&M University, College Station, TX, USA2005 13 9 2005 6 59 59 1 7 2005 13 9 2005 Copyright © 2005 Santillano 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 fetal cortical neuroepithelium is a mosaic of distinct progenitor populations that elaborate diverse cellular fates. Ethanol induces apoptosis and interferes with the survival of differentiating neurons. However, we know little about ethanol's effects on neuronal progenitors. We therefore exposed neurosphere cultures from fetal rat cerebral cortex, to varying ethanol concentrations, to examine the impact of ethanol on stem cell fate.
Results
Ethanol promoted cell cycle progression, increased neurosphere number and increased diversity in neurosphere size, without inducing apoptosis. Unlike controls, dissociated cortical progenitors exposed to ethanol exhibited morphological evidence for asymmetric cell division, and cells derived from ethanol pre-treated neurospheres exhibited decreased proliferation capacity. Ethanol significantly reduced the numbers of cells expressing the stem cell markers CD117, CD133, Sca-1 and ABCG2, without decreasing nestin expression. Furthermore, ethanol-induced neurosphere proliferation was not accompanied by a commensurate increase in telomerase activity. Finally, cells derived from ethanol-pretreated neurospheres exhibited decreased differentiation in response to retinoic acid.
Conclusion
The reduction in stem cell number along with a transient ethanol-driven increase in cell proliferation, suggests that ethanol promotes stem to blast cell maturation, ultimately depleting the reserve proliferation capacity of neuroepithelial cells. However, the lack of a concomitant change in telomerase activity suggests that neuroepithelial maturation is accompanied by an increased potential for genomic instability. Finally, the cellular phenotype that emerges from ethanol pre-treated, stem cell depleted neurospheres is refractory to additional differentiation stimuli, suggesting that ethanol exposure ablates or delays subsequent neuronal differentiation.
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Background
Children exposed to alcohol during gestation can exhibit a spectrum of abnormalities that range from Alcohol Related Neurodevelopmental Disorders (ARND) to Fetal Alcohol Syndrome (FAS), based upon the severity of symptoms. These abnormalities can include facial anomalies, growth deficits, mental retardation, attention deficit/hyperactivity disorders, motor difficulties, learning and memory impairment and psychological disorders such as depression [1-9]. Ethanol is teratogenic and exerts pleiotrophic effects in the differentiating nervous system, including induction of cell-death mechanisms [10-13], disruption of trophic support [14-17] and deregulation of neurotransmitter networks like the GABA, glutamate and serotonergic systems [18-24].
During prenatal period of neurogenesis, the number of neuroepithelial cells expands rapidly to generate most of the neurons of the adult brain [25] requiring, as with other tissues [26], the conversion of un-committed stem cells to more fate-restricted neuroblasts, and ultimately neurons. Ethanol exposure during gestation may alter the number and types of neuronal stem and blast precursors available for normal development, potentially producing irreversible damage to the developing brain. For example, previous research using BrdU incorporation analyses has shown that ethanol suppresses cell-cycle in the ventricular zone while promoting proliferation in the more mature subventricular zone [27]. The opposing effect of ethanol on these two cell populations indicates that ethanol disrupts the normal balance of precursor populations and suggests that ethanol may not affect all immature precursors similarly. We know little about the molecular heterogeneity of cortical neuroepithelial cells, though emerging evidence suggests that the neuroepithelium is quite heterogeneous with respect to differentiation and gene expression states of its constituent cells [28-30]. Cell surface markers like CD133/prominin-1, Sca-1 (Ly6A/E), CD117/c-kit and ABCG2 (ATP-binding cassette, sub-family G (WHITE), member 2) have been used successfully to monitor stem cell heterogeneity in a variety of tissues [31-34], and we therefore used these markers to monitor neuronal stem cell heterogeneity following ethanol exposure. We hypothesized that if ethanol influenced the proliferation of neuroepithelial cells, it would also alter the numbers of stem cells within the cortical neuroepithelium.
Finally, mechanisms that maintain genomic stability are important during neurogenesis because the frequency of DNA synthesis errors and aberrant chromatin assembly are increased during periods of robust proliferation. These errors must be limited, so that neural stem cells do not accumulate and transmit genetic damage to daughter cells. The telomerase complex, a reverse transcriptase enzyme complex, maintains telomeres during DNA replication [35-37], thereby preventing genomic instability and cellular senescence. Telomerase is active in all germline tissues, transformed cells and most human cancers [38,39], but is particularly robust during neurogenesis where it has been shown to function as an anti-apoptotic factor in developing neurons [40,41]. The activation of telomerase during neurogenesis, and the anti-apoptotic role of telomerase in neurons, led us to hypothesize that telomerase is a molecular target for ethanol during neuroblast expansion.
To examine the effects of ethanol on cortical precursor growth and survival, neurosphere cultures were generated from gestational day 15 rat fetuses. Contrary to our initial hypothesis, prolonged ethanol exposure promoted cell-cycle activity without concomitant increases in telomerase activity, or significant apoptosis. However, despite directly increasing cell cycle activity, ethanol suppressed expression of several stem cell markers, and decreased the future proliferation and differentiation potential of exposed cortical-derived neurospheres. Collectively, these data indicate that stem cell diversity, stability, and maturation are likely to be important components of the prenatal brain damage caused by maternal ethanol consumption.
Results
Neuronal progenitor cells, including multi-potent stem, and more committed blast cells, can be isolated from the developing cortex, expanded in-vitro as neurosphere aggregates, and subsequently used to generate neurons and glia [42-44]. Acutely dissociated cortical progenitors were cultured as neurosphere aggregates in serum-free mitogenic media to model the period of neuroblast precursor expansion during neurogenesis in the rodent. Cultured neurospheres were immuno-positive for nestin (Figure 1A), consistent with the hypothesis that neurospheres were comprised of immature cells. In contrast, immunofluorescence for the neuronal nuclear antigen (NeuN) was expressed within the soma of a few progenitor cells at the periphery of the neurosphere, but did not co-localize to any nucleus (Figure 1B). The presence of nestin [45] indicates that neurospheres are immature and multi-potent, and the lack of nuclear labeling for NeuN (Figure 1B) indicates that these cells are not yet committed to neuronal differentiation. In contrast, differentiation, induced by removal of Epidermal Growth Factor and culturing neurosphere-derived cells on a laminin substrate, leads to clear nuclear expression of NeuN (Figure 1C) and suppression of nestin immunoreactivity (data not shown), suggesting that neurosphere-derived cells can be committed to a neuronal lineage. Additionally, our flow cytometric data (Figure 2) indicate that, on average, less than 4% of cells in neurosphere cultures are apoptotic, suggesting that a majority of cells are viable in this culture model.
Ethanol exposure activates cell cycle activity in primary cortical neurosphere cultures and does not lead to apoptosis
Figure 2A (i-iii) illustrates the proportion of cells in cell cycle in response to ethanol treatment measured by flow cytometry. Exposure to ethanol for 4 days induced cell cycle activity at 120 mg/dl and 620 mg/dl, respectively. The proportion of cells in S-phase increased significantly (by 1.8–1.9 fold) after exposure to both doses of ethanol, relative to controls (Figure 2Bi). Furthermore, the ethanol-stimulated increase in the S-phase fraction was mirrored by a significant increase (1.8 – 2.5 fold) in the percentage of cells progressing to the G2 phase of the cell cycle (Figure 2Bii). The ratio of G2/S reflects the proportion of cells completing DNA synthesis and cellular division. At low ethanol concentrations (120 mg/dl), the G2/S ratio was similar to controls, but the ratio increased with higher ethanol concentrations (620 mg/dl) suggesting that the increase in DNA synthesis did result in progression to the G2 phase of the cell cycle (Figure 2Biii). Ethanol did not alter the number of cells with less than G0 DNA content (a marker of apoptosis, [46]), indicating that ethanol did not induce significant apoptosis in cortical-derived neurosphere cultures (Figure 2Biv).
Consistent with the above data, morphometric analyses indicate that ethanol significantly increased the density of neurospheres per field (Figure 3A–D). Analysis of variance followed by post-hoc statistical analyses indicated that ethanol induced a dose-related increase in the density of neurospheres (p < 0.001, Figure 3E). We observed a moderate but statistically significant correlation (Pearson product moment correlation (r) of 0.52 (p < 0.001)) between ethanol dose and density of neurospheres in the culture dish. When we controlled for the effect of neurosphere size on the density of neurospheres (i.e., the fact that an increase in the number of large neurospheres would effectively decrease the density of neurospheres within a field), the partial correlation coefficient between ethanol dose and neurosphere number increased (r = 0.71, p < 0.0001). While an overall analysis of variance indicated that ethanol did not statistically increase neurosphere size (Figure 3F), ethanol did lead to a ~2-fold increase in variation in neurosphere size at 120 mg/dl and a ~3-fold increase at 320 mg/dl within the culture dish, as indicated by an increase in the variance measure (i.e., Standard Deviation, Figure 3G). Therefore, ethanol-treated cultures exhibited a high degree of variability in neurosphere size compared to untreated cultures, and some of the neurospheres in ethanol treated cultures were extremely large (Figure 3C, D compared to 3A&B). It is possible that the ethanol-associated increased size and growth rate of neurospheres may translate into decreased viability of stem and progenitor pools within the neurosphere. However, this scenario is unlikely to be true since the overall rate of cell death was unchanged by treatment condition (Figure 2). Hence, these data collectively support the hypothesis that ethanol induced cell cycle in neurosphere cultures. One interesting observation was that ethanol-exposed neurospheres loose their spherical shape and exhibit irregular edges, suggesting either a structural disorganization of the neurosphere, perhaps due to variable growth rates within different parts of an individual neurosphere, or alternatively, the merging together of smaller neurospheres.
Our observation that ethanol-treated neurospheres exhibited increased heterogeneity in size and assumed a disorganized shape over time suggested that ethanol may promote differential responses among cells within an individual neurosphere. We therefore dissociated neurospheres (Figure 4A), and cultured individual cells in mitogenic medium with or without ethanol (120 mg/dl) to determine if individual cortical precursors could regenerate neurospheres. Over a four-day treatment period, control (Figure 4D) and ethanol-treated neural progenitors (Figure 4B) proliferated symmetrically, to generate morphologically similar daughter cells (symmetrical division, Figure 4E), and ultimately to generate new neurospheres over a 72-hour period (Figure 4G). Under the mitogenic conditions used (see cell culture methods), we only observed symmetrical cell division in control cultures. However, in contrast to control cultures, ethanol-treated neurospheres also exhibited an asymmetric mode of division, where one daughter cell assumed a more differentiated morphology compared to its mitotic partner (Figure 4C). One of the asymmetrically dividing pair of cells tended to be non-motile, while the second daughter cell exhibited extensive somatic motility (Figure 4F, H) over a period of 72 hours. In a majority of cases, the more morphologically differentiated member of the asymmetrically dividing pair assumed a stellate morphology. Occasionally, one of the pair of asymmetrically dividing daughter cells transiently expressed elongated, radial-glia-like processes (e.g., Figure 4F), before assuming a stellate appearance, despite the continued presence of mitogenic medium.
The presence of asymmetric cell division events in ethanol-treated cultures suggested that ethanol forces progenitor cell maturation, potentially depleting the numbers of self-replicating progenitors in neurosphere cultures. Therefore, we next determined the extent to which ethanol altered the future proliferation capacity of neural progenitor cells. We treated neurospheres with ethanol for four days; then dissociated the neurospheres into single cells that were cultured in mitogenic medium for two days. Prior exposure to ethanol led to a statistically significant (p < 0.0001, Figure 5) dose-related decline in the numbers of clonal colonies that were formed from dissociated cells, suggesting that a prior episode of ethanol ultimately depletes the proliferative capacity of cortical progenitors.
Ethanol suppresses cell-surface stem cell marker expression but not nestin mRNA expression in neurospheres
Neurosphere cultures consist of a heterogeneous mixture of immature neuronal stem cells and more differentiated daughter neuroblasts [47]. Based on our observations that ethanol promoted cell cycle activity, we hypothesized that ethanol would also lead to an aberrant expansion of the stem cell pool in neurosphere cultures. In the initial set of experiments, we utilized flow-cytometry to examine the expression of three stem cell markers, Sca-1 (Ly6A/E), CD117/c-kit and CD133/prominin-1. Figure 6 shows that these stem cell antigens are expressed in vitro in control proliferating neurosphere cultures. However, contrary to our hypothesis, we observed a large and statistically significant decrease in the numbers of cells expressing Sca-1 (~23-fold), CD117/C-kit (~9-fold) and CD133/prominin-1 (~19-fold) on their cell surface, after treatment with ethanol at 120 mg/dl (p < 0.05, N = 9 samples per stem cell antigen group). Increasing the dose of ethanol did not lead to a further reduction in stem cell antigen expression.
We wanted to examine the extent to which ethanol suppression of cell-surface stem cell marker expression was a generalized phenomenon that could be observed with other, more selective, stem-cell markers. Therefore, in our next experiment we examined the expression of the ATP-binding multidrug transporter ABCG2, an integral membrane protein that identifies stem cells in many tissues including the nervous system [48], and is considered a reliable marker for stem cells [49-52]. We examined the cellular expression of ABCG2 by flow cytometry in neurosphere cultures exposed to ethanol at the same dosages and time period. Figure 7A shows that ethanol significantly reduced the number of live cells that expressed ABCG2 immunofluorescence after 4 days (p < 0.05). These data suggest that ethanol does indeed suppress the expression of a specific cell-surface stem cell marker in neurosphere cultures.
In contrast to the above cell-surface markers, nestin is an intermediate filament protein that is expressed by both stem and progenitor cells within neurospheres ([45] and Figure 1A), and levels decline in differentiated post-mitotic neurons [53]. We hypothesized that if ethanol suppressed the expression of cell-surface stem cell markers, it would similarly suppress the expression of mRNA for nestin. However, semi-quantitative RT-PCR analysis (Figure 7B) showed that there was no change in nestin mRNA expression (normalized to cyclophilin-A) following ethanol exposure. These data suggest that while ethanol may not decrease the overall size of the precursor pool (the combined stem and blast pool, indicated by constant nestin mRNA expression), it decreases the diversity of stem cells within this pool (indicated by decreased numbers of cells expressing Sca-1, c-kit, CD133 and ABCG2).
Ethanol reduces telomerase reverse transcriptase (TERT) mRNA levels in neurosphere cultures
Telomerase reverse transcriptase (TERT) has been shown by others to function as a neuroprotective factor in developing neurons. Therefore, we tested the hypothesis that ethanol targets TERT transcription in progenitor neuroblasts. Reverse transcription PCR (Figure 8A, B) shows that TERT mRNA levels were modestly but statistically significantly increased (p < 0.05) at the low ethanol dosage (120 mg/ml). Conversely, high levels of ethanol (620 mg/dl) produced a statistically significant decrease in TERT mRNA levels. We hypothesized that the ethanol-induced changes in TERT mRNA levels would lead to changes in the activity of telomerase complex itself. However, quantitative measurements of telomerase activity indicate that low and high doses of ethanol do not affect the activity of the telomerase complex (Figure 8C). The cycle threshold values in the low and high ethanol-treated groups were identical, reflecting equal amounts of telomerase activity in each sample. The increase in TERT transcription following low doses of ethanol (120 mg/dl) was not followed by a measurable increase in telomerase activity, an unexpected observation in light of the marked induction of DNA synthesis and cell-cycle progression illustrated in Figure 2A, B. These results suggest that ethanol may uncouple telomerase activation from neuroepithelial cell proliferation.
Ethanol prevents subsequent differentiation of cells derived from neurosphere cultures
In the final set of experiments, we examined the effect of ethanol on the subsequent differentiation potential of cortical progenitor cells. We hypothesized that ethanol induction of cell proliferation, would alter the ability of cortical progenitors to differentiate into neurons. Rat neurosphere cultures we maintained in control medium or exposed to the low or moderate doses of ethanol (120 or 320 mg/dl) for 4 days. Following this period of ethanol exposure, we dissociated neurospheres and cultured the constituent cells at low density in the presence of 10 nM retinoic acid. Neurosphere-derived cells were cultured at low density so that individual cells would have a low probability of contacting another cell. This protocol was followed to reduce the impact of cell-cell interactions and target-derived trophic support mechanisms on neuronal differentiation. We selected retinoic acid as the differentiation stimulus, since previous work in our laboratory showed that retinoic acid is a strong trigger for cortical neuroepithelial differentiation [54]. Overall, ethanol pre-exposure led to a significant dose-related decline in the number of first-order (p < 0.0001) and second-order (p < 0.003) branches that were induced following retinoic acid exposure (Figure 9A). Naïve cells, and cells derived from neurospheres pre-exposed to the low dose of ethanol (120 mg/dl) exhibited a strong differentiation phenotype in response to retinoic acid (Figure 9B, C), including the expression of second-order neurite branching. However, cells derived from neurospheres pre-exposed to 320 mg/dl exhibited a marked reduction in neurite branching in response to retinoic acid (Figure 9D).
Discussion
Our data show that ethanol did not induce appreciable apoptosis in embryonic cortical-derived neurosphere cultures. These data are somewhat surprising because the activation of cell death mechanisms is intuitively consistent with ethanol's adverse effects on brain development. However, ethanol induction of cell death may be differentiation stage-specific. For example, previous research in our laboratory demonstrated that ethanol concentrations equal to those used in this study induced apoptosis in more differentiated postnatal cortical [10], and cerebellar explant cultures [11], perhaps because tumor-suppressor genes like p53, and down-stream pro-apoptotic genes like Bax are suppressed in proliferating cortical cells and only induced during differentiation [54]. Clearly, more research is needed to identify genes and mechanisms that confer apoptosis-resistance to precursors, but not differentiated neuronal cells.
Ethanol promotes cell-cycle activity
Rather than killing cells, ethanol stimulated DNA synthesis and promoted cell-cycle progression in cerebral cortical precursors as indicated by the increase in the size variation and number of neurospheres, induction of S-phase, and increased progression through G2/M phases of the cell cycle. On the other hand, ethanol induced asymmetric division in progenitor cells and decreased the cell surface expression of a number of stem cell markers, i.e., c-kit/CD117, Sca-1 (Ly6A/E), CD133/prominin-1 and the ABCG2 transporter. In contrast to the induction of cell cycle, asymmetric cell division and loss of stem cell antigens are both indicative of neuroepithelial maturation. These apparently antagonistic data are best explained within the context of normal cerebral cortex development. Neurogenesis in the developing cerebral cortex, occurs within two distinct germinal zones, the earlier developing ventricular zone, and the later developing subventricular zone. Proliferation within the ventricular zone serves to replenish stem and blast pools (by symmetrical division) and to generate more mature, fate-restricted daughter progeny (by asymmetrical division, [55]). The ethanol-induced asymmetric division and loss of stem cell markers supports the notion that ethanol induces maturation of the stem cell pool. The daughter blast cells or 'proto-neurons' that exit the ventricular zone, migrate in turn to the subventricular zone where they may continue to proliferate and generate neurons, principally by symmetrical division [55]. The increased proliferation that we observed in ethanol-treated cultures may therefore reflect the expansion of a 'subventricular-zone-like phenotype'. Recent evidence suggests that the first blast cells that migrate out of the ventricular zone are in fact radial glia, and that these cells are also precursors for mature neurons (for review, see [56]). This view is consistent with our observations that asymmetric division occasionally resulted in the appearance of polarized cells with long (radial-like) processes that fit morphological criteria for radial glia. Further support for this 'two-zone' hypothesis of ethanol's action comes from our observations that ethanol treatment does ultimately deplete the regenerative capacity of cortical neuroepithelial precursors. Cells derived from ethanol-pretreated neurospheres exhibited a decreased ability to form new colonies, implying that the loss of stem cell markers does indeed mean that ethanol depletes stem cells from the pool of immature precursors.
Overall, this interpretation of these data in terms of stem cell maturation is also consistent with previously published work from other laboratories using alternate labeling indices, showing that ethanol decreases cell proliferation within the ventricular zone, while increasing cell proliferation within the more differentiated subventricular zone [27,57,58]. Increased cell proliferation is a requirement for differentiation in other tissues as well. For example, in the hematopoietic system, the differentiation of CD34+ stem cells into pro-erythroblasts is accompanied by a significant increase in cell cycle. This induction of cell cycle supports successive stages of blast maturation, till the formation of the orthochromatophilic erythroblast, the immediate blast precursor to the differentiated erythrocyte [26].
The consequences of enhanced proliferation on neural differentiation remain to be determined. The diversity of cellular subpopulations that contribute to the lamination of the cortical plate is likely to be disrupted, leading to an overabundance of some neuroblast populations at the expense of others. Such population imbalances may be one cause of phenomena like cellular heterotopias that have been observed in brains of children with FAS [59], and in animal models of FAS [60]. While in vitro models suggest that migration defects contribute to the formation of heterotopias [61], the role of aberrant ethanol-driven expansion of specific progenitor pools in heterotopia formation cannot be ruled out.
Ethanol limits cortical stem cell diversity in neurosphere cultures
Neurosphere cultures consist of a heterogeneous mixture of multi-potent neuronal stem cells and daughter neuroblasts [47]. CD133/prominin-1 identifies stem cell groups with multi-potent properties in the developing central nervous system of humans and rodents [62-66]. Sca-1 (Ly6A/E) identifies hematopoietic cells with the potential to form neurons, while CD117/C-kit [67-69] and the ABCG2 multi drug-resistance transporter [48] are expressed in stem cell precursors of the rodent central nervous system. We demonstrate that populations of cortical precursors do express these stem cell markers. Nestin mRNA was also expressed in cultured cortical precursors, but nestin mRNA levels did not change with ethanol exposure, indicating that despite maturational pressure, immature, possibly blast-type cells continue to persist in ethanol-treated cultures. However nestin mRNA expression is not informative about the extent to which ethanol affects the heterogeneity of stem cells in culture, because nestin expression is a common phenotype of stem and more differentiated blast cells. In contrast, ethanol significantly decreased the numbers of cells that expressed cell-surface Sca-1, c-kit, CD133 and ABCG2. The absence of an appreciable degree of apoptosis in ethanol-treated cultures, suggests that ethanol influences the diversity of the stem cell pool, rather than cell survival per se. In the context of the previously noted requirement for cell cycle induction as a component of stem cell to blast cell transformation [26], it is interesting to note that the suppression of the stem cell marker Sca-1 [70]) promotes myoblast proliferation. Consequently, our observations that ethanol induces cell cycle and decreases the expression of stem cell markers is mutually consistent with the hypothesis that ethanol drives stem cell to blast cell transformation in cortical neuroepithelial precursors.
The functions ascribed to the stem cell markers that we utilized in this study are particularly relevant to the issue of ethanol's impact on cell fate determination. For example, CD133 and c-kit are important components of lipid raft micro-domains [71,72], that sequester cell-signaling machinery. In bone marrow, Stem Cell Factor/c-kit interactions form a critical component of stromal-driven differentiation of several hematopoietic lineages [73]. Therefore, changes in the composition of lipid rafts are likely to result in significant alterations to signaling mechanisms that drive stem cell differentiation, and in the brain, lead to aberrant development. Sca-1 is also a signaling molecule, linked to the cell-surface by a phosphatidyl-inositol anchor, and is important for stimulating hematopoietic stem cell renewal [74]. Sca-1 expression is suppressed in hematopoietic stem cells, during the process of lymphocyte differentiation [75]. While Sca-1 may well exhibit different developmental-stage associated kinetics in the neuroepithelium, ethanol-dependent depletion of Sca-1 in the neuroepithelium may similarly drive precursor maturation and prevent the renewal of a stem cell pool. Interestingly, both Sca-1 [70] and CD117/c-kit [76,77] share the Src-like tyrosine kinase Fyn as a common signaling intermediary, suggesting that the two cell-surface molecules mediate common functions during development. Fyn in turn, mediates ethanol-sensitivity and dependence in the adult animal, and a Fyn polymorphism is predictive of alcohol dependence in human populations [78-80], further suggesting that ethanol's effects in the developing and adult brain are likely to be mediated by common mechanisms. Finally, ethanol suppression of the expression of multi-drug resistance transporters like ABCG2 is also likely to be clinically significant. Multi-drug resistance transporters are thought to protect stem cells from damage by non-selectively extruding a wide variety of cytotoxic compounds [52]. Ethanol exposure, by suppressing ABCG2 expression, may decrease the survivability of neural stem cells in the face of subsequent toxic insults, including perhaps, subsequent episodes of ethanol exposure.
Ethanol's effect on Telomerase activity and TERT transcription
Rapidly dividing tissues are susceptible to errors in DNA synthesis that can lead to DNA damage, chromosomal instability and cell death. The telomerase complex maintains chromosomal ends, and consequently ensures chromosome stability during DNA synthesis (reviewed in [81-83]). Telomerase activity is robust during embryogenesis [84] and is particularly elevated in the proliferating neuroepithelium. Though activity is decreased following cellular differentiation, the catalytic component of telomerase (TERT) continues to be expressed widely in post-mitotic neurons where it appears to function as a neuroprotective factor [40,41,85]. The lower dose of ethanol (120 mg/dl) resulted in increased TERT mRNA levels, perhaps as a compensatory protective response. In contrast, the suppression of TERT mRNA expression at high doses (620 mg/dl) is inconsistent with the observed increase in cell cycle progression at that dose. Interestingly, TERT physically associates with the anti-apoptotic kinase AKT [86], and a reduction in TERT expression levels has the potential to render dividing cortical precursors susceptible to future apoptotic stimuli, (for example, see [41,87,88]), even if ethanol itself does not induce apoptosis.
Because we observed that ethanol increased cell cycle activity in neurosphere cultures, we hypothesized that ethanol would also increase telomerase activity. Surprisingly, ethanol had no effect on telomerase activity. The lack of coordination between telomerase activity and changes in cell cycle activity suggests that DNA synthesis is likely to be incomplete in telomeric regions, increasing genomic instability in proliferating, ethanol-exposed neuroblasts. Recent research indicates that the prenatal rodent neuroepithelium normally exhibits a significant degree of genetic mosaicism in which 33% of all cortical precursors display some degree of chromosomal aneuploidy [89] and loss of heterozygosity [90]. It is surprising that the developing neuroepithelium tolerates such a significant degree of genetic instability without negative sequellae. However, given a high tolerance for mosaicism, any increases in genetic instability among ethanol-exposed cells of the neuroepithelium may not result in apoptosis, but in altered patterns of gene expression due to mechanisms like gene shedding [90], consequently altering neural differentiation. An intriguing possibility, supported by the discrepancy between telomerase and cell cycle data, is that the accumulation of progenitors with greater than G0 DNA content in ethanol-treated cultures represents increased aneuploidy rather than increased progression through cell cycle per se.
Ethanol pre-treatment prevents stimulus-dependent neuronal differentiation
The loss of stem cell markers, and evidence for increased cell division led us to hypothesize that ethanol exposure would promote the further maturation of the cortical stem and blast cells. However, contrary to our hypothesis, ethanol pre-treated neural progenitor cells were refractory to retinoic acid stimulation. Though retinoic acid is a potent stimulator of neuronal differentiation [54], it appears to have growth-stimulatory effects in part, by stimulating neurotrophic signaling via factors like glial-derived neurotrophic factor and neurotrophin-3 [91]. Since ethanol has been previously shown to interfere with growth factor signaling mechanisms in a variety of neural differentiation models [11,14-17,92], it is likely that ethanol disrupts growth factor signaling to interfere with the response to retinoic acid as well. The developmental consequences of delays in neuronal differentiation are likely to be profound. Even if the effect of ethanol is transient, the inside-out lamination of the cortical plate is likely to be significantly disrupted, because later-generated neural precursors do not appear to be able to populate earlier-developing cortical plate laminae, after the critical period for the generation of that specific lamina has been passed [93].
Conclusion
In toto, our data support the hypothesis that the elimination of neural stem cell diversity contributes to the etiology of prenatal brain damage caused by maternal ethanol consumption. Rather than inducing apoptosis, ethanol stimulates cell-cycle activity and eliminates stem cell antigen expression. It is likely therefore, that ethanol drives the stem cell to blast cell transformation, ultimately depleting the reserve regenerative capacity of cerebral cortical neuroepithelium. Secondly, the uncoupling of telomerase activity from cell-cycle induction suggests that ethanol exposure may hasten telomere decay and cellular senescence, rendering maturing neuroblasts susceptible to genetic damage as they proceed through cycles of maturation-driven proliferation. Finally, if the ventricular and subventricular zones are indeed 'proto-maps' of the mature cortical plate [28], restrictions in the diversity of the stem cell pool are likely to translate into loss of diversity of neuronal and glial phenotypes that populate the mature cortical plate. While the developmental consequences of perturbing the cortical stem cell pool have not been adequately assessed in experimental systems of ethanol-induced brain damage, one likely sequel to such perturbations is a permanent transformation in the phenotypic composition of the neural network of the cerebral cortex.
Methods
Isolation of embryonic neural precursors
The University Laboratory Animal Care Committee approved all animal procedures. Timed-pregnant Sprague-Dawley rats (gestational day [GD] 13) were purchased from Harlan, Houston, Texas, and maintained in the animal housing facility at Texas A&M University System Health Sciences Center, College of Medicine for two days until the pregnancy matured to GD15. At GD 15, pregnant females were anesthetized with ketamine (0.09 mg/gram)/xylazine (0.106 mg/gram) by intraperitoneal injection. Under aseptic conditions, the gravid uterus was delivered through a midline transverse abdominal incision. The gravid uterus was rinsed in chilled PBS containing 1% penicillin/streptomycin. Eight to ten fetuses were isolated and rinsed three times in chilled PBS. Anesthesia was achieved by placing fetuses in ice-cold Gey's Balanced Salt Solution. Fetuses were rapidly decapitated, and whole brains were removed and placed in chilled Gey's Balanced Salt Solution supplemented with glucose and magnesium chloride. Brains remained suspended in this solution throughout the remainder of the microdissection procedure. Meningeal tissue was removed, regions of the rat fetal brain corresponding to the structural precursor of the neocortex were isolated, and care was taken to exclude the structural precursors to the striatum and hippocampus. Individual cortical fragments were collected in sterile 15 ml conical tubes and gently triturated in trypsin/EDTA. Trypsin was inactivated with DMEM containing 10% fetal bovine serum. The cell suspension was centrifuged for 5 minutes at 18°C, 1000 rpm (300 × g). Cell pellets were resuspended in chilled PBS containing 0.5% BSA, Fraction-V, (#1526037, Invitrogen) and 2.0 mM EDTA. Total cell counts were determined using a hemocytometer
Cell culture
Neurosphere cultures were established from acutely dissociated cortices (described above). Precursor cultures were established at an initial density of 106 cells in T-25 flasks containing 5–6 ml of serum-free mitogenic media (DMEM/F12 (#11330-032 Invitrogen), 20 ng/ml bFGF (#13256-029 Invitrogen), 20 ng/ml hEGF (#53003-018 Invitrogen), ITS-X supplement (#51500-056 Invitrogen), 0.85 Units/ml heparin (#15077-019 Invitrogen), and 20 nM progesterone (#P6149 Sigma)). Cultures were incubated at 37°C, 5% CO2 in a humidified environment for 48–72 hours before the commencement of ethanol treatments to allow for stabilization of culture and for the generation of small neurospheres, a sign of precursor expansion. For some experiments, following ethanol treatment, neurosphere cultures were dissociated and differentiated for 4.5 days in the presence of 10 nM retinoic acid (in DMEM/F12 and 1%N2 supplement).
Ethanol treatment
Cultures were assigned to three treatment groups: (I) a control group containing no ethanol; (II) a low dose group containing 120 mg/dl (26.07 mM); (III) a high dose group containing 620 mg/dl (134.78 mM, prepared from 95% ethanol, molecular biology grade-Sigma). For some experiments, an intermediate, dose of ethanol, 320 mg/dl (69.56 mM) was used in place of the high dose. Gas chromatographic analyses indicated that the low and high doses resulted in ethanol levels of 97–106 mg/dl (21.09 mM–23.04 mM) and 367–557 mg/dl (79.35 mM–121.09 mM) respectively, while the moderate dose of ethanol resulted in measured levels between 182–227 mg/dl (39.57 mM–49.35 mM). These concentrations are within the range that has been previously observed in chronic alcoholics [94,95]. Doses of 150 mg/dl and 200 mg/dl have been used previously in cell culture models of chronic ethanol exposure [96,97]. The ethanol concentrations used in this study are expected to reflect the levels in the fetus during prenatal exposure, as it has been shown in rodent models that maternal blood alcohol levels produce equivalent concentrations in the fetus [98]. The ethanol treatment lasted 4 days. Ethanol containing media was replaced daily throughout the duration of the treatment period.
Labeling for stem cell surface antigen expression
Neurosphere cultures were dissociated in Accumax solution (Innovative Cell Technologies) by gentle trituration through a fire polished pipette. Precursors were labeled with phycoerythrin-conjugated antibodies to Sca-1/Ly6A/E (1 ug, Caltag Labs, MM4104), CD117/c-kit (1 ug, BD Pharmingen, 555714), CD133/prominin-1 (0.55 ug, Miltenyi Biotec, AC141), BCRP1 (ABCG2)-FITC (1 ug, Chemicon International MAB4155F) or isotype-matched IgG. 106 live cells were incubated separately with each of the above antibodies for 10 minutes at 4°C and immediately analyzed by flow cytometry. In each group, an equal amount of unlabeled IgG isotype-matched antibody was added to the labeling solution to serve as a blocking reagent for potential Fc-Receptor sites on the cell surface. Between 10,000 and 100,000 cells were counted in each sample. Staining levels from cells labeled with phycoerythrin-conjugated IgG isotype-matched antibodies served as a measure of background fluorescence. This background was subtracted from all groups labeled with stem cell antigen markers and flow cytometric data were collected for this population of cells. The proportion of labeled cells was expressed as the percentage of total cells gated.
Propidium iodide staining for DNA content
Neurosphere cultures were dissociated in Accumax solution (Innovative Cell Technologies) as mentioned above. Dissociated cells were collected by brief centrifugation at 1000 rpm, 18°C and resuspended in cold PBS. An equal volume of 2% phosphate-buffered paraformaldehyde was added to yield a final concentration of 1% paraformaldehyde. Cells were fixed for 45 minutes at 4°C. Fixed cells were washed twice in PBS and resuspended in PBS containing 0.5 mM EDTA, 0.1% Triton X-100, 0.05 mg/ml RNAse A (#R4642 Sigma). Cells were incubated at 37°C for 30 minutes to allow for RNA degradation. Propidium iodide (#1348639 Roche) was added to the solution at a final dilution of 1:50 and incubated for at least 30 minutes at 4°C. At least 10,000 cells were analyzed by flow cytometry.
Flow cytometry
Cell cycle analysis and measurements of stem cell antigen levels were conducted on a FACS Calibur Flow Cytometer (Beckton Dickinson). Excitation wavelength was set at 488 nm (Argon laser) and emission spectra for phycoerythrin and propidium iodide were 575 nm and 630 nm, respectively. Cell-cycle histograms were generated using Cell Quest software for Macintosh.
Immuno-fluorescence analysis
Neurosphere cultures were assayed for the expression of the neuroepithelial marker Nestin, and neuronal marker NeuN (neuronal nuclear antigen), according to previously published protocols [54]. After 3.5–4 days, media was removed and cultures were washed in PBS, fixed in 1% phosphate-buffered paraformaldehyde for 45 minutes at room temperature. Cultures were rinsed twice with PBS and once with TBS, followed by incubation in blocking solution (2% normal serum, 0.1% BSA, 0.2% triton x-100, in TBS) for 1 hour at room temperature. Antibodies (all from Chemicon) against nestin 1:100 (MAB3353) and NeuN 1:100 (MAB377), were diluted in staining solution (TBS 0.1% BSA), and incubated with cells overnight at 4°C. Following three washes in TBS, cells were labeled with rat-adsorbed, biotinylated secondary horse anti-mouse antibodies (1:250) (Vector Laboratories) in TBS according to manufacturers instructions, and antibody binding was visualized with by conjugation with avidin-rhodamine 1:250 (Vector Laboratories). Cells were mounted in Fluorescence Mounting Media containing DAPI (Vector Laboratories).
RNA extraction and cDNA synthesis
RNA was extracted using the Trizol reagent in accordance with the manufacturer's instructions (Invitrogen). RNA was purified with the SV Total RNA Isolation System (#Z3100 Promega). 2.5 micrograms of total RNA was used to synthesize cDNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen #18080-051) and random hexamers according to the manufacturer's protocol.
Polymerase chain reaction (PCR)
PCR was performed by conventional thermocycling methods. 2 uls of cDNA mixture from above was combined with the primers (200 nM each) and PCR Supermix (#10572-014, Invitrogen) in a total volume of 50 ul. Primers for TERT were those used in (Holzmann et al., 2003). PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. The cyclophilin-A (Peptidyl-prolyl cis-trans isomerase A) gene served as an internal control measure [99,100]. Forward and reverse primer sequences are as indicated in Table 1.
Measurement of telomerase activity by TRAP Assay
Telomerase is a reverse transcriptase ribonucleoprotein complex that catalyzes the addition of six base pair repeats to telomeric ends [101-103]. The PCR-based Telomeric Repeat Amplification Protocol (TRAP), to measure telomerase activity in biological samples, was adapted from [38], as described in [104], based on SYBR-Green I fluorescence and real-time PCR. PCR reactions were performed with SYBR Green JumpStart TaqReady Mix (Sigma #S4438). Cell pellets were homogenized in 200 uL of cold lysis buffer and chilled on ice for 30 minutes, then re-sedimented by centrifugation at 16,000 × g for 20 minutes at 4°C, then lysed in CHAPS lysis buffer [85]. Lysates were quantified for total protein content and diluted to a working concentration of 100 ng/ul and stored at -80°C until use. The TS and ACX primers used for both the telomerase elongation and the amplification of telomerase elongation products are as described in [105]. The following components were mixed in a 25 ul volume and analyzed for telomerase activity; 13 ul SYBR Jumpstart Mix, 0.1 ug (1 ul) TS primer, 0.05 ug (1 ul) ACX primer (for primer sequence, see Table 1), 0.2 ul T4 Gene 32 Protein (1 unit, Roche 972983), 1 ul extract (100–500 ng) and water to 25 ul final volume. SYBR Green fluorescence intensity was quantified during the 60°C annealing/elongation step and expressed graphically as a function of cycle number. Cycle threshold values (Ct) are an indirect measurement of telomerase activity and represent the cycle number at which the generated product reaches a preset threshold level. Thresholds were set at 10 standard deviations above background fluorescence.
Data analysis
TERT and nestin mRNA levels were quantified by densitometric measurement, and were expressed as a ratio of cyclophilin-A mRNA expression (Molecular Analyst for Windows, BioRad). For TRAP assays, Sybr-Green fluorescence intensities were plotted against cycle number, and cycle threshold (ct) determined. Neurosphere number and size were computed using a Java version of NIH image (ImageJ, V1.32j (NIH)). Data were analyzed using a standard statistical package, SPSS for Windows (Version 11). Analysis of Variance (ANOVA) and the Fischer's Least Significant Difference post-hoc test were used to identify statistically significant differences between groups. Alternatively, Pearson's correlations, and two-tailed tests of significance were computed to determine relationships between ethanol dose and response. Statistical significance was set at p < 0.05.
List of abbreviations
CD117 C-kit
CD133 prominin-1
ABCG2 ATP-binding cassette, sub-family G (WHITE), member 2; member of the ATP-dependent multi-drug resistance transporter family; aka. BCRP1
FAS Fetal Alcohol Syndrome
NeuN Neuronal nuclear antigen
Sca-1 Stem Cell Antigen-1 or Ly6A/E
TERT Telomerase reverse transcriptase subunit
Authors' contributions
DRS and RCM contributed to the design of the experiments, data analysis and manuscript preparation. DRS, LSK, TLP, CC and JDT contributed to the cell culture, immunofluorescence and flow cytometric analyses. DRS and JDT contributed the RT-PCR and telomerase activity assay. All authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank Dr. Jane Miller for technical assistance with flow cytometry, and Drs. James West and Wei-Jung Chen for critical review of this manuscript. This research was supported by grants from National Institute of Alcohol Abuse & Alcoholism (#AA13440) and The Texas Tobacco Endowment Fund to RCM.
Figures and Tables
Figure 1 Photomicrographs depicting immunofluorescence analysis of neurospheres labeled for intermediate filament proteins nestin (A), and the neuronal lineage marker, the neuronal-specific nuclear antigen, NeuN (B and lower magnification inset). Cell nuclei were counter-stained with DAPI (blue). Control neurosphere cultures are immuno-positive for nestin. However, cultures do not exhibit nuclear localization of NeuN, showing that neurosphere cultures were comprised of immature cells. (C) Differentiation following removal of the mitogen EGF, and dispersion of cells onto a laminin substrate, results in the upregulation and nuclear localization of NeuN. Photomicrograph represents a digitally merged immunofluorescence and phase contrast microscopic image showing NeuN immunofluorescence overlying nuclei (white arrows) of early differentiating neurons. Scale bars: 50 uM.
Figure 2 (A) Cell-cycle analysis of cortical progenitors treated with ethanol for 4 days. (Ai-iii) Flow-cytometric frequency histograms of progenitors stained with propidium iodide (PI) for DNA content. (Aii,iii) Ethanol stimulates DNA synthesis and cell-cycle progression, as indicated by the increase in area under the S-phase and G2/M peaks, relative to controls. Bi-iii, Quantitative analyses of cell-cycle. (Bi,ii) Ethanol significantly increased the number of cells entering S and G2/M-phases of the cell-cycle. (Biii) The G2/S ratio was unchanged at low ethanol doses, but significantly increased with the high dose. (Biv) Ethanol did not induce apoptosis at either dose used, and very little DNA fragmentation was observed in the sub-G0/1 range in (Ai-iii). Asterisks indicate statistical-significance, p < 0.05.
Figure 3 Ethanol increases neurosphere number and increases variation in the size of neurospheres. (A-D) Representative photomicrographs of control (A,B) and ethanol-treated (C,D) neurospheres showing that ethanol (at 5 days exposure) increases both the density and size variation of neurospheres. Arrows mark the appearance of large neurospheres in ethanol-treated cultures. (E-G) Morphometric analyses show that ethanol induces a significant increase in the density of neurospheres (E), without altering the mean area per neurosphere (in square pixels, F). However, ethanol induced a dose-related increase in variation (Standard Deviation) in neurosphere size (G). Asterisks indicate statistical-significance, p < 0.05. Scale bar: A-D, 100 uM.
Figure 4 Ethanol induces asymmetric division of neural progenitor cells. Dissociated neural progenitor cells (A) cultured under mitogenic conditions divide symmetrically to generate two similar daughter cells (D, representative examples of cells undergoing cytokinesis), and regenerate new neurospheres (data not shown). Ethanol (120 mg/dl)-treated neural progenitors also generate daughter cells by symmetrical division (B, examples of cells in different stages of cytokinesis), to regenerate neurospheres over a period of 44 (E) and 72 (G) hours, under mitogenic conditions. However, despite being cultured under mitogenic conditions, ethanol-treated progenitors also exhibit asymmetric division to generate morphologically dissimilar daughter cells (C, examples of asymmetrical division, showing daughter cells in different stages of cytokinesis). Over the period of 44 to 72 hours, one daughter cell exhibits somatic mobility (e.g., F,H, cell marked by a *), while the second daughter cells remains stationary (e.g., F,H, cell marked by +). The motile daughter cell transiently exhibits long filamentous processes (F,*), though these processes retract over a period of 72 hours (H,*), in mitogenic medium.
Figure 5 Quantitative analysis of the effect of ethanol-pretreatment on the regenerative capacity of neural progenitor cells. Neurosphere cultures were treated with varying doses of ethanol for five days, then dissociated and cultured as individual progenitor cells, in ethanol-free mitogenic medium. Ethanol pre-treatment leads to a dose-related decline in the subsequent ability of neural progenitors to undergo cell division (mean number of dividing cells per well ± SEM) and generate clonal colonies of cells. Asterisks indicate statistical-significance, p < 0.05.
Figure 6 Quantitative analysis of flow-cytometric data for stem-cell antigen expression in cortical progenitors. Progenitors express stem-cell antigens (control). After 4 days of treatment, low doses of ethanol significantly reduced the proportion of cells expressing Sca-1, CD117/C-kit and CD133. The high dose of ethanol did not further reduce stem-cell antigen expression. Asterisks indicate statistical-significance, p < 0.05. Data were collected from at least 10,000 events/sample.
Figure 7 (A) ABCG2 Transporter expression in cortical progenitors after ethanol treatment. Cultures w ere exposed to ethanol and live, immuno-labeled cells were processed for FACS analysis (see methods) with antibodies to surface ABCG2. Ethanol significantly reduced the expression of ABCG2 (p < 0.05). Data were collected from 100,000 events/sample. (B) RT-PCR analysis for nestin in cortical progenitors treated with ethanol for 4 days. The levels of each transcript were unchanged by ethanol. Transcript levels were normalized to cyclophilin (data not shown).
Figure 8 (A,B) TERT mRNA expression and telomerase activity in cortical-derived neurospheres treated with ethanol for 4 days. (A), RT-PCR for the TERT transcript. (B), Quantification of TERT levels in A, expressed as a ratio to cyclophilin. Low doses of ethanol modestly, but significantly, increased TERT transcript levels. However, high doses of ethanol decreased TERT mRNA expression. (C), TRAP assays revealed no differences in telomerase activity between treated and control groups. The cycle threshold value (Ct) indirectly measures activity; a lower Ct corresponds to higher telomerase activity. Inset table indicates Ct ± Standard Deviation (SD). Color code: Black, control; blue, 120 mg/dl; red, 620 mg/dl; pink, no-telomerase control. Asterisks indicate statistical-significance, p < 0.05.
Figure 9 Pre-treatment with ethanol disrupts retinoic acid-mediated differentiation. Neurosphere cultures were exposed to a dose range of ethanol for 4 days, dissociated into a single cell suspension, plated and then differentiated with retinoic acid (A) Quantitative analysis of the mean number (± SEM) of primary and secondary branches per cell, formed in the presence of retinoic acid, as a function of ethanol pre-treatment dose. Asterisks indicate statistically significant differences from non-ethanol exposed, control cultures, * = p < 0.003; ** = p < 0.0001. (B-E) Representative photomicrographs of branching patterns observed in the presence of retinoic acid, in control (B), low (C) and moderate doses (D,E) of ethanol. Arrows point to second-order branches, which are missing in cells derived from neurosphere cultures exposed to 320 mg/dl ethanol. Scale bar, B-E, 25 uM
Table 1 List of PCR primers
Primer Name Sequence Product Size Accession #/Reference
TS primer 5'-ATTCCGTCGAGCAGAGTT-3' [105]
ACX primer 5'-GCGCGG [CTTACC]3CTAACC-3'
R-Tert_Forward GGTCTTCCGCACGTTGGTTG 349bp [106]
R-Tert_Reverse CAGCAGGTAGAGCGCACAGT
Rat_Nestin_Forward TGCAGCCACTGAGGTATCTG 1061bp Acc#: M34384
Rat_Nestin_Reverse AGTTCCCACTCCTGTGGTTG
Cyclophilin A_Forward TGGTCAACCCCACCGTGTTCTTCG 372bp Acc#: M19533
Cyclophilin A_Reverse TGCCATCCAGCCACTCAGTCTTGG
==== Refs
Coles CD Platzman KA Raskind-Hood CL Brown RT Falek A Smith IE A comparison of children affected by prenatal alcohol exposure and attention deficit, hyperactivity disorder. Alcohol Clin Exp Res 1997 21 150 161 9046388
Kodituwakku PW May PA Clericuzio CL Weers D Emotion-related learning in individuals prenatally exposed to alcohol: an investigation of the relation between set shifting, extinction of responses, and behavior Neuropsychologia 2001 39 699 708 11311300 10.1016/S0028-3932(01)00002-1
Mattson SN Riley EP Delis DC Stern C Jones KL Verbal learning and memory in children with fetal alcohol syndrome Alcohol Clin Exp Res 1996 20 810 816 8865953
Mattson SN Goodman AM Caine C Delis DC Riley EP Executive functioning in children with heavy prenatal alcohol exposure. Alcohol Clin Exp Res 1999 23 1808 1815 10591598
Roebuck TM Simmons RW Mattson SN Riley EP Prenatal exposure to alcohol affects the ability to maintain postural balance. Alcohol Clin Exp Res 1998 22 252 258 9514315
Roebuck TM Mattson SN Riley EP Behavioral and psychosocial profiles of alcohol-exposed children. Alcohol Clin Exp Res 1999 23 1070 1076 10397293
Schonfeld AM Mattson SN Lang AR Delis DC Riley EP Verbal and nonverbal fluency in children with heavy prenatal alcohol exposure. J Stud Alcohol 2001 62 239 246 11327190
Thomas SE Kelly SJ Mattson SN Riley EP Comparison of social abilities of children with fetal alcohol syndrome to those of children with similar IQ scores and normal controls. Alcohol Clin Exp Res 1998 22 528 533 9581664
Streissguth AP O'Malley K Neuropsychiatric implications and long-term consequences of fetal alcohol spectrum disorders Semin Clin Neuropsychiatry 2000 5 177 190 11291013 10.1053/scnp.2000.6729
Cheema ZF West JR Miranda RC Ethanol induces Fas/Apo [apoptosis]-1 mRNA and cell suicide in the developing cerebral cortex. Alcohol Clin Exp Res 2000 24 535 543 10798591 10.1097/00000374-200004000-00029
McAlhany REJ West JR Miranda RC Glial-derived neurotrophic factor (GDNF) prevents ethanol-induced apoptosis and JUN kinase phosphorylation Developmental Brain Research 2000 119 209 216 10675770 10.1016/S0165-3806(99)00171-6
Mooney SM Miller MW Ethanol-induced neuronal death in organotypic cultures of rat cerebral cortex Brain Res Dev Brain Res 2003 147 135 141 14741758 10.1016/j.devbrainres.2003.08.012
Mooney SM Miller MW Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus Brain Res 2001 911 71 81 11489446 10.1016/S0006-8993(01)02718-4
Heaton MB Mitchell JJ Paiva M Walker DW Ethanol-induced alterations in the expression of neurotrophic factors in the developing rat central nervous system Developmental Brain Research 2000 121 97 107 10837897 10.1016/S0165-3806(00)00032-8
Luo J West JR Pantazis NJ Ethanol exposure reduces the density of the low-affinity nerve growth factor receptor (p75) on pheochromocytoma (PC12) cells Brain Research 1996 737 34 44 8930347 10.1016/0006-8993(96)00657-9
McAlhaney REJ Miranda RC Finnell RH West JR Ethanol decreases Glial-Derived Neurotrophic Factor (GDNF) protein release but not mRNA expression and increases GDNF-stimulated Shc phosphorylation in the developing cerebellum. Alcohol Clin Exp Res 1999 23 1691 1697 10550003
Miller R King MA Heaton MB Walker DW The effects of chronic ethanol consumption on neurotrophins and their receptors in the rat hippocampus and basal forebrain Brain Research 2002 950 137 147 12231238 10.1016/S0006-8993(02)03014-7
Olney JW Wozniak DF Farber NB Jevtovic-Todorovic V Bittigau P Ikonomidou C The enigma of fetal alcohol neurotoxicity Annals of Medicine 2002 34 109 119 12108574 10.1080/07853890252953509
Zhou FC Sari Y Li TK Goodlett C Azmitia EC Deviations in brain early serotonergic development as a result of fetal alcohol exposure Neurotox Res 2002 4 337 342 12829423 10.1080/10298420290030532
Hsiao SH DuBois DW Miranda RC Frye GD Critically timed ethanol exposure reduces GABAAR function on septal neurons developing in vivo but not in vitro Brain Res 2004 1008 69 80 15081384 10.1016/j.brainres.2004.02.020
Hsiao SH Parrish AR Nahm SS Abbott LC McCool BA Frye GD Effects of early postnatal ethanol intubation on GABAergic synaptic proteins Brain Res Dev Brain Res 2002 138 177 185 12354645 10.1016/S0165-3806(02)00470-4
Hsiao SH Acevedo JL DuBois DW Smith KR West JR Frye GD Early postnatal ethanol intubation blunts GABA(A) receptor up-regulation and modifies 3alpha-hydroxy-5alpha-pregnan-20-one sensitivity in rat MS/DB neurons Brain Res Dev Brain Res 2001 130 25 40 11557091 10.1016/S0165-3806(01)00194-8
Savage DD Queen SA Sanchez CF Paxton LL Mahoney JC Goodlett CR West JR Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring Alcohol 1992 9 37 41 1346364 10.1016/0741-8329(92)90007-W
Savage DD Montano CY Otero MA Paxton LL Prenatal ethanol exposure decreases hippocampal NMDA-sensitive [3H]-glutamate binding site density in 45-day-old rats Alcohol 1991 8 193 201 1648928 10.1016/0741-8329(91)90806-8
Bayer SA Altman J Neocortical Development 1991 New York, Raven Press
Dai MS Mantel CR Xia ZB Broxmeyer HE Lu L An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2 Blood 2000 96 3985 3987 11090089
Miller MW Effects of prenatal exposure to ethanol on neocortical development: II. Cell proliferation in the ventricular and subventricular zones of the rat. The Journal of Comparative Neurology 1989 287 326 338 2778108 10.1002/cne.902870305
Donoghue MJ Rakic P Molecular gradients and compartments in the embryonic primate cerebral cortex Cereb Cortex 1999 9 586 600 10498277 10.1093/cercor/9.6.586
Nery S Fishell G Corbin JG The caudal ganglionic eminence is a source of distinct cortical and subcortical cell populations Nat Neurosci 2002 5 1279 1287 12411960 10.1038/nn971
Zecevic N Specific characteristic of radial glia in the human fetal telencephalon Glia 2004 48 27 35 15326612 10.1002/glia.20044
Bunting KD ABC Transporters as Phenotypic Markers and Functional Regulators of Stem Cells Stem Cells 2002 20 11 20 11796918 10.1634/stemcells.20-3-274
Miraglia S Godfrey W Yin AH Atkins K Warnke R Holden JT Bray RA Waller EK Buck DW A Novel Five-Transmembrane Hematopoietic Stem Cell Antigen: Isolation, Characterization, and Molecular Cloning Blood 1997 90 5013 5021 9389721
Yin AH Miraglia S Zanjani ED Almeida-Porada G Ogawa M Leary AG Olweus J Kearney J Buck DW AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells Blood 1997 90 5002 5012 9389720
Zhu G Chang Y Zuo J Dong X Zhang M Hu G Fang F Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-regulated and can up-regulate the expression of GAPDH Biochem Biophys Res Commun 2001 281 951 956 11237753 10.1006/bbrc.2001.4439
Broccoli D Young JW De Lange T Telomerase activitiy in normal and malignant hematopoetic cells PNAS 1995 92 9082 9086 7568077
Hiyama K Hirai Y Kyoizumi S Akiyama M Hiyama E Piatyszek MA Shay JW Ishioka S Yamakido M Activation of telomerase in human lymphocytes and hematopoietic progenitor cells Immunol 1995 155 3711 3715
Wright WE Piatyszek MA Rainey WE Byrd W Shay JW Telomerase activity in human germline and embryonic tissues and cells Developmental Genetics 1996 18 173 179 8934879 10.1002/(SICI)1520-6408(1996)18:2<173::AID-DVG10>3.0.CO;2-3
Kim NW Piatyszek MA Prowse KR Harley CB West MD Ho PL Coviello GM Wright WE Weinrich SL Shay JW Specific association of human telomerase activity with immortal cells and cancer Science 1994 266 2011 2015 7605428
Rhyu MS Telomeres, telomerase and immortality J Nat Cancer Inst 1995 87 884 894 7666477
Fu W Begley JG Mattson MP Anti-apoptotic role of telomerase in pheochromocytoma cells J Biol Chem 1999 274 7264 7271 10066788 10.1074/jbc.274.11.7264
Fu W Killen M Culmsee C Dhar S Pandita TK Mattson MP The catalytic subunit of telomerase is expressed in developing brain neurons and serves a cell survival-promoting function Journal of Molecular Neuroscience 2000 14 3 15 10854032 10.1385/JMN:14:1-2:003
Reynolds BA Tetzlaff W Weiss S A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes J Neurosci 1992 12 4565 4574 1432110
Reynolds BA Weiss S Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992 255 1707 1710 1553558
Vescovi AL Reynolds BA Fraser DD Weiss S bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 1993 11 951 966 8240816 10.1016/0896-6273(93)90124-A
Lendahl U Zimmerman LB McKay RD CNS stem cells express a new class of intermediate filament protein. Cell 1990 60 585 595 1689217 10.1016/0092-8674(90)90662-X
Cheema ZF Santillano DR Wade SB Newman JM Miranda RC The extracellular matrix, p53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors, and reciprocally, Fas-ligand modifies estrogen control of cell-cycle proteins BMC Neurosci 2004 5 11 15038834 10.1186/1471-2202-5-11
Kukekov VG Laywell ED Suslov O Davies K Scheffler B Thomas LB O'Brien TF Kusakabe M Steindler DA Multipotent Stem/Progenitor Cells with Similar Properties Arise from Two Neurogenic Regions of Adult Human Brain Experimental Neurology 1999 156 333 344 10328940 10.1006/exnr.1999.7028
Cai J Cheng A Luo Y Lu C Mattson MP Rao MS Furukawa K Membrane properties of rat embryonic multipotent neural stem cells J Neurochem 2004 88 212 226 14675165
Zhou S Schuetz JD Bunting KD Colapietro AM Sampath J Morris JJ Lagutina I Grosveld GC Osawa M Nakauchi H Sorrentino BP The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype Nat Med 2001 7 1028 1034 11533706 10.1038/nm0901-1028
Kim M Turnquist H Jackson J Sgagias M Yan Y Gong M Dean M Sharp JG Cowan K The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells Clin Cancer Res 2002 8 22 28 11801536
Kim M Morshead CM Distinct populations of forebrain neural stem and progenitor cells can be isolated using side-population analysis J Neurosci 2003 23 10703 10709 14627655
Sarkadi B Ozvegy-Laczka C Nemet K Varadi A ABCG2 -- a transporter for all seasons FEBS Lett 2004 567 116 120 15165903 10.1016/j.febslet.2004.03.123
Dahlstrand J Lardelli M Lendahl U Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system Developmental Brain Research 1995 84 109 129 7720210 10.1016/0165-3806(94)00162-S
Wade SB Oommen P Conner W Earnest D Miranda RC Overlapping and divergent actions of estrogen and the neurogrophins on cell fate and p-53 dependent signal transduction in conditionally immortalized cerebral cortical neuroblasts J Neurosci 1999 15 6994 7006
Noctor SC Martinez-Cerdeno V Ivic L Kriegstein AR Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases Nat Neurosci 2004 7 136 144 14703572 10.1038/nn1172
Ever L Gaiano N Radial 'glial' progenitors: neurogenesis and signaling Curr Opin Neurobiol 2005 15 29 33 15721741 10.1016/j.conb.2005.01.005
Miller MW Nowakowski RS Effect of prenatal exposure to ethanol on the cell cycle kinetics and growth fraction in the proliferative zones of fetal rat cerebral cortex Alcohol Clin Exp Res 1991 15 229 232 2058800
Miller MW Limited ethanol exposure selectively alters the proliferation of precursor cells in the cerebral cortex Alcohol Clin Exp Res 1996 20 139 143 8651443
Clarren SK Alvord ECJ Sumi SM Streissguth AP Smith DW Brain malformations related to prenatal exposure to ethanol J Pediatr 1978 92 64 67 619080
Kotkoskie LA Norton S Prenatal brain malformations following acute ethanol exposure in the rat. Alcohol Clin Exp Res 1988 12 831 836 3064646
Mooney SM Siegenthaler JA Miller MW Ethanol Induces Heterotopias in Organotypic Cultures of Rat Cerebral Cortex Cereb Cortex 2004 14 1071 1080 15166098 10.1093/cercor/bhh066
Corbeil D Roper K Fargeas CA Joester A Huttner WB Prominin: A Story of Cholesterol, Plasma Membrane Protrusions and Human Pathology Traffic 2001 2 82 91 11247306 10.1034/j.1600-0854.2001.020202.x
Corbeil D Fargeas CA Huttner WB Rat prominin, like its mouse and human orthologues is a pentaspan membrane glycoprotein Biochem Biophys Res Commun 2001 285 939 944 11467842 10.1006/bbrc.2001.5271
Sawamoto K Nakao N Kakishita K Ogawa Y Toyama Y Yamamoto A Yamaguchi M Mori K Goldman SA Itakura T Okano H Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene J Neurosci 2001 21 3895 3903 11356877
Uchida N Buck DW He D Reitsma MJ Masek M Phan TV Tsukamoto AS Gage FH Weissman IL Direct isolation of human central nervous system stem cells PNAS 2000 97 14720 14725 11121071 10.1073/pnas.97.26.14720
Vogel W Gruneback F Messam CA Kanz L Brugger W Buhring HJ Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica 2003 88 126 133 12604402
Jin K Mao XO Sun Y Xie L Greenberg DA Stem cell factor stimulates neurogenesis in vitro and in vivo. J Clin Invest 2002 110 311 319 12163450 10.1172/JCI200215251
Lee JB Kuroda S Shichinohe H Ikeda J Seki T Hida K Tada M Sawada K Iwasaki Y Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice Neuropathology 2003 23 169 180 14570283 10.1046/j.1440-1789.2003.00496.x
Sanchez-Ramos J Song S Cardozo-Pelaez F Hazzi C Stedeford T Willing A Freeman TB Saporta S Janssen W Patel N Adult Bone Marrow Stromal Cells Differentiate into Neural Cells in Vitro Experimental Neurology 2000 164 247 256 10915564 10.1006/exnr.2000.7389
Epting CL Lopez JE Shen X Liu L Bristow J Bernstein HS Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells J Cell Sci 2004 117 6185 6195 15546912 10.1242/jcs.01548
Giebel B Corbeil D Beckmann J Hoehn J Freund D Giesen K Fischer J Koegler G Wernet P Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells Blood 2004 [Epub ahead of print]
Jahn T Seipel P Coutinho S Urschel S Schwarz K Miething C Serve H Peschel C Duyster J Analysing c-kit internalization using a functional c-kit-EGFP chimera containing the fluorochrome within the extracellular domain Oncogene 2002 21 4508 4520 12085229 10.1038/sj.onc.1205559
Broudy VC Stem cell factor and hematopoiesis Blood 1997 90 1345 1364 9269751
Ito CY Li CY Bernstein A Dick JE Stanford WL Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice Blood 2003 101 517 523 12393491 10.1182/blood-2002-06-1918
Chen HC Frissora F Durbin JE Muthusamy N Activation induced differential regulation of stem cell antigen-1 (Ly-6A/E) expression in murine B cells Cell Immunol 2003 225 42 52 14643303 10.1016/j.cellimm.2003.09.006
Sette C Paronetto MP Barchi M Bevilacqua A Geremia R Rossi P Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase Embo J 2002 21 5386 5395 12374739 10.1093/emboj/cdf553
Paronetto MP Venables JP Elliott DJ Geremia R Rossi P Sette C Tr-kit promotes the formation of a multimolecular complex composed by Fyn, PLCgamma1 and Sam68 Oncogene 2003 22 8707 8715 14647465
Schumann G Rujescu D Kissling C Soyka M Dahmen N Preuss UW Wieman S Depner M Wellek S Lascorz J Bondy B Giegling I Anghelescu I Cowen MS Poustka A Spanagel R Mann K Henn FA Szegedi A Analysis of genetic variations of protein tyrosine kinase fyn and their association with alcohol dependence in two independent cohorts Biol Psychiatry 2003 54 1422 1426 14675807 10.1016/S0006-3223(03)00635-8
Yaka R Tang KC Camarini R Janak PH Ron D Fyn kinase and NR2B-containing NMDA receptors regulate acute ethanol sensitivity but not ethanol intake or conditioned reward Alcohol Clin Exp Res 2003 27 1736 1742 14634488 10.1097/01.ALC.0000095924.87729.D8
Yaka R Phamluong K Ron D Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol J Neurosci 2003 23 3623 3632 12736333
Bouffler SD Involvement of telomeric sequences in chromosomal aberrations. Mutation Research 1998 404 199 204 9729384
Holt SE Shay JW Role of telomerase in cellular proliferation and cancer. J Cell Physiol 1999 180 10 18 10362013 10.1002/(SICI)1097-4652(199907)180:1<10::AID-JCP2>3.0.CO;2-D
Oulton R Harrington L Telomeres, telomerase, and cancer: life on the edge of genomic stability. Curr Opin Oncol 2000 12 74 81 10687733 10.1097/00001622-200001000-00013
Yamaguchi Y Nozawa K Savoysky E Hayakawa N Nimura Y Yoshida S Change in telomerase activity of rat organs during growth and aging Experimental Cell Research 1998 242 120 127 9665809 10.1006/excr.1998.4102
Klapper W Shin T Mattson MP Differential regulation of telomerase activity and TERT expression during brain development in mice J Neurosci Res 2001 64 252 260 11319769 10.1002/jnr.1073
Haendeler J Hoffmann J Rahman S Zeiher AM Dimmeler S Regulation of telomerase activity and anti-apoptotic function by protein-protein interaction and phosphorylation FEBS Letters 2003 536 180 186 12586360 10.1016/S0014-5793(03)00058-9
Zhu H Fu W Mattson MP The catalytic subunit of telomerase protects neurons against amyloid beta-peptide-induced apoptosis Journal of Neurochemistry 2000 75 117 124 10854254 10.1046/j.1471-4159.2000.0750117.x
Lu C Fu W Mattson MP Telomerase protects developing neurons against DNA damage-induced cell death. Brain Res Dev Brain Res 2001 131 167 171 11718848 10.1016/S0165-3806(01)00237-1
Rehen SK McConnell MJ Kaushal D Kingsbury MA Yang AH Chun J Chromosomal variation in neurons of the developing and adult mammalian nervous system PNAS 2001 98 13361 13366 11698687 10.1073/pnas.231487398
Kaushal D Contos JJ Treuner K Yang AH Kingsbury MA Rehen SK McConnell MJ Okabe M Barlow C Chun J Alteration of gene expression by chromosome loss in the postnatal mouse brain J Neurosci 2003 23 5599 5606 12843262
Thang SH Kobayashi M Matsuoka I Regulation of glial cell line-derived neurotrophic factor responsiveness in developing rat sympathetic neurons by retinoic acid and bone morphogenetic protein-2 J Neurosci 2000 20 2917 2925 10751444
Dohrman DP West JR Pantazis NJ Ethanol reduces expression of the nerve growth factor receptor, but not nerve growth factor protein levels in the neonatal rat cerebellum. Alcohol Clin Exp Res 1997 21 882 893 9267539
Desai AR McConnell SK Progressive restriction in fate potential by neural progenitors during cerebral cortical development Development 2000 127 2863 2872 10851131
Adachi J Mizoi Y Fukunaga T Ogawa Y Ueno Y Imamichi H Degrees of alcohol intoxication in 117 hospitalized cases. Journal of Studies on Alcohol 1991 52 448 453 1943100
Perper JA Twerski A Wienand JW Tolerance at High Blood Alcohol Concentration: A study of 110 Cases and Review of the Literature. Journal of Forensic Sciences 1986 31 212 221 3511175
Netzeband JG Schneeloch JR Trotter C Caguioa-Aquino JN Gruol DL Chronic ethanol treatment and withdrawal alter ACPD-evoked calcium signals in developing Purkinje neurons. Alcohol Clin Exp Res 2002 26 386 393 11923593
Webb B Walker DW Keaton MB Nerve growth factor and chronic ethanol treatment alter calcium homeostasis in developing rat septal neurons Developmental Brain Research 2003 143 57 71 12763581 10.1016/S0165-3806(03)00100-7
Gottesfeld Z Morgan B Perez-Polo JR Prenatal alcohol exposure alters the development of sympathetic synaptic components and of nerve growth factor receptor expression selectivity in lymphoid organs Journal of Neuroscience Research 1990 26 308 316 2168949 10.1002/jnr.490260307
Miranda R Sohrabji F Singh M Toran-Allerand D Nerve growth factor (NGF) regulation of estrogen receptors in explant cultures of the developing forebrain. Journal of Neurobiology 1996 31 77 87 9120438 10.1002/(SICI)1097-4695(199609)31:1<77::AID-NEU7>3.0.CO;2-C
Sohrabji F Miranda RCG Toran-Allerand CD Identification of a Putative Estrogen Response Element in the Gene Encoding Brain-Derived Neurotrophic Factor PNAS 1995 92 11110 11114 7479947
Greider CW Blackburn EH Identification of a specific telomere terminal transferase activity in Tetrahymena extracts Cell 1985 43 405 413 3907856 10.1016/0092-8674(85)90170-9
Greider CW Blackburn EH A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis Nature 1989 337 331 337 2463488 10.1038/337331a0
Lingner J Hughes TR Shevchenko A Mann M Lundblad V Cech TR Reverse transcriptase motifs in the catalytic subunit of telomerase Science 1997 267 567
Wege H Chui MS Le HT JM T Zern MA SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity. Nucleic Acids Research 2003 31 E3 3 12527792 10.1093/nar/gng003
Kim NW Wu F Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP) Nucleic Acids Research 1997 25 2595 2597 9185569 10.1093/nar/25.13.2595
Holzmann K Berger W Mejri D Cerni C Sasgary S Detection and quantification of transcripts for the catalytic subunit TERT and the RNA component of telomerase in rat tissue. Analytical Biochemistry 2003 317 120 123 12729609 10.1016/S0003-2697(03)00091-5
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-6-611618802710.1186/1471-2202-6-61Research ArticleRLIP76, a non-ABC transporter, and drug resistance in epilepsy Awasthi Sanjay [email protected] Kerri L [email protected] Vince [email protected] Sharad S [email protected] Luca [email protected] Yogesh C [email protected] Gabriele [email protected] Damir [email protected] Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX USA2 Cerebrovascular Research, Department of Neurological Surgery, Cleveland Clinic Foundation, Cleveland, OH USA3 Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland, OH USA4 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, Galveston, TX USA2005 27 9 2005 6 61 61 5 4 2005 27 9 2005 Copyright © 2005 Awasthi 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
Permeability of the blood-brain barrier is one of the factors determining the bioavailability of therapeutic drugs and resistance to chemically different antiepileptic drugs is a consequence of decreased intracerebral accumulation. The ABC transporters, particularly P-glycoprotein, are known to play a role in antiepileptic drug extrusion, but are not by themselves sufficient to fully explain the phenomenon of drug-resistant epilepsy. Proteomic analyses of membrane protein differentially expressed in epileptic foci brain tissue revealed the frequently increased expression of RLIP76/RALBP1, a recently described non-ABC multi-specific transporter. Because of a significant overlap in substrates between P-glycoprotein and RLIP76, present studies were carried out to determine the potential role of RLIP76 in AED transport in the brain.
Results
RLIP76 was expressed in brain tissue, preferentially in the lumenal surface of endothelial cell membranes. The expression was most prominent in blood brain barrier tissue from excised epileptic foci. Saturable, energy-dependent, anti-gradient transport of both phenytoin and carbamazepine were demonstrated using recombinant RLIP76 reconstituted into artificial membrane liposomes. Immunotitration studies of transport activity in crude membrane vesicles prepared from whole-brain tissue endothelium showed that RLIP76 represented the dominant transport mechanism for both drugs. RLIP76-/- knockout mice exhibited dramatic toxicity upon phenytoin administration due to decreased drug extrusion mechanisms at the blood-brain barrier.
Conclusion
We conclude that RLIP76 is the predominant transporter of AED in the blood brain barrier, and that it may be a transporter involved in mechanisms of drug-resistant epilepsy.
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Background
Each year approximately 160,000 persons are diagnosed with epilepsy, and about 10,000 of these individual develop drug-resistant epilepsy [1]. The causes of drug-resistant epilepsy are numerous, many due to ontogenic abnormalities in brain maturation, severe brain injuries with resultant irreversible changes to cerebral neuroglial organization and inhibitory neuron function, kindling phenomenon, seizure-induced disturbances of oxygen supply, as well as acquired (or hereditary) changes in transporter proteins of the blood-brain barrier which function in the efflux of anti-epileptic drugs (AEDs) from the brain. The latter mechanisms have been the focus of intense efforts to develop new rationally designed AEDs that could bypass these transport mechanisms. Unfortunately, the identity of the multiple transport mechanisms of the blood brain barrier, and the individual role of each in mediating drug-resistant epilepsy is as yet incompletely defined.
The ABC-family transporters have been the subject of considerable interest in the mechanisms of drug-resistant epilepsy [2,3]. The prototypical ABC transporter, P-glycoprotein (Pgp, MDR1, or mdr1 gene product), as well as MRP2, and BCRP are expressed in the blood-brain barrier [2]. Endothelial expression of Pgp has been demonstrated, and the role of Pgp in regulating brain drug-concentrations has been established in knockout mouse studies [4]. These studies evaluated the transport of a number of centrally acting drugs including antipsychotics, ant emetics, and natural product antineoplastic agents. The only AED examined, phenytoin (PHE), appeared to be a weak substrate of Pgp in intact cell transport experiments. Carbamazepine (CBZ) has been found not to inhibit Pgp mediated transport, thus would be a poor substrate [5]. Recently, studies examining the ability of AED to competitively inhibit the transport activity of Pgp have found little effect of AEDs on Pgp activity, and then only at concentrations well above clinically achieved therapeutic levels [6]. Although Pgp appears to mediate some minor transport activity towards most CNS active agent, the greatest CSF/plasma ratios (6.6–17 fold) were seen with antipsychotics and antiemetics rather than AEDs, evidence has been presented which suggests that there have been no reports directly demonstrating transport of any AED by Pgp in an isolated system [7].
In epileptic patients, expression of MDR1 has a complex pattern that does not directly support a significant pharmacokinetic role for MDR1 in human epilepsy since MDR1 expression was found in both blood-brain barrier and parenchymal cells in epileptic brain [8-10]. This is based on the possibility that expression of MDR1 in glia may actually favor drug interactions with neuronal by reducing the accumulation of drug in the glial syncitium. Conversely, parenchymal expression may shift concentrations in the extracellular and intracellular space or may mediate compartmentalization, and thus may reduce concentrations of antiepileptic drugs at their target sites. The fact that MDR1 may not be a crucial determinant for multiple drug resistance to antiepileptic drugs has been recently challenged in a number of reports [11-13]. Other ABC-transporters, most notably MRP2 and BCRP have also been localized to the blood-brain barrier, but similarly, direct evidence for the ability of these proteins to transport AEDs in isolated systems has been lacking. Because of the lack of information regarding the kinetic parameters of transport, the relative contributions of different ABC transporters in mediating drug-resistant epilepsy remains unknown and a major impediment to development of novel targeted anti-epileptic agents.
We have recently described a novel non-ABC mutispecific transporter, RLIP76, a multifunctional modular protein found ubiquitously from Drosophila to humans [14-17]. It is encoded in humans on chromosome 18p11.3 by a gene with 11 exons and 9 introns. RLIP76 is a 76 kDa protein product of this gene, but splice-variants including a 67 kDa peptide and longer 80 or 102 kDa peptide, cytocentrin have been identified [18]. RLIP76 was cloned as a Ral-binding protein and predicted to be an effector involved in regulation of membrane plasticity, movement, and endocytosis [17,19-21]. RLIP76 was identified as a highly active efflux mechanisms for removing glutathione-electrophile conjugate (GS-E, i.e. LTC4) from cells [22]. In addition to GS-E, the exceptionally broad substrate specificity of RLIP76 extends to Pgp substrates including anthracycline and vinca alkaloids, towards which it mediates resistance [23-27]. Studies on proteomic screening of epileptic foci led to the identification of RLIP76 as being frequently up-regulated. Present studies were carried out to examine the role of RLIP76 in AED transport in the blood-brain barrier.
Results and discussion
RLIP76 was expressed in all normal human tissues examined, more prominently in breast, heart, liver and erythrocytes, and less so in colon and brain parenchyma (Fig. 1a, b &1c). Significant expression was seen in malignant human cell lines including the PC-3 human prostate cancer and H1299 human non-small cell lung cancer cell lines. Whereas RLIP76 was barely detectable in normal brain parenchyma or vessels, blood vessels from epileptic patients has a markedly increased expression of RLIP76 (Fig. 1b &1c).
Epileptic brain sections obtained from multiple drug resistant patients also revealed substantial differences compared to non-epileptic brain. By immunocytochemical analysis, we discovered widespread RLIP76 expression in the cerebral vasculature from epileptic brain. We used double label immunofluorescence to reveal that RLIP76 co-localized with the multidrug resistance transporter P-glycoprotein (MDR1) but not with NeuN or GFAP (Fig. 1d, e, f, and 1h). However, while MDR1 was expressed in both parenchymal and endothelial cells [8,9], RLIP76 immunoreactivity was limited to the vasculature. No overlapping expression of RLIP76 was observed in GFAP positive astroglia (Fig. 1e) or NeuN positive neurons (Fig. 1d). By confocal analysis, RLIP76 expression was found in capillary endothelial cells, penetrating pial vessels, and larger (>100 μm) vessels (Fig. 1d, e, and 1f). In capillary endothelial cells, MDR1 expression was both lumenal (endothelial) and abluminal (glial endfeet), whereas RLIP76 expression appeared to be predominantly lumenal and did not co-localize with GFAP immunoreactivity (Fig. 1g and 1h). Additional studies are needed to confirm this finding.
Since the predominant endothelial localization of RLIP76 suggested a strategic role in determining multiple drug resistance to antiepileptic drugs, we tested the ability of RLIP76 to extrude the classic antiepileptic drug PHE [28] (Fig. 2). This was examined in an isolated system consisting of asolectin-cholesterol artificial liposomes reconstituted in the presence of purified human RLIP76 [23,24]. Uptake of PHE by liposomes with RLIP76 or without (control) was examined in the presence or absence of ATP. Presence of ATP in the transport medium increased the uptake of 14C-PHE by RLIP76-liposomes in a dose dependent fashion, whereas ATP had no effect on uptake by control liposomes (Fig. 2a). We also determined that the intra-vesicular concentration of PHE in control liposomes with or without ATP and in RLIP76 liposomes without ATP were near the extra-vesicular drug-concentration (1 μM), while RLIP76-liposomes in the presence of ATP had intra-vesicular PHE concentrations of 5.7 μM, demonstrating that in the presence of ATP, RLIP76 liposomes are able to concentrate PHE against a gradient, the hallmark of active transport.
Similar findings were obtained when investigating ATP-dependent transport of another classic antiepileptic drug carbamazepine (CBZ). While it is commonly believed that PHE is an MDR1 substrate, uncertainty exists on CBZ extrusion by MDR1 [5,29]. Uptake of PHE or CBZ by inside-out vesicles was a time dependent process with kinetics consistent with a single compartment filling model (Fig. 2b), and the initial velocity of transport could be reasonably estimated by measuring uptake at 2 min after addition of ATP. Both antiepileptic drugs (AED) were transported by RLIP76 liposomes (Fig. 2d) Initial velocity kinetics performed with varying either substrate (ATP or CBZ/PHE), while holding the other constant showed that the Km for PHE and CBZ was 0.43 and 0.25 μM respectively (Fig. 2e) and for ATP 1.33 mM (PHE) and 3.3 mM (CBZ) (Fig. 2c).
We then compared function and levels of expression of RLIP76 in non-epileptic brain resected during cerebrovascular surgery unrelated to epileptic pathology or drug resistance vs. epileptic brain. Expression of RLIP76 was determined by Western blot of tissue blocks and mRNA analysis in isolated and cultured brain microvascular endothelial cells (Fig. 2f–h). We found that RLIP76 mRNA expression was greater in epileptic brain or endothelial cells isolated from the same tissue and that RLIP76 protein levels correlated with PHE transport activity measured in inside-out vesicles prepared from brain tissue (Fig. 2h).
Functional RLIP76 expression and AED transport activity were further studied in freshly collected human brain samples from epileptic patients. To determine the relative contribution of RLIP76 toward total PHE/CBZ efflux capacity, we examined drug transport in the absence or presence of anti-RLIP76 antibodies [23] in crude membrane vesicles prepared from freshly collected human epileptic brain tissue (n = 8; Fig. 3a, b). Anti-RLIP76 inhibited total transport of PHE and CBZ by 64 ± 6 and 74 ± 1.82% respectively (p < 0.01). When we repeated the same experiments with anti-MDR1 antibodies, we found inhibition by 21 ± 9 (PHE) and 13 ± 1.86 (CBZ) %; exposure to both antibodies resulted in a cumulative inhibition. Interestingly, the amount of PHE extrusion by MDR1 determined by antibody-mediated inactivation was comparable to that described in a previous study after pharmacological blockade by XR9576, a specific MDR1 blocker [30]. These findings indicated that RLIP76 is the predominant AED transporter in brain tissue.
For the above experiments, we directly obtained vesicles from acutely isolated cortical samples. Under these conditions, culture artifacts are avoided but the individual contribution of a given cell type remains undetectable. To determine the cell type involved in the process of RLIP76-mediated PHE extrusion we compared 14C-PHE transport in vesicles obtained from endothelial cells and astrocytes isolated and cultured from either control (n = 4) or epileptic (n = 6) brain [31] (Fig. 3c, d). PHE transport was significantly higher in endothelial cells as compared to astrocytes, and greater in both cell types from epileptic tissues. These data are in agreement with the immunocytochemical results showing predominant expression of RLIP76 in human epileptic endothelial cells and not brain parenchymal cells.
PHE has been described as an MDR1 substrate based on experiments performed on knock out mice lacking Pgp [32]. We performed experiments in RLIP76+/+ and RLIP76-/- C57B mice injected i.p. with PHE (33 or 83 mg/kg, 6 animals per group; Fig. 3e). At both doses, brain PHE levels were higher in RLIP76-/- mice as compared with RLIP76+/+ (p < 0.05). However, this was more prominent at the 83 mg/kg level. A statistically significant increase in PHE accumulation in brain was observed at both concentrations (Fig. 3f). RLIP76-/- mice were characterized by higher levels of PHE in both brain and serum compared with the wild-type, consistent with a role of RLIP76 in renal excretion of PHE (not shown). To account for this, a group of wild-type animals was injected with elevated (4166 mg/kg) doses of PHE to achieve serum levels comparable to those seen in knock out animals injected with much lower quantities (83 mg/kg). RLIP76-/- mice demonstrated greater neurotoxicity after administration of PHE; side effects in these animals included lethargy and status-epilepticus (Fig. 3g).
Conclusion
Taken together, our results show that RLIP76 is an important PHE and CBZ transporter at the human blood brain barrier, and its expression is increased in the BBB from patients with drug-resistant epilepsy. RLIP76 fulfilled many of the predicted properties for a mediator of CNS pharmacoresistance, including: 1) presence at the anatomical interface between brain and blood; 2) transport of the antiepileptic drugs PHE and CBZ; 3) functional expression in brain microvascular endothelial cells but not in parenchymal glia or neurons; and 4) increased CNS accumulation of PHE in RLIP76-/- mice. These results also demonstrate for the first time that the putative mediator of multiple drug resistance in epilepsy, MDR1 [33], is in fact overshadowed in potency by another ATP-dependent transporter. Our results are also in accord with previous reports which questioned the relevance of MDR1 as a multiple drug resistance mechanism, while rather suggesting a role in neuroglial protection [10]. We also confirmed that CBZ is a poor MDR1 substrate [34].
Several important questions remain unanswered. For example, is the widespread distribution of MDR1, MRPs (in particular MRP2 [35]) and RLIP76 in epileptic brain also linked to the pathology itself? Are these transporters expressed as a response to a hostile environment or are they regulated exclusively by chemotherapy? Both MDR1 and MRP are involved in cell survival, and recent evidence by this laboratory [8-10] have shown that apoptotic mechanisms are lacking in epileptic brain. Interestingly, the original proposed role for RLIP76 was indeed that of a molecule involved in detoxification or protection of cells living in hostile environments [36,37]. More recently, Yadav et al. have confirmed a dual role for RLIP76, consisting of anti-apoptotic and drug resistance functions [38]. Thus, purely on the bases of overall function, MDR1 and RLIP76 are indistinguishable. It is possible that multiple drug resistance molecules, in addition to cooperating in drug extrusion, also play a role in the control of cellular homeostasis.
In conclusion, we report a novel, non-ABC transporter mediated mechanism of antiepileptic drug resistance that may synergistically cooperate with MDR1. The relative contribution of each transporter as determined in vitro and ex situ suggests a predominant role for RLIP76. Our results are consistent with a predominant role of the blood-brain barrier in determining multiple drug resistance to antiepileptic drugs.
Methods
Reagents
CNBr-activated Sepharose 4B, 1-chloro-2,4-dinitrobenzene (CDNB), PMSF, β-mercaptoethanol (BME), EGTA, EDTA, ATP, butylated hydroxytoluene (BHT), and polidocanol (C12E9), were purchased from Sigma Chemical Co., St. Louis, MO. DE-52 (diethylaminoethyl cellulose) anion exchanger was purchased from Whatman International Ltd. Maidstone, England. Bio-Beads (SM-2 adsorbent) and Chelex-100 resin were purchased from Bio-Rad Laboratories (Hercules, CA). Tryptone and yeast extract for preparing culture media were purchased from DIFCO laboratories, Detroit, MI. PHE was purchased from Pfizer New York, NY. [4-14C]-5,5-diphenylhydantoin (specific activity 49.37 mCi/mmol) and [14C]-CBZ (specific activity 22.6 mCi/mmol) were purchased from Perkin Elmer Life Sciences, Boston, MA and Sigma Chemical Co., St. Louis, MO, respectively. Source of anti-RLIP76 IgG used in these studies was the same as previously described [39]. DNP-SG and DNP-SG-Sepharose-4B affinity resin were prepared as previously described [39].
Tissue procurement and inside-out vesicle preparation (IOV)
Human subjects (see Table 1 for details) were used for these studies as donors of tissue samples. The investigation conforms to the principles outlined in the Declaration of Helsinki. For freshly isolated surgical samples, patient consent was obtained as per the Institutional Review Board instructions before collection of the specimens; autopsy materials were obtained from organ donors. Endothelial cells and glia were isolated from brain specimens from patients undergoing a temporal lobectomy to relieve medically intractable seizures (n = 12) or cortices of patients undergoing surgery for removal of vascular malformations (n = 3). Tissue from autopsy material was used for Fig. 1a. Blood vessels were isolated from resected tissue by manually pulling out a combination of penetrating pial and superficial pial vessels. The methods are described in detail elsewhere [9,34]. The plasma membrane vesicles of human brain cells were prepared as described elsewhere [21,23,25]. Briefly, the cells were separated form the suspension buffer by centrifugation and lysed by incubation in hypotonic buffer (0.5 mM sodium phosphate, pH 7.0, containing 0.1 mM EDTA and 0.1 mM PMSF for 1.5 h, followed by homogenization. After centrifugation of the homogenate at 12,000g (10 min at 4°C), the postnuclear supernatant was further centrifuged at 100,000g for 40 min at 4°C. The resulting pellet was suspended in the reconstitution buffer (250 mM sucrose-10 mM Tris-HCl, pH 7.4) homogenized with tight fitting Dounce homogenizer and layered over 38% sucrose in 5 mM Hepes-KOH, pH 7.4. After centrifugation at 28,000g for 2 h at 4°C the interphases were collected, washed by centrifugation in the reconstitution buffer (100,000g), and passed 20 times through a 27-gauge needle for vesicle formation.
Anti-RLIP76 and anti-MDR1 antibodies
Goat-anti-human Pgp antibody C-19 was purchased from Santa Cruz Biotech, CA. Polyclonal rabbit-anti-RLIP76 IgG as well as pre-immune IgG were prepared and purified as described previously [39,40]. Briefly, recombinant human RLIP76 expressed in E. coli and purified by DNP-SG-Sepharose affinity purification as previously described [39] was injected (75 μg) into New Zealand White rabbit after obtaining preimmune serum. After booster doses of 50 μg each at two week intervals, post-immune serum was obtained. The IgG fraction from pre- and post-immunized, heat-inactivated serum was purified by DE-52 anion exchange chromatography, followed by protein-A-Sepharose affinity chromatography. The purity of the antibody was checked by SDS-PAGE as well as Western blotting against goat anti-rabbit IgG. Aliquots of the antibody were stored at -86°C and checked regularly by aerobic and anaerobic cultures for contamination. The specificity of anti-RLIP76 and other antibodies has been stringently established: purified recombinant RLIP76 used for raising polyclonal antibodies was demonstrated to be homogenous by amino acid composition analysis demonstrating amino acid yields within 96% of those expected according to its sequence SELDI-MS demonstrating a pattern of [M+H] peaks consistent with homogenous preparations of RLIP76 [39].
Immunocytochemistry
To investigate the expression of RLIP76 protein and its localization in both various human tissues and in human epileptic brain, slide mounted sections of 10 μm thickness from frozen brain tissue were labeled as previously described [8,9]. Purified anti-RLIP76 IgG was used as primary antibody. FITC conjugated purified donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania) was the secondary antibody.
Preparation of liposomes containing purified recombinant RLIP76
The 1968 bp full length open reading frame cDNA of human RLIP76 was cloned from a λgt11 human bone marrow library by immuno-screening, using anti-DNP-SG ATPase antibodies and was subcloned into the prokaryotic expression vector, pET30a(+) (Novagen, Madison, Wisconsin), creating the pET30-RLIP76 plasmid free of extraneous sequences. This plasmid was transformed into E. coli BL21 (DE3). Protein was expressed in E. coli BL21 (DE3) grown at 30°C after induction with 0.4 mM IPTG [39]. DNP-SG affinity chromatography was used as described previously [39,40] to obtain purified RLIP76. ATPase activity was performed as previously described to monitor purification [26]. Purity was checked by SDS-PAGE, Western blot analysis and amino acid composition analysis as previously described [39]. Purified protein was dialyzed against liposome reconstitution buffer (10 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 1 mM EGTA, 100 mM KCl, 40 mM sucrose, 2.8 mM BME, 0.05 mM BHT, and 0.025% polidocanol). An aqueous emulsion of soybean asolectin (40 mg/ml) and cholesterol (10 mg/ml) was prepared in the reconstitution buffer by sonication. This emulsion was diluted 10 fold by addition of dialyzed RLIP76 in reconstitution buffer to achieve a final RLIP76 concentration of 0.1 mg/ml. The reaction mixture was sonicated at 15 s at 50 W. Vesiculation was initiated by addition of SM-2 Bio-beads (200 mg/ml) pre-equilibrated in the reconstitution buffer (without polidocanol). Vesiculation was carried out for 4 h at 4°C, followed by removal of SM-2 Bio-beads by centrifugation. The vesicles were collected and analyzed for protein content, transport activity, and microbial contamination. Control vesicles to measure non-specific transport, were prepared using an equal amount of crude protein from E. coli not expressing RLIP76.
Transport studies
For transport studies, crude membrane inside-out vesicles (IOV) were prepared from different brain tissues and cells by the method as previously described [23,25]. Briefly, stock solutions of 40 mM MgCl2 and 40 mM of ATP were prepared in buffer containing 40 mM sucrose and 10 mM Tris-HCl, pH 7.4. The reaction mixture (120 μL) consisted of IOV protein (80 μg), 10 mM Tris-HCl, pH 7.4, 40 mM sucrose, 4 mM MgCl2 and appropriate volume of radio-labeled 14C-PHE and 14C-CBZ were added to attain a final concentration of 1 μM. To start the reaction, buffer with or without ATP was added to achieve a final concentration of 0 or 4 mM ATP. The uptake was stopped by rapid filtration of a fixed aliquot (30 μL) of the reaction mixture through 96 well nitrocellulose plates (0.45 μm pore size). After filtration, the bottoms of the nitrocellulose membranes were blotted dry with filter paper and punched out, and the associated radioactivity was measured by placing in liquid scintillation counting vials. Scintillation vials were vortexed thoroughly, allowed standing for 1 h at room temperature, and counted in a liquid scintillation counter. Each determination was performed in triplicate. ATP-dependent uptake of 14C-PHE and 14C-CBZ were determined by subtracting the radioactivity of the control without ATP from that of the experimental containing ATP and the transport was calculated in terms of pmol/min/mg protein.
cDNA array and mRNA
The details of the tissue culture procedures and mRNA extraction have been described previously [5]. Briefly, surgically obtained specimens were washed in phosphate buffered saline (PBS) and incubated in collagenase type II (2 mg/ml, Worthington chemicals) at 37°C for 20 min to dissociate the endothelial cells. Collagenase was then washed off with the medium used for growing ECs (1.5 g/100 ml, MCDB 105 supplemented with Endothelial Cell Growth Supplement, 15 mg/100 ml, heparin 800 units/100 ml, 10% fetal bovine serum, and penicillin/streptomycin 1%, Sigma chemicals), and ECs were harvested using a sterile cotton swab soaked in the medium. EC stained positive for Von Willebrand factor and were negative for glial fibrillary acidic protein. ECs were purged from the culture dishes by gentle enzymatic dissociation (collagenase) and collected by centrifugation. Equal amounts of the pellet were used for total RNA isolation and protein extraction. Total RNA was extracted with the Trizol reagent (Gibco Labs). Integrity of the isolated RNA was confirmed by agarose formaldehyde gels. For gene expression analysis, human GENEFILTERS™ (Research Genetics Inc., Huntsville, Alabama) were used for this study. Each filter membrane contains approximately 4,000 known human genes. 33P-dCTP was used to label probe used for hybridization to produce clean and sharp signals, as recommended by the manufacturer.
RLIP76 knockout animals
RLIP76+/- heterozygous knockout animals were commissioned from Lexicon genetics, and prepared by the strategy described previously [41]. Briefly, C57B mice (12 wk old), born of RLIP76+/- × RLIP76+/- mating, were genotyped by PCR strategy. We generated C57B mice which carry heterozygous (+/-) or homozygous (-/-) disruption of the RLIP76 gene, and established colonies of RLIP76+/+, RLIP76+/-, and RLIP76-/- C57B mice by segregation and mating of animals based on genotyping by polymerase chain reaction (PCR) on tail DNA. Western-blot analysis of mouse tissues using anti-RLIP76 antibodies confirmed decreased RLIP76 levels in the RLIP76+/- mouse, and its absence in tissues from the RLIP76-/- mouse [41]. Consistent with the observed function of RLIP76 as a transporter of GS-E and doxorubicin (DOX) in cell culture studies [23,25], GS-E and DOX transport in membrane vesicles was decreased in a stepwise fashion from the RLIP76+/+, to RLIP76+/-, to RLIP76-/- mice [41].
Measurement of PHE concentration in wild type and RLIP76 knockout mouse serum and brain tissues
Twelve week old C57B mice born of heterozygous × heterozygous mating were genotyped by PCR on mouse tail DNA using forward, reverse and LTR primers [41]. PHE measurements were performed on 5 wild type (RLIP76+/+) and 5 RLIP76 knockout (RLIP76-/-) animals sacrificed 2 h after a single i.p.-injection of phosphenytoin or PHE. A 10% homogenate of mouse brain tissues was prepared and centrifuged at 28,000 × g for 45 min at 4°C. PHE levels in homogenate and plasma was measured using the Dade Behring Clinical Multichannel Analyzer with the PHE Flex® reagent cartridge, a method based on PETINIA technology.
Abbreviations used
RALBP1, official human genome designation for human Ral-binding protein-1, synonymous with RALBP1, and homologous to rat RALBP1 and mouse RIP1. We refer to the mouse, rat and human proteins as RLIP76 in the present communication; AED, anti-epileptic drug; BBB, blood-brain barrier; CBZ, carbamazepine; EC, endothelial cells; GSH, glutathione; GS-E, glutathione electrophile conjugates; DOX, doxorubicin; MCDB, modified Czapek Dox broth; PHE, phenytoin.
Authors' contributions
Designed the study, participated in its design and coordination and helped to draft the manuscript: Sanjay Awasthi, Damir Janigro
Performed pharmacological experiments, including knock out animal studies: Sharad S. Singhal
Contributed to designing of pharmacological experiments and assisted in the writing of the discussion section: Yogesh C. Awasthi
Performed immunocytochemical, mRNA, and Western blotting analysis: Kerri L Hallene, Vince Fazio, Luca Cucullo, Gabriele Dini
Acknowledgements
This work was supported in part by the National Institutes of Health (NIH-NS43284, NIH-HL51614, NIH-NS46513 and NIH-NS38195) to DJ and ES 012171 to YCA and CA 77495 and CA 104661 to SA. We would also like to thank Dr. William Bingaman, MD, the section head of Epilepsy Surgery at the Cleveland Clinic Foundation for expertise in procuring human tissue for this study.
Figures and Tables
Figure 1 Human expression of RLIP76. A) Immunocytochemical detection was performed on tissue arrays with histological sections from normal human tissue. RLIP76 expression was virtually absent from normal brain autopsies; both gray and white matter were analyzed; cumulative data for brain and blood tissue are presented in b). RLIP76 immunoreactivity was observed in breast lobules of primary duct elements, in cardiac myocytes and liver sinusoids. No expression was found in colon tissue. Intense immunoreactivity was present in cell lines of tumor origin. Bars indicate 60 μm with the exception of the cell culture data, where the bars reflect 10 μm. The inset in a) shows a negative control of a brain section incubated with secondary but not primary antibodies. B) Cerebrovascular expression of RLIP76 in epileptic brain. Blood vessels from multiple drug resistant epileptic brain were characterized by high levels of RLIP76 expression. Both vascular and intravascular cells were RLIP76 immunopositive. RLIP76-positive intravascular cells were anucleated and did not react with the nuclear stain DAPI (in blue, C). Note that erythrocytes were found to be RLIP76 immunopositive in both normal and epileptic tissue. RLIP76 is expressed exclusively in epileptic endothelial cells and does not localize to glia or neurons. D) NeuN expression is segregated from RLIP76 immunoreactivity, which is limited to cortical vessels. E) Widespread GFAP immunoreactivity in epileptic brain does not co-localize with RLIP76 (in green). Both large (arrow) and capillary-size (asterisk) vessels express RLIP76. Note the large region of GFAP positive reactive gliosis (limited by a dotted line) characterized by the absence of RLIP76 expression. F) MDR1 and RLIP76 co-localize in "epileptic" blood vessels but MDR1 expression extends to parenchymal cells. Three large pial vessels are shown to demonstrate the predominant vascular expression of RLIP76. Note that MDR1 expression was more predominant in parenchymal glia. G-H) High power demonstration of endothelial co-expression of RLIP76/MDR1. Note that MDR1 expression co-localized with RLIP76 expressed at the lumenal surface, while MDR1 expression was also observed in RLIP76-negative ablumenal structures reflecting glial endfeet (arrowheads in H). We studied a total of 41 patients, including samples from 6 autopsies. The average age of the patients was 32 ± 18 years, range 3 months – 59 years old. No data are available on the autopsy material, besides the fact that these were adults of either sex. The non-epileptic patients were either undergoing surgery for aneurysm clipping or to remove arteriovenous or other vascular malformations. None of these patients had seizures prior to surgery or received antiepileptic drug treatment. The surgical epileptic patients studied were resistant to the following drugs: CBZ = 61%; PHE = 77%; Pentobarbital = 55%; Tompiramate = 50%; valproic acid = 44%; other AEDs, less than 5%. Most patients were resistant to > two drugs; 1 patients (a 3 months old infant) did not undergo any drug treatment, while another patient (8 month old) attempted ketogenic diet treatment).
Figure 2 Phenytoin Transport by RLIP76. A) Requirement of both ATP and RLIP76 for increased liposomal uptake of 14C-PHE. Experiments in a-e were performed four times and triplicate determinations were performed for each data point. B) Time dependent uptake of 14C-PHE and 14C-CBZ by RLIP76-proteoliposomes in the presence of ATP. Data were fitted by y = y0+A1e(-x/t). C) Saturable kinetics of 14C-PHE and 14C-CBZ transport by purified recombinant human RLIP76 with respect to ATP concentrations. Data were fitted by a single exponential. D) Transport of PHE and CBZ by RLIP76-proteoliposomes. Radiolabeled drugs were incubated in the absence or presence of 4 mM ATP with RLIP76 liposomes containing variable amounts of RLIP76. Unless both ATP and RLIP76 were present, drug-uptake was close to the detection limit. Data points were fitted by a Sigmoid y = A2 + (A1-A2)/(1 + exp(x-x0)/dx). E) Determination of Km values for RLIP76-mediated transport of 14C-PHE and 14C-CBZ. F) RLIP76 mRNA expression in normal (aneurysm, HUVEC) and "epileptic" endothelial cells isolated from tissue resections [31,34]. Note that RLIP76 was significantly (p < 0.05) increased in endothelial cells isolated from multiple drug resistant patients (n = 6) compared to control tissue (n = 8). G) Analysis of specimens by Western-blot analysis confirmed these findings. We compared protein expression in dysplastic or normal cortex within the same patient [42,43]. Note that the actively epileptic cortex was characterized by gross abnormality and increased expression of RLIP76. The Coomassie stained band shows the migration pattern of purified human RLIP76 to emphasize the increased levels of signal in both bands. The arrows point to histological sections from the same regions used to isolate protein. Note that abnormal clustering of cells is evident on H&E stained sections and in neighboring samples stained with DAPI. The bars indicate 100 μm; the dotted and dashed lines show the extent of abnormal nuclear clustering while the asterisks refer to abnormal vascular structure present in these grossly malformed cortices. H) RLIP76 is up-regulated in epileptic brain and RLIP76 protein expression levels correlate with transport activity in inside-out membrane vesicles prepared from the same tissues. Data were fitted linearly (R value of 0.99).
Figure 3 Phenytoin transport in epileptic brain is mediated by RLIP76. A-B) Relative contribution of RLIP76 and MDR1 to total AED transport capacity in IOV prepared from brain tissue of non-epileptic, non-multiple drug resistant and multiple drug resistant epileptic patients. This was determined using anti-RLIP76 and anti-MDR1 antibodies [23,25]. In this figure, * and ** represent p < 0.05 and 0.01 respectively. C-D) Time dependent PHE uptake by IOV prepared from primary cultures of astrocytes or endothelial cells from normal (diamonds) or epileptic brain (squares). E-F) Brain PHE levels in RLIP76+/+ and RLIP76-/- mice 2 h after IP administration of Phosphenytoin at 33 or 83 mg/kg (3 animals/group). G) After exposure to the antiepileptic drug, RLIP76+/+ animals (left panel) appeared relatively unaffected compared with RLIP76-/- mice (right panel) where a severe neurological toxicity including extensor posturing, lethargy and status epilepticus were observed.
Table 1 Human Tissue Donor Information
ID Age Sex Notes AED failed Use
1 0.8 M EPi- Hemispherectomy None WB
2 0.3 M Epi-Hemispherectomy Ketogenic Diet WB, AED transport studies
3 57 F Temporal Lobe Epilepsy PHE, CBZ,TPM WB
4 33 F Control (AVM) None WB
5 61 F Temporal Lobe epilepsy VAL,CBZ,TPB WB, AED transport studies
6 30 M Control Parasaggital Cist None WB, AED transport studies
7 0.9 F Frontal/Parietal Epilepsy VAL, PBT, TPM WB
8 14 M Temporal Lobe Epilepsy CBZ, Keppra, PBT,TPM, VAL WB
9 42 M Temporal Lobe Epilepsy VAL, PBT, primidone, tiagabine, AED transport studies
10 27 F Temporal Lobe Epilepsy PHE,VAL,gabapentin,CBZ, TPM AED transport studies
11 7 M R. Lateral Parietal/Occipital/Temporal Lobes PBT, PHE, TPM, CBZ, DZP, LMT AED transport studies
12 14 M Temporal Lobe Epilepsy VAL,Keppra, CBZ, other AED transport studies
13 31 F Temporal Lobe Epilepsy gabapentin, CBZ,VAL,PHE,PBT AED transport studies
14 60 F Temporal Lobe Epilepsy PBT, MBL, TGL, DPT, VLM, MSL, FBT In vitro cells AED
15 43 F Focal Epi N/A In vitro cells AED
16 56 M Focal Epi PHE, PBT, CBZ, VAL, ETS In vitro cells AED
17 43 F Temporal Lobe Epilepsy PHE, CBZ, VAL, GBP mRNA analysis
18 40 F Control (Aneurysm) NONE mRNA analysis
20 58 F Focal Epi PHE, PBT, VAL, PRM, FBT, GBP, TPM mRNA analysis
21 35 M Temporal Lobe Epilepsy PHE, CBZ, VAL, PRM, VGB mRNA analysis
22 28 M Temporal Lobe Epilepsy PHE, PBT, CBZ, VAL, TPM, CZP, GBP mRNA analysis
23 38 M Temporal Lobe Epilepsy PHE, CBZ, VAL, FBM, GBP mRNA analysis
24 39 M Temporal Lobe Epilepsy PHE, PBT, VAL, PRM, MSD mRNA analysis
25 31 M Temporal Lobe Epilepsy PHE, PBT, VAL, TPM mRNA analysis
26 23 M Temporal Lobe Epilepsy PHE, PBT, CBZ, VAL, GBP mRNA analysis
27 45 F Control (Aneurysm) NONE mRNA analysis
28 22 F Control (Aneurysm) NONE mRNA analysis
36 7 M Temporal Lobe Epilepsy PHE, CBZ, VAL, GBP ICC
37 28 F Temporal Lobe Epilepsy PHE, PBT, CBZ, VAL ICC
38 7 M Temporal Lobe Epilepsy ? ICC
39 4 F Frontal and Parietal Epilepsy PHE, CBZ, VAL, PRM, ICC
40 8 F Temporal lobe epilepsy CBZ, PHE, VAL ICC
41 24 M Control (AVM) NONE ICC
42 1 M Frontal Epilepsy CBZ, PHE, VAL ICC
43 25 M Temporal Lobe Epilepsy Phe CBZ ICC
44 1 M Hemispherectomy None ICC
45 23 F Contol (AVM) None ICC
46 23 M Temporal lobe epilepsy gabapentin, CBZ,VAL,PHE, ICC
47 23 M Control (AVM) None ICC
48 7 M TSC ? ICC
49 3 F Epilepsy Right Hemi ? ICC
50 Autopsy ICC
51 Autopsy ICC
52 Autopsy ICC
53 Autopsy ICC
54 Autopsy ICC,WB
55 Autopsy ICC,WB
==== Refs
Sander JW The problem of the drug-resistant epilepsies Novartis Found Symp 2002 243 4 12 11990781
Sisodiya SM Mechanisms of antiepileptic drug resistance Curr Opin Neurol 2003 16 197 201 12644749 10.1097/00019052-200304000-00013
Begley DJ ABC transporters and the blood-brain barrier Curr Pharm Des 2004 10 1295 1312 15134482 10.2174/1381612043384844
Schinkel AH Wagenaar E Mol CA van Deemter L P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs J Clin Invest 1996 97 2517 2524 8647944
Owen A Pirmohamed M Tettey JN Morgan P Chadwick D Park BK Carbamazepine is not a substrate for P-glycoprotein Br J Clin Pharmacol 2001 51 345 349 11318771 10.1046/j.1365-2125.2001.01359.x
Weiss J Kerpen CJ Lindenmaier H Dormann SM Haefeli WE Interaction of antiepileptic drugs with human P-glycoprotein in vitro J Pharmacol Exp Ther 2003 307 262 267 12954800 10.1124/jpet.103.054197
Doran A Obach RS Smith BJ Hosea NA Becker S Callegari E Chen C Chen X Choo E Cianfrogna J Cox LM Gibbs JP Gibbs MA Hatch H Hop CE Kasman IN Laperle J Liu J Liu X Logman M Maclin D Nedza FM Nelson F Olson E Rahematpura S Raunig D Rogers S Schmidt K Spracklin DK Szewc M Troutman M Tseng E Tu M Van Deusen JW Venkatakrishnan K Walens G Wang EQ Wong D Yasgar AS Zhang C The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model Drug Metab Dispos 2005 33 165 174 15502009 10.1124/dmd.104.001230
Marroni M Marchi N Cucullo L Abbott NJ Signorelli K Janigro D Vascular and parenchymal mechanisms in multiple drug resistance: a lesson from human epilepsy Curr Drug Targets 2003 4 297 304 12699350 10.2174/1389450033491109
Marroni M Agarwal M Kight K Hallene K Hossain M Cucullo L Signorelli K Namura S Janigro D Relationship between expression of multiple drug resistance proteins and p53 tumor suppressor gene proteins in human brain astrocytes Neuroscience 2003 121 605 617 14568021 10.1016/S0306-4522(03)00515-3
Marchi N Hallene KL Kight KM Cucullo L Moeddel G Dini G Bingaman W Vezzani A Janigro D Significance of MDR1 and multiple drug resistance in refractory human epileptic brain BMC Med 2004 2 37 15473912 10.1186/1741-7015-2-37
Sills GJ Mohanraj R Butler E McCrindle S Collier L Wilson EA Brodie MJ Lack of association between the C3435T polymorphism in the human multidrug resistance (MDR1) gene and response to antiepileptic drug treatment Epilepsia 2005 46 643 647 15857428 10.1111/j.1528-1167.2005.46304.x
Kwan P Brodie MJ Potential role of drug transporters in the pathogenesis of medically intractable epilepsy Epilepsia 2005 46 224 235 15679503 10.1111/j.0013-9580.2005.31904.x
Maines LW Antonetti DA Wolpert EB Smith CD Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells Neuropharmacology 2005 49 610 617 15961125
Park SH Weinberg RA A putative effector of Ral has homology to Rho/Rac GTPase activating proteins Oncogene 1995 11 2349 2355 8570186
Jullien-Flores V Mahe Y Mirey G Leprince C Meunier-Bisceuil B Sorkin A Camonis JH RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis J Cell Sci 2000 113 ( Pt 16) 2837 2844 10910768
Cantor SB Urano T Feig LA Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases Mol Cell Biol 1995 15 4578 4584 7623849
Jullien-Flores V Dorseuil O Romero F Letourneur F Saragosti S Berger R Tavitian A Gacon G Camonis JH Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity J Biol Chem 1995 270 22473 22477 7673236 10.1074/jbc.270.38.22473
Quaroni A Paul EC Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus J Cell Sci 1999 112 ( Pt 5) 707 718 9973605
Rosse C L'Hoste S Offner N Picard A Camonis J RLIP, an effector of the Ral GTPases, is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis J Biol Chem 2003 278 30597 30604 12775724 10.1074/jbc.M302191200
Sharma R Sharma A Yang Y Awasthi S Singhal SS Zimniak P Awasthi YC Functional reconstitution of Ral-binding GTPase activating protein, RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal Acta Biochim Pol 2002 49 693 701 12422239
Sharma R Singhal SS Cheng J Yang Y Sharma A Zimniak P Awasthi S Awasthi YC RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes Arch Biochem Biophys 2001 391 171 179 11437348 10.1006/abbi.2001.2395
Sharma R Singhal SS Wickramarachchi D Awasthi YC Awasthi S RLIP76 (RALBP1)-mediated transport of leukotriene C4 (LTC4) in cancer cells: implications in drug resistance Int J Cancer 2004 112 934 942 15386349 10.1002/ijc.20516
Awasthi S Singhal SS Singhal J Yang Y Zimniak P Awasthi YC Role of RLIP76 in lung cancer doxorubicin resistance: III. Anti-RLIP76 antibodies trigger apoptosis in lung cancer cells and synergistically increase doxorubicin cytotoxicity Int J Oncol 2003 22 721 732 12632061
Awasthi S Singhal SS Sharma R Zimniak P Awasthi YC Transport of glutathione conjugates and chemotherapeutic drugs by RLIP76 (RALBP1): a novel link between G-protein and tyrosine kinase signaling and drug resistance Int J Cancer 2003 106 635 646 12866021 10.1002/ijc.11260
Awasthi S Singhal SS Singhal J Cheng J Zimniak P Awasthi YC Role of RLIP76 in lung cancer doxorubicin resistance: II. Doxorubicin transport in lung cancer by RLIP76 Int J Oncol 2003 22 713 720 12632060
Singhal SS Singhal J Sharma R Singh SV Zimniak P Awasthi YC Awasthi S Role of RLIP76 in lung cancer doxorubicin resistance: I. The ATPase activity of RLIP76 correlates with doxorubicin and 4-hydroxynonenal resistance in lung cancer cells Int J Oncol 2003 22 365 375 12527936
Stuckler D Singhal J Singhal SS Yadav S Awasthi YC Awasthi S RLIP76 transports vinorelbine and mediates drug resistance in non-small cell lung cancer Cancer Res 2005 65 991 998 15705900
Treiman DM Woodbury DM Levy RH, Mattson RH and Meldrum BS Absorption, distribution and excretion of phenytoin Antiepileptic Drugs 1998 Raven Press
Potschka H Fedrowitz M Loscher W P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain Neuroreport 2001 12 3557 3560 11733711 10.1097/00001756-200111160-00037
Martin C Berridge G Mistry P Higgins C Charlton P Callaghan R The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein Br J Pharmacol 1999 128 403 411 10510451 10.1038/sj.bjp.0702807
Marroni M Kight KM Hossain M Cucullo L Desai SY Janigro D Dynamic in vitro model of the blood-brain barrier. Gene profiling using cDNA microarray analysis Methods Mol Med 2003 89 419 434 12958437
Schinkel AH P-Glycoprotein, a gatekeeper in the blood-brain barrier Adv Drug Deliv Rev 1999 36 179 194 10837715 10.1016/S0169-409X(98)00085-4
Abbott NJ Khan EU Rollinson CM Reichel A Janigro D Dombrowski SM Dobbie MS Begley DJ Drug resistance in epilepsy: the role of the blood-brain barrier Novartis Found Symp 2002 243 38 47 11990780
Dombrowski S Desai S Marroni M Cucullo L Bingaman W Mayberg MR Bengez L Janigro D Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy Epilepsia 2001 42 1504 1507 10.1046/j.1528-1157.2001.12301.x
Potschka H Fedrowitz M Loscher W Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity J Pharmacol Exp Ther 2003 306 124 131 12663688 10.1124/jpet.103.049858
Awasthi S Sharma R Yang Y Singhal SS Pikula S Bandorowicz-Pikula J Singh SV Zimniak P Awasthi YC Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76) Acta Biochim Pol 2002 49 855 867 12545192
Awasthi YC Sharma R Cheng JZ Yang Y Sharma A Singhal SS Awasthi S Role of 4-hydroxynonenal in stress-mediated apoptosis signaling Mol Aspects Med 2003 24 219 230 12893000 10.1016/S0098-2997(03)00017-7
Yadav S Zajac E Singhal SS Singhal J Drake K Awasthi YC Awasthi S POB1 over-expression inhibits RLIP76-mediated transport of glutathione-conjugates, drugs and promotes apoptosis Biochem Biophys Res Commun 2005 328 1003 1009 15707977 10.1016/j.bbrc.2005.01.055
Awasthi S Cheng J Singhal SS Saini MK Pandya U Pikula S Bandorowicz-Pikula J Singh SV Zimniak P Awasthi YC Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin Biochemistry 2000 39 9327 9334 10924126 10.1021/bi992964c
Singhal SS Singhal J Cheng J Pikula S Sharma R Zimniak P Awasthi YC Awasthi S Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes Acta Biochim Pol 2001 48 551 562 11732624
Awasthi S Singhal SS Yadav S Singhal J Drake K Nadkar A Zajac E Wickramarachchi D Rowe N Yacoub A Boor P Dwivedi S Dent P Jarman WE John B Awasthi YC RLIP76 is a major determinant of radiation sensitivity Cancer Res 2005 65 6022 6028 16024601 10.1158/0008-5472.CAN-05-0968
Boonyapisit K Najm I Klem G Ying Z Burrier C LaPresto E Nair D Bingaman W Prayson R Luders H Epileptogenicity of focal malformations due to abnormal cortical development: direct electrocorticographic-histopathologic correlations Epilepsia 2003 44 69 76 12581232 10.1046/j.1528-1157.2003.08102.x
Marusic P Najm IM Ying Z Prayson R Rona S Nair D Hadar E Kotagal P Bej MD Wyllie E Bingaman W Luders H Focal cortical dysplasias in eloquent cortex: functional characteristics and correlation with MRI and histopathologic changes Epilepsia 2002 43 27 32 11879383 10.1046/j.1528-1157.2002.00801.x
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BMC OphthalmolBMC Ophthalmology1471-2415BioMed Central London 1471-2415-5-221615938610.1186/1471-2415-5-22Research ArticleRelationship between visual field loss and contrast threshold elevation in glaucoma Tochel CM [email protected] JS [email protected] JL [email protected] JD [email protected] Institute of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Glasgow, Scotland, UK2 Tennent Institute of Ophthalmology, Gartnavel General Hospital, 1053 Great Western, Road, Glasgow, Scotland, UK2005 13 9 2005 5 22 22 9 5 2005 13 9 2005 Copyright © 2005 Tochel et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
There is a considerable body of literature which indicates that contrast thresholds for the detection of sinusoidal grating patterns are abnormally high in glaucoma, though just how these elevations are related to the location of visual field loss remains unknown. Our aim, therefore, has been to determine the relationship between contrast threshold elevation and visual field loss in corresponding regions of the peripheral visual field in glaucoma patients.
Methods
Contrast thresholds were measured in arcuate regions of the superior, inferior, nasal and temporal visual field in response to laser interference fringes presented in the Maxwellian view. The display consisted of vertical green stationary laser interference fringes of spatial frequency 1.0 c deg-1 which appeared in a rotatable viewing area in the form of a truncated quadrant extending from 10 to 20° from fixation which was marked with a central fixation light. Results were obtained from 36 normal control subjects in order to provide a normal reference for 21 glaucoma patients and 5 OHT (ocular hypertensive) patients for whom full clinical data, including Friedmann visual fields, had been obtained.
Results
Abnormally high contrast thresholds were identified in 20 out of 21 glaucoma patients and in 2 out of 5 OHT patients when compared with the 95% upper prediction limit for normal values from one eye of the 36 normal age-matched control subjects. Additionally, inter-ocular differences in contrast threshold were also abnormally high in 18 out of 20 glaucoma patients who had vision in both eyes compared with the 95% upper prediction limit. Correspondence between abnormally high contrast thresholds and visual field loss in the truncated quadrants was significant in 5 patients, borderline in 4 patients and absent in 9 patients.
Conclusion
While the glaucoma patients tested in our study invariably had abnormally high contrast thresholds in one or more of the truncated quadrants in at least one eye, reasonable correspondence with the location of the visual field loss only occurred in half the patients studied. Hence, while contrast threshold elevations are indicative of glaucomatous damage to vision, they are providing a different assessment of visual function from conventional visual field tests.
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Background
The visual field loss caused by glaucoma has been divided into 5 stages ranging from relative defects in sensitivity to almost complete visual loss [1]. The pattern of loss which alerts awareness of the disease, however, is often an arcuate scotoma occurring with approximately equal frequency in the superior or inferior hemifield within 20 degrees of the fovea and with or without a nasal step. These losses are generally detected in the clinical environment by visual field tests involving presentation of spots of light within a hemi-spherical dome at various locations within the central visual field. However, by the time visual field defects are detected, considerable loss of retinal ganglion cells may already have occurred, as exemplified by the case of a glaucoma suspect who, despite an absence of local field defects prior to death, showed ganglion cell losses post-mortem of 63% and 44% in left and right retinae, respectively [2]. Accordingly, there has been an impetus to develop alternative tests to visual field testing capable of detecting glaucoma at earlier stages. One such test is the measurement of contrast sensitivity in response to sinusoidal grating patterns generated by a variety of methods-oscilloscope, TV or computer monitor, printed paper, laser interferometry, or Snellen letters. The most usual protocol has involved measurements of contrast sensitivity in response to foveal viewing of the display [3-14], though more involved studies have additionally determined contrast sensitivities at peripheral locations in the visual field [15-22]. The consensus has been, with one exception [13], that contrast sensitivity in response to low spatial frequencies is impaired in glaucoma, leading to strong advocacy of its usefulness in glaucoma screening. While most studies reported the results of visual field tests, correlations between impaired contrast sensitivity and the location of visual field loss were generally not undertaken. In the one case in which this was done on a limited scale [17], contrast sensitivity deficits were described as occurring in both the visual hemifield showing no visual loss and the hemifield which did show visual loss.
We have therefore set out to answer the question whether regions of glaucomatous visual field loss are associated with elevations of contrast threshold which was employed rather than its reciprocal, the contrast sensitivity. For this purpose, we have designed a streamlined method of measuring contrast thresholds at different peripheral locations in the visual field based on the advantages offered by viewing laser interference fringes in the Maxwellian view. This provides a large visual field which can be viewed without the need for refraction of the viewer and has a sense of proximity, rather than the unavoidable remoteness of an externally generated grating display. Our results were then compared with visual field data obtained in the conventional manner. A preliminary report of our results has been made previously [23].
Methods
Patients
The experiments were undertaken with the informed consent of the participants and with the approval of the Ethics Committee of the North Glasgow University Hospitals NHS Trust and were performed in conformity with the Declaration of Helsinki. All subjects and patients underwent the Snellen test to determine their best visual acuity with refraction if necessary.
A total of 26 patients ages 37 to 83 yr (mean 69 ± 10 S.D. yr) were recruited from the Tennent Institute of Ophthalmology, Gartnavel General Hospital. They were diagnosed according to their intra-ocular pressure (IOP) determined by applanation tonometry, the appearance of the optic disc on ophthalmoscopic examination and the extent of visual field loss determined with the Friedmann Visual Field Analyzer Mark II (Clement Clarke International Ltd., London). Twenty one patients were diagnosed as having glaucoma (Table 1): the most common condition was primary open angle glaucoma (POAG) in which IOP exceeded 22 mmHg on at least 2 occasions before treatment, there was cupping of the optic disc and visual field defects characteristic of glaucoma were present. Normal pressure glaucoma (NPG) was diagnosed according to the same criteria as POAG except that IOP was less than 22 mmHg. In addition, one patient had each of primary chronic angle closure glaucoma, post-traumatic glaucoma and sarcoid-induced glaucoma. Within the glaucoma group, since 6 patients had a fellow eye which showed no visual field defects or definite pathological cupping of the optic disc and had an IOP of less than 22 mmHg, these eyes were designated normal, though were not used as controls. The 5 ocular hypertensives (OHT) were defined on the basis of an IOP which exceeded 21 mmHg but with no visual field loss and were subdivided into those with and without pathological cupping of the optic disc (Table 1). All patients were selected on the basis of having stable visual fields through being superimposable in terms of their position, extent and depth, tested usually on a six monthly basis over the 2 years preceding the study. Five patients who were subsequently retested over intervals of 1 day to 6 months showed reproducible visual fields.
As controls, we examined 36 non-glaucoma subjects ages 48–78 yr (mean 62 ± 8 S.D. yr.) who were recruited from within the University and from personal acquaintances. They were deemed to be normal on the basis of a reported absence of visual problems, a Snellen acuity of 6/6 or better in each eye and a report of normal vision from a recent visit to their optometrist. While 28 subjects had 2 normal eyes, 8 subjects had one normal eye and a non-glaucomatous problem in the fellow eye, which is dealt with separately. Of the 36 subjects, 19 were emmetropic while the remainder had refractive errors ranging from +1.50 DS to -7.00 DS with astigmatism of up to +5.00 DC and all were presbyopic to varying extents. Twenty three subjects ages 55–78 yr, including all subjects above 69 yr, were also examined with the Central 24-2 threshold test with the Humphrey II Visual Field Analyzer model 750 (Humphrey Instruments, San Leandro, CA, USA) to which we had ready access at times convenient to our subjects.
Apparatus
Contrast thresholds were measured with a modification of a laser interferometer described previously [24,25] and is shown schematically in Figure 1. Monochromatic green light (λ = 543 nm) from a 0.95 mW He-Ne laser (Uniphase 1652P) was passed through a spatial filter (SF), a -10 DS lens (L1) to expand the beam and a +10 DS lens (L2) to collimate the beam. The beam was then divided by a 2 inch cube beamsplitter (B1), the 2 beams reflected from λ/20 front silvered mirrors (M1 and M2) and recombined by a second 2 inch cube beamsplitter (B2). The two beams were equated in intensity by the neutral density filter N1 and the polarization shift caused by the beamsplitters was compensated by the rotatable sheet polarizer (P1, extinction = 10-4). The combined beams were passed through a second polarizer (P2) to sharpen the polarization prior to combination at the plate beamsplitter B3 with the background beam which consisted of non-coherent light from a tungsten filament microscope lamp. The latter beam was passed through heat absorbing filters, a green interference filter (C, peak λ = 546 nm), a polarizer (P3) to polarize the light at 90° to the laser beams and a neutral density filter (N2) to equate the intensity to that of the laser beams. The combined laser and background beams were passed through a rotatable polarizer of 2 inch diameter (P4, Coherent-Ealing 22-9161). The emergent beam passed through an iris diaphragm (I) which controlled the overall field size, an aperture consisting of a rotatable truncated quadrant (Q) and the Maxwellian lens assembly which consisted of 38 mm diameter +20 DS plano-convex and +32 DS biconvex high refractive index lenses mounted in the same housing as the rotatable aperture. The total power of the Maxwellian lens assembly was +42 DS. The subject or patient looked into the Maxwellian lens with the chin supported by a chin rest. The observed visual display consisted of an attenuated green central fixation light of subtense 2° and the truncated quadrant which was rotatable to the 4 chosen visual field positions viz. temporal, superior, inferior and nasal, as shown for right eye viewing in Figure 2.
The truncated quadrant extended from 10° to 20° from the central fixation point. The angular dimensions were calibrated from determination of the location of the nasal and temporal margins of the blind spot from the axis of fixation. These were determined first in angular subtenses for viewing a sheet of graph paper from 25 cm. The linear dimensions of the blind spot from the axis of fixation were then obtained for viewing a needle mounted on a micrometer, coplanar with the iris diaphragm, through the Maxwellian lens. The factor to convert linear dimensions into angular dimensions was then used to convert the linear dimension of the Maxwellian display into angular subtenses. The procedure was undertaken in both eyes in 3 subjects with close agreement between the results.
By translation of mirror M1 to increase the pathlength of the laser beam reflected from M1, interference fringes were generated at a spatial frequency directly related to the pathlength difference. By adjustment, the spatial frequency was set to 1.0 c deg-1 which is readily detected for peripheral viewing at our eccentricities [26]. The contrast of the display was varied by rotation of the polarizer P4, since contrast is proportional to sin2θ where θ is the angle of rotation from the position of zero contrast. The intensities of the laser beam and background beam were equated using a UDT S370 Optometer at the position of the eye. Likewise, the intensities of the 4 quadrants were also equalized. The final viewing intensity was determined psychophysically to be 3.2 log units above foveal threshold.
Contrast threshold determinations
Prior to the determinations, each subject or patient was given a standard explanation with the aid of diagrams as to the nature of the test, which was then carried out under standardized subdued illumination. The subject or patient viewed the display through the Maxwellian lens without external refraction. With the direction of gaze determined by the central fixation spot, the subject or patient increased the contrast of the grating display in the peripherally-located truncated quadrant until the pattern became just visible and no more. This gives results for contrast thresholds similar to those for the more time consuming 50% of seeing [27]. The angle of rotation was recorded and readings repeated. First, the subject or patient had practice runs with the truncated quadrant in each of its 4 positions. This was then followed by 6 contrast threshold determinations for each of the 4 truncated quadrant positions for each eye in turn. The tests including the preliminary Snellen test and explanation took 20–40 min to complete. Repeat determinations in 2 normal subjects and one glaucoma patients on different days confirmed the consistency of the contrast threshold values. The determinations for the glaucoma patients were undertaken prior to the release of the visual field charts for analysis and the time interval between the tests ranged from the same day to 36 weeks (mean 64 ± 55 S.D. days).
Data analysis
Statistical analyses were undertaken with the Minitab statistical package [28]. The basic method of analysis involved the calculation of 95% prediction limits for the contrast thresholds and for the inter-ocular differences in contrast threshold between companion normal eyes, against which the results for the glaucoma patients were compared. After conversion of the data to cumulative probabilities, the sensitivity (the percentage of correctly classified glaucoma patients) was plotted against 1 – specificity (the percentage of incorrectly classified normal subjects) to obtain the Receiver Operating Characteristic (ROC) curve [29,30]. The area under the ROC curve is a measure of the sensitivity over a range of criterion levels and varies from 0.5 when there is an equal likelihood of glaucoma patients and normal subjects exceeding the specified criterion to 1.0 when there is perfect discrimination of glaucoma patients from normal subjects.
Comparisons between the mean contrast threshold for each truncated quadrant against visual field loss were undertaken by linear regression analysis after quantifying the visual field loss from the patient's Friedmann visual field chart [31]. A template containing an aperture corresponding to the dimensions of the truncated quadrant was laid over the Friedmann visual field chart and the following scores allocated to each point tested according to whether the point was visible through the applied neutral density filter: 3 – densest filter appropriate to age (usually 1.2 log units attenuation), 2 – next densest filter (usually 0.8 log units attenuation), 1 – no filter and 0 – not visible. The summed values were expressed as a percentage of the maximum possible score for that truncated quadrant and the difference from 100% was the visual field loss score. This was repeated for each of the 4 positions shown in Figure 2 and no allowance was made for the presence of the blind spot in the temporal quadrant. From the regression analysis, the value of R2, the coefficient of determination, was obtained. This gives as a percentage the amount of the variation in contrast threshold against visual field loss score which is accounted for by the line of best fit (regression line); so R2 ranges from 0% when the data are randomly distributed to 100% when the data points fall exactly on the regression line. The value of R2 is amenable to statistical testing and significance was taken as p < 0.05.
Results
Control group
The contrast thresholds for the 64 normal eyes were normally uniformly low for each of the superior, temporal, inferior and nasal truncated quadrants with no significant differences between the mean values of the 4 quadrants (p = 0.81). The mean contrast threshold and the 95% upper prediction limit were then calculated for the 4 truncated quadrants for the normal eyes of the group of 8 subjects together with one eye chosen at random from the remaining 28 subjects to give equal numbers of left and right eyes. The contrast threshold values for the group conformed closely to a normal distribution and had a mean value of 0.027 ± 0.011 S.D. contrast units, giving a 95% upper prediction limit of 0.045 contrast units. Inter-ocular comparisons were made between the mean values for left and right eyes in the group of 28 subjects with 2 normal eyes. The mean of the modulus of the difference was 0.0029 ± 0.0020 S.D. contrast units, giving a 95% upper prediction limit of 0.0060 contrast units. These upper prediction limits were then used to assess the contrast thresholds of the glaucoma patients and of the abnormal eyes of the non-glaucoma group.
The Humphrey Central 24-2 test for the 40 normal eyes in 23 subjects (the remaining 6 eyes had non-glaucomatous abnormalities), missed 5 blindspots, gave mean deviations outside normal limits in 8 eyes and gave abnormal or borderline results with the glaucoma hemifield test in 12 eyes. In all cases, the abnormal results were explained by drooping eyelids, obstruction by spectacles or trial frames and subject error, which are common hazards of automated perimetry [32].
Glaucoma patients
The first comparison was to determine the extent to which the contrast threshold for a truncated quadrant exceeded the upper prediction limit of 0.045 contrast units, irrespective of the location of the quadrant. Abnormally high values occurred in 29 out of 33 glaucomatous eyes (87%) and, on a patient by patient basis, in 20 out of 21 patients (95%). There were no apparent differences among the different types of glaucoma (Table 1). Examples of patients with elevated contrast thresholds are shown in Figures 3 &4.
Second, a comparison was made of the modulus of the inter-ocular difference of the mean contrast threshold for left and right eyes against the upper prediction limit of 0.0060 contrast units. This was abnormally high in 18 out of 20 glaucoma patients (90%) for whom the comparison was possible, including the patient with bilateral normal contrast thresholds. However, when identification was made on the basis of the combination of either an abnormal contrast threshold or abnormal inter-ocular difference, 100% correct identification was achieved. The optimal sensitivity and specificity and the area under the ROC curve are given in Table 2 which also shows data for tests of contrast threshold restricted to the superior and inferior truncated quadrants and to the nasal and temporal truncated quadrants. Both these assessments show reduced sensitivity compared with testing of all 4 truncated quadrants. It is thus clear contrast threshold elevations, especially in combination with intra-ocular differences were effective in detecting glaucoma.
OHT patients
In the 5 OHT patients, contrast thresholds were elevated in 4 out of 10 eyes, corresponding to 2 patients, with no differences between those with and without pathological cupping of the optic disc. The inter-ocular difference was elevated in 3 out of 5 OHT patients. Follow-up of the OHT patients over the next 12 months showed that those with normal contrast thresholds retained normal visual fields while the 2 patients with elevated thresholds developed marked visual field loss in both eyes (Table 1).
Comparison of locations of contrast threshold elevation and visual field loss
Our main assessment was to determine whether those parts of the visual field which showed abnormally high contrast thresholds also corresponded to the location of the visual field loss. This was undertaken by regression analysis of contrast threshold against the corresponding visual field loss score for each of the truncated quadrants in both eyes. In order to achieve a reasonable distribution of data points, patients were required to have a range of visual field loss scores in excess of 20% together with an absence of visibility in no more than one of the truncated quadrants. This provided 18 glaucoma patients whose results are shown in Table 1. Contrast threshold and visual field loss were strongly related in 5 patients (R2 = 55–85%, p < 0.05) as illustrated by Figure 3, showed a semblance of s direct relationship in 4 patients (R2 = 46–49%, 0.05 <p < 0.10) and showed an absence of a relationship in 9 patients (R2 = 0–26%, p > 0.1) as illustrated by Figure 4. There was no correlation between the form of the relationship and the type of glaucoma (Table 1). We also repeated the analysis in 8 patients with minimal visual field loss in one eye for which a single mean value was calculated (i.e. giving n = 5 for regression analysis). The R2 values were unaffected in the new analysis (p = 0.84, paired t-test) and a significant or borderline relationship was still present in 5 patients.
Control subjects with abnormal fellow eye
There were 8 subjects in the non-glaucoma control group with an abnormality of one eye. In the subject with the retinal scar, the contrast thresholds were elevated in the truncated quadrant containing the scar as confirmed by the Humphrey Visual Field. On the other hand, the contrast thresholds for the subjects with repaired retinal detachment, mild cataract and solar damage and for 2 amblyopes were normal. The third amblyope had abnormally high contrast thresholds while the subject with macular degeneration was unable to fixate with that eye.
Discussion
Contrast thresholds were readily measured with our Maxwellian view interferometer in normal subjects, even in the presence of refractive errors as large as -7.00 DS and +5.00 DC, which is a positive advantage over externally viewed displays which depend on the accuracy of the refraction and factors like spectacle slippage or obstruction by the edges of frames. In a limited number of cases, contrast thresholds were readily obtained in the presence of minor lens opacities and in amblyopes, though the method is impracticable in cases where fixation cannot be achieved. Generally, the contrast thresholds of normal subjects were uniformly low. While evidence of the blind spot in the form of elevated contrast thresholds in the temporal truncated quadrant occurred infrequently, this was probably the result of stimulation of a large area of retina compared with the well circumscribed extent of the blind spot of which there is a perceptual lack of awareness [33]. The use of relatively large truncated quadrants may suggest intuitively that localisation of visual loss was being sacrificed though there is evidence to suggest that this is not the case. Threshold values between neighbouring test points tend to be correlated with the result that the central visual field is organized into discrete clusters or sectors which are related to the projection of retinal ganglion cell axons [34,35]. Another issue is the variation in the number of cycles between superior/inferior and nasal/temporal truncated quadrants. We chose the stimulus spatial frequency to be 1.0 c deg-1 to ensure high sensitivity in the peripheral retina [26] and a number of cycles which did not limit sensitivity, at least for foveal viewing [36]. It is conceivable that, at a peripheral location, differences in the number of cycles may affect contrast sensitivity though such an effect is likely to be small [37]. Frequency Doubling Technology (FDT) perimetry employs stimulus fields not dissimilar to those employed in the present study though the grating pattern is phase modulated at a high temporal frequency to produce an illusory doubling of the spatial frequency [38]. Reduction of the dimension of the square stimulus field from 10° to 4° produced only a modest increase in contrast threshold [39]. These results are thus not inconsistent with the absence of a significant differences between the contrast thresholds for the different truncated quadrants.
In terms of correct identification of our glaucoma patients, contrast thresholds were elevated in one or more truncated quadrants in all but one of our 21 patients while inter-ocular differences were elevated in all but 2 out of 20 patients, which is consistent with previous studies [4,15-18,21]. These elevations were present irrespective of the type of glaucoma, which is consistent with a common mechanism for ganglion cell loss in chronic glaucoma [40]. In terms of detection of glaucoma, our method has performed soundly with an area under the ROC curve of 0.956 which compares well with other methodologies employed in the early detection of glaucoma viz. short wave length automated perimetry (SWAP), frequency doubling technology, resolution acuity perimetry, detection acuity perimetry and temporal modulation perimetry [41] and considerably better than for contrast sensitivities with central viewing [42]. Of especial interest was the performance of our test with the OHT patients. Initially, it seemed that two patients would be incorrectly diagnosed in both eyes which had markedly elevated contrast thresholds; however within the next 12 months they each developed appreciable visual field loss in their eyes (Table 1). While the sample size is small, it indicates a potential predictive function which has also been described for FDT, high-pass resolution perimetry and SWAP [43,44]. A further improvement in the area under the ROC curve to 0.993 occurred when the assessment of contrast thresholds was combined with that of the inter-ocular difference in contrast threshold. This is a simple extension of the analysis which uses pre-existing data and its value has been remarked upon previously [38].
The central aim of our study, however, was whether the contrast threshold results reflected the amount of visual field loss in the same region of the visual field. While there was a reasonable correspondence in 50% of our 18 glaucoma patients (Figure 3), in the other half of the patients, there was a marked mismatch so that some patients had normal contrast thresholds in quadrants showing high visual field loss while others had elevated contrast thresholds in seemingly normal parts of the visual field (Figure 4). We are confident that the results were not caused by abrupt shifts of visual field loss between quadrants since the visual fields were stable over the 2 years preceding our tests and indeed subsequent to our tests. While automated perimetry is widely used to ascertain visual field loss, our preference was firmly for the Friedmann test on the basis that it was less disconcerting to elderly patients and is consistent with the transferability of visual field data between different machines [45]. While our identification of the misalignment with visual field loss relates to the detection of a stationary grating pattern, allusions have also been made to FDT error scores which occurred in the hemifield opposite to that containing the visual field loss [46,47]. This lack of correspondence between contrast threshold elevation and visual field loss can reasonably be attributed to the different attrition rates for the different modalities of visual function in different individuals which is well known in glaucoma [12,38,48-53]. To this, we can further add that different modalities, at least in terms of light detection and contrast detection, are affected differently in different parts of the retina. The sentiment has been expressed that it does not matter whether two tests do not agree so long as the disease is detected during screening [47]. To an extent this is understandable though it would clearly be desirable to have a rational basis for whatever testing regime is adopted. One possible explanation is that conventional perimetry detects light detection defects [54] while tests like FDT, SWAP and contrast threshold measurements detect different manifestations of ganglion cell damage [40,41]. Certainly there are several tests, particularly FDT and SWAP, which could be used profitably alongside visual field testing [48] and, to these, contrast threshold determinations in the peripheral visual field may be added.
Conclusion
Our results therefore do confirm that contrast thresholds in response to sinusoidal grating patterns presented to peripheral regions of the visual field are invariably abnormally elevated in cases of glaucomatous visual field loss. The regions of threshold elevations, however, did not map onto the regions of visual field loss, which indicates contrast threshold testing cannot be considered as a substitute for visual field tests. It may however provide valuable supplementary information which may be obtained simply and rapidly with the type of interferometric apparatus we have described.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JLJ carried out the clinical examination of the patients. The scientific experiments were carried out and analyzed by CMT, JSM and JDM who also undertook the construction of the apparatus and drafting of the manuscript which all authors approved.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Our appreciation is expressed to our subjects and patients for their participation in the experiments. We especially thank Mr. W.H. Biddlecombe for manufacturing the Maxwellian lens housing. We also thank Dr. T. Maddess for his detailed comments on the manuscript and the W.H. Ross Foundation for the Prevention of Blindness for financial support.
Figures and Tables
Figure 1 Schematic representation of optical apparatus used to generate vertical sinusoidal laser interference fringes which are observed in the Maxwellian view. Abbreviations: A aperture, B beam splitter, C interference filter, I iris diaphragm, L lens, M front silvered mirror, N neutral density filter, P polarizer, SF spatial filter. Further explanation is given in Methods.
Figure 2 Schematic representation of the truncated quadrants in the temporal, superior, inferior and nasal positions as seen by the right eye. The central fixation light was of subtense 2 deg and the truncated quadrant extended from 10–20 deg. The sinusoidal interference fringe pattern had a spatial frequency of 1.0 c deg-1.
Figure 3 Patient 9 (Table 1) A & B Histograms of contrast threshold (mean S.E.M.) for superior (s), temporal (t), inferior (i) and nasal (n) truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken horizontal line. Contrast thresholds were abnormal in the left eye. C & D Friedmann visual fields showing: unmarked letters-normal detection (1.2 logarithmic units attenuation), reduced sensitivity (○ 0.8 logarithmic units attenuation and ø zero attenuation) and ● zero detection. The main visual field loss was in the inferior hemifield of the left eye. E Contrast threshold against visual field loss score taken from Friedmann charts for each truncated quadrant, showing best fitting relationship as broken line. Relationship was significant: R2 = 85%, p = 0.001.
Figure 4 Patient 6. Same conventions as in Figure 3 but with ○ as 1.0 logarithmic units attenuation. A & B Contrast thresholds were abnormal in left and right eyes. C & D Visual field loss was diffuse in the left eye and occurred in inferior temporal quadrant of right eye. E Relationship between contrast threshold and visual field loss score was not significant: R2 = 25%, p = 0.12.
Table 1 Clinical data for glaucoma and OHT patients
Pat no. Age (yr) LE Condition LE VA LE VF loss LE CTmax RE Condition RE VA RE VF loss RE CTmax I-O diff R2
1 72 POAG 6/5 74% 0.051 POAG 6/4 7% 0.023 0.025 79%*
2 73 POAG 6/6 60% 0.098 POAG 6/7.5 51% 0.168 0.045 0%°
3 67 POAG 6/6 4% 0.099 NPG 6/9 53% 0.107 0.004 48%+
4 76 POAG 6/6 7% 0.075 POAG 6/7.5 44% 0.078 0.001 1%°
5 71 POAG 6/6 36% 0.142 POAG 6/6 30% 0.067 0.045 0%°
6 66 POAG 6/6 17% 0.075 POAG 6/12 7% 0.091 0.014 -25%°
7 77 POAG 6/24 55% 0.055 POAG 6/6 67% 0.044 0.012 14%°
8 68 POAG 6/6 23% 0.069 normal 6/5 4% 0.049 0.019 55%*
9 75 POAG 6/9 44% 0.102 normal 6/5 1% 0.041 0.034 85%*
10 72 normal 6/6 2% 0.023 POAG 6/9 42% 0.031 0.006 47%+
11 37 POAG 6/18 14% 0.089 blind nil 100% n/s na na
12 83 POAG pxf 6/9 1% 0.066 POAG pxf 6/9 51% n/s na na
13 83 POAG pxf 6/18 69% 0.058 POAG pxf 6/12 46% 0.074 0.025 -24%°
14 74 POAG pxf 6/5 26% 0.067 OHT 6/6 3% 0.056 0.008 9%°
15 73 NPG 6/5 17% 0.055 NPG 6/5 47% 0.033 0.014 0%°
16 76 NPG 6/9 18% 0.150 NPG 6/12 47% 0.060 0.021 46%+
17 73 NPG 6/9 53% 0.048 normal 6/9 4% 0.027 0.010 49%+
18 54 RD 6/12 2% 0.046 NPG 6/5 41% 0.083 0.023 85%*
19 61 PCACG 6/9 11% 0.078 ACG 6/9 3% 0.072 0.012 na
20 79 PTG 6/9 55% 0.147 normal 6/9 6% 0.077 0.055 26%°
21 55 normal 6/6 7% 0.044 SIG 6/6 52% 0.058 0.017 73%*
22 70 OHT 6/5 0%(2%) 0.031 OHT 6/5 0% (6%) 0.018 0.002 na
23 78 OHT 6/5 0% (7%) 0.022 OHT 6/9 3% (8%) 0.033 0.0064 na
24 57 OHT 6/5 6% (13%) 0.054 OHT 6/5 4%(13%) 0.051 0.0006 na
25 62 OHT 6/5 2% (2%) 0.034 OHT pdc 6/12 4% (6%) 0.042 0.010 na
26 72 A OHT pdc 6/60 8% (29%) 0.155 OHT pdc 6/12 8% (35%) 0.059 0.068 na
Pat no. Patient number, LE left eye, RE right eye, A amblyopic, CTmax highest contrast threshold value (to be compared against the upper prediction limit of 0.045), I-O diff inter-ocular difference (to be compared against the upper prediction limit of 0.006), NPG normal pressure glaucoma, OHT ocular hypertensive, na not applicable, n/s not seen, PCACG primary chronic angle closure glaucoma, pdc pathological cupping of optic disc, POAG primary open angle glaucoma, PTG post-traumatic glaucoma, pxf pseudoexfoliation, RD retinal detachment, SIG sarcoid-induced glaucoma, VA visual acuity, VF visual field. Negative R2 values denote inverse relationship. *p < 0.05, +0.05 <p < 0.10, °p > 0.10. Some eyes denoted normal show visual field loss due to the blind spot while some glaucomatous eyes showing minimal visual loss had additional loss either more peripheral or more central to the truncated quadrant. OHT patients also show visual field loss score in parenthesis at 12 months follow-up examinations.
Table 2 Signal detection data
Criterion n Sensitivity Specificity Area under ROC
CTmax by eye 33 93.9% 91.0% 0.956
CTmax S/I 33 87.8% 91.0% 0.953
CTmax N/T 33 81.8% 91.0% 0.923
CTmax by patient 21 90.4% 95.0% 0.954
I-O diff 20 89.5% 85.7% 0.897
CTmax or I-O diff 20 95.0% 100% 0.993
CTmax highest contrast threshold value, S/I superior and inferior truncated quadrants, N/T nasal and temporal truncated quadrants, I-O diff inter-ocular difference.
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Aulhorn E Karmeyer H Frequency distribution in early glaucomatous visual field defects Doc Ophthalmol Proc Ser 1977 14 75 83
Quigley HA Dunkelberger GR Green WR Chronic human glaucoma causing selectively greater loss of large optic nerve fibres Ophthalmology 1988 95 357 363 3174003
Arden GB Jacobson JJ A simple grating test for contrast sensitivity: preliminary results indicate value in screening for glaucoma Invest Ophthalmol 1978 17 23 32
Atkin A Wolkstein M Bodis-Wollner I Anders M Kels B Podos SM Interocular comparisons of contrast sensitivities in glaucoma patients and suspects Br J Ophthalmol 1980 64 858 862 7426557
Isayama Y Mizokami K Tagami Y Spatial contrast sensitivity in optic nerve disorders. Its relationship to the visual fields and atrophy of retinal nerve fibre layer Jap J Ophthalmol 1980 34 293 300
Tagami Y Onuma T Mizokami K Isayama Y Comparison of spatial contrast sensitivity with visual field in optic neuropathy and glaucoma Doc Ophthalmol Proc Ser 1981 26 147 154
Hitchings RA Powell DJ Arden GB Carter RM Contrast sensitivity gratings in glaucoma family screening Br J Ophthalmol 1981 65 515 517 7295611
Vaegan Halliday BL A forced choice test improves clincal contrast sensitivity testing Br J Ophthalmol 1982 66 477 491 7104264
Ross JE Bron AJ Clarke DD Contrast sensitivity and visual disability in chronic simple glaucoma Br J Ophthalmol 1984 68 821 827 6498136
Motolko MA Phelps CD Contrast sensitivity in asymmetric glaucoma Int Ophthalmol 1984 7 45 50 6706474 10.1007/BF00138269
Lustgarten JS Marx MS Podos SM Bodis-Wollner I Campeas D Serle JB Contrast sensitivity and computerised perimetry in early detection of glauco-matous change Clin Vis Sci 1990 5 407 413
Teoh SL Allan D Dutton GN Foulds WS Brightness discrimination and contrast sensitivity in chronic glaucoma – a clinical study Br J Ophthalmol 1990 74 215 9 2186795
Sponsel WE DePaul KL Martone JF Shields MB Ollie AR Stewart WC Association of Vistech contrast sensitivity and visual field findings in glaucoma Br J Ophthalmol 1991 75 558 60 1911660
Swindale NV Fendick MG Drance SM Graham SL Hnik P Contrast sensitivity for flickering and static latters at isoluminance in glaucoma J Glaucoma 1996 5 156 169 8795753
Lundh BL Lennerstrand G Eccentric contrast sensitivity loss in glaucoma Acta Ophthalmologica 1981 59 21 24 7211277
Neima D Leblanc R Regan D Visual field defects in ocular hypertension and glaucoma Arch Ophthalmol 1984 102 1042 1045 6743082
Lundh BL Central and peripheral contrast sensitivity for static and dynamic sinusoidal gratings in glaucoma Acta Ophthalmologica 1985 63 487 492 4072627
Falcão-Reis F O'Donoghue E Buceti R Hitchings RA Arden GB Peripheral contrast sensitivity in glaucoma and ocular hypertension Br J Ophthalmol 1990 74 712 716 2275933
Zulauf M Flammer M Correlation of spatial contrast sensitivity and visual fields in glaucoma Graefe's Arch Clin Exp Ophthalmol 1993 231 146 150 8462886 10.1007/BF00920937
Horn FK Korth M Martus P Quick full field flicker test in glaucoma diagnosis: correlations with perimetry and papillometry J Glaucoma 1994 3 206 13
Lundh BL Gottvall E Peripheral contrast sensitivity for dynamic sinusoidal gratings in early glaucoma Acta Ophthalmol Scand 1995 73 202 206 7493229
Ansari EA Morgan JE Snowden RJ Psychophysical characterisation of early functional loss in glaucoma and ocular hypertension Br J Ophthalmol 2002 86 1131 1135 12234893 10.1136/bjo.86.10.1131
Tochel CM Jay JL Morrison JD Comparison of contrast thresholds and brightness discrimination between visual quadrants and hemifields in normal and glaucomatous subjects J Physiol 1999 521 8 9P
Morrison JD McGrath C Assessment of the optical contributions to the age-related deterioration in vision Quart J Exp Physiol 1985 70 249 269
Webb RM Sahal A Morrison JD The optical quality of the human eye revisited Ophthal Physiol Opt 1997 17 516 21 10.1016/S0275-5408(97)00046-X
Kelly DH Retinal inhomogeneity. I Spatiotemporal contrast sensitivity J Opt Soc Am A 1984 1 107 113 6699746
Morrison JD Reilly J An assessment of decision-making as a possible factor in the age-related loss of contrast sensitivity Perception 1986 15 541 552 3588214
Ryan BE Joiner BL Minitab Handbook 1994 3 Belmont, California: Duxbury Press
Stanislaw H Todorov N Calculation of signal detection measures Behav Res Meth Instr Comp 1999 31 137 149
Cello KE Nelson-Quigg J Johnson CA Frequency doubling technology perimetry for detection of glaucomatous field loss Am J Ophthalmol 2000 129 314 322 10704546 10.1016/S0002-9394(99)00414-6
Sponsel WE Visual field quantification in the diagnosis and assessment of chronic open angle glaucoma Thesis M572 1985 Bristol University
Anderson DR Standard perimetry Ophthalmol Clin N Am 2003 16 205 212 10.1016/S0896-1549(03)00005-1
Ramachandran VS Blind spots Sci Am 1972 266 44 49
Suzuki Y Mathematical and optimal clustering of test points of the central 30-degree visual field of glaucoma J Glaucoma 2001 10 121 128 11316094 10.1097/00061198-200104000-00009
De la Rosa MG González-Hernández M Abraldes M Azuara-Blanco A Quantification of interpoint topographic correlations of threshold values in glaucomatous visual fields J Glaucoma 2002 11 30 34 11821687 10.1097/00061198-200202000-00007
Hoekstra J Van Der Goot DPJ Van Den Brink G Bilsen FA The influence of the number of cycles upon the visual contrast threshold for spatial sine wave patterns Vis Res 1973 14 365 368 10.1016/0042-6989(74)90234-X
Robson JG Grahan N Probability summation and regional variation in contrast sensnitivity across the visual field Vis Res 1981 21 409 418 7269319 10.1016/0042-6989(81)90169-3
Maddess T Goldberg I Dobinson J Wine S Welsh AH James AC Testing for glaucoma with spatial frequency doubling illusion Vis Res 1999 39 4258 4273 10755162 10.1016/S0042-6989(99)00135-2
Spry PGD Johnson CA Within-test variability of frequency-doubling perimetry using a 24-2 test pattern J Glaucoma 2002 11 315 320 12169968 10.1097/00061198-200208000-00007
Quigley HA Neuronal death in glaucoma Prog Retinal Eye Res 1999 18 39 57 10.1016/S1350-9462(98)00014-7
Spry PGD Johnson CA Mansberger SL Cioffi GA Psychophysical investigation of ganglion cell loss in early glaucoma J Glaucoma 2005 14 11 19 15650598 10.1097/01.ijg.0000145813.46848.b8
Ivers RQ Macaskill P Cumming RG Mitchell P Sensitivity and specificity of tests to detect eye disease in an older population Ophthalmology 2001 108 968 975 11320029 10.1016/S0161-6420(00)00649-7
Iester M Altieri M Vittione P Calabria G Zingirian M Traverso CE Detection of glaucomatous visual field defect by nonconventional perimetry Am J Ophthalmol 2003 135 35 39 12504694 10.1016/S0002-9394(02)01818-4
Landers JA Goldberg I Graham SL Detection of early visual field loss in glaucoma using frequency-doubling perometry and short-wavelength automated perimetry Arch Ophthalmol 2003 121 1705 1710 14662589 10.1001/archopht.121.12.1705
Anderson DR Feuer WJ Alward WL Skuta GL Threshold equivalence between perimeters Am J Ophthalmol 1989 83 1396 1402
Quigley HA Identification of glaucoma-related abnormality with screening protocol of frequency doubling technology Am J Ophthalmol 1998 125 819 829 9645719 10.1016/S0002-9394(98)00046-4
Allen CS Sponsel WE Trigo Y Dirks MS Flynn WJ Comparison of the frequency doubling technology screening algorithm and the Humphrey 24-2 SITA-FAST in a large eye screening Clin Exp Ophthalmol 2002 30 8 14 10.1046/j.1442-9071.2002.00478.x
Sample PA Bosworth CF Blumenthal EZ Girkin C Weinreb RN Visual function-specific perimetry for indirect comparisons of different ganglion cell populations in glaucoma Invest Ophthalmol Vis Sci 2000 41 1783 1790 10845599
Anderson RS O'Brien C Psychophysical evidence for a selective loss of M ganglion cells in glaucoma Vis Res 1989 15 493 505
Bosworth CF Sample PA Gupta N Bathija R Weinreb RN Motion automated perimetry (MAP) identifies early glaucomatous field defects Arch Ophthalmol 1998 116 1153 8 9747672
Casson EJ Johnson CA Shapiro LR Longitudinal comparison of temporal-modulation perimetry with white-on-white and blue-on-yellow perimetry in ocular hypertension and early glaucoma J Opt Soc Am A 1993 10 1792 1806
Pacheco-Cutillas M Sahraie A Edgar DF Acquired colour vision defects in glaucoma – their detection and clinical significance Br J Ophthalmol 1999 83 1396 1402 10574822
Pearson P Swanson WH Fellman RL Chromatic and achromatic defects in patients with progressing glaucoma Vis Res 2001 41 1215 1227 11292509 10.1016/S0042-6989(00)00311-4
Janssen P Naskar R Moore S Thanos H Thiel H Evidence for glaucoma-induced horizontal cell alterations in the human retina Ger J Ophthalmol 1997 5 378 385
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BMC Oral HealthBMC Oral Health1472-6831BioMed Central London 1472-6831-5-81618802410.1186/1472-6831-5-8Research ArticleEthnic variations in orthodontic treatment need in London schoolchildren Alkhatib Mhd Nour [email protected] Raman [email protected] Claire [email protected] Pooja [email protected] Sue [email protected] Department of Dental Public Health, Guy's King's & St Thomas' Dental Institute, Floor 2, Caldecot Road, Denmark Hill Campus, London SE5 9RW, UK2 WHO Collaborating Centre for Research, Education and Service in oral health, disability and culture, UK3 North West London Community Dental Services, Hammersmith and Fulham Primary Care Trust. Parsons Green Centre, 5-7 Parsons Green, London. SW6 4UL, UK2005 27 9 2005 5 8 8 28 6 2005 27 9 2005 Copyright © 2005 Alkhatib et al; licensee BioMed Central Ltd.2005Alkhatib 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 study was carried out to determine the prevalence of orthodontic treatment need in children from minority ethnic groups and compare the need to the white population. The second objective was to explore variations in agreement between subjective and objective treatment need in a multiethnic context using the aesthetic component of Orthodontic Treatment Need Index (IOTN AC).
Methods
A cross-sectional study in North West London, 14 schools were randomly selected from the 27 schools in the two boroughs of Harrow and Hillingdon. Comparison between objective and subjective treatment need was carried out using IOTN AC index. Clinical orthodontic treatment need was also recorded using the dental health component of Orthodontic Treatment Need Index (IOTN DHC).
Results
2,788 children were examined and completed the questionnaire. 16% of the study population were already wearing appliances or had finished orthodontic treatment. Of the remaining children; 15% had definite need for treatment using the dental health component of the IOTN. There was no significant variation in the need for orthodontic treatment between different ethnic backgrounds (P > 0.05) whether using the AC or DHC components of the IOTN index. However, poor agreement was detected between professional and subjective assessment of ethnic minority of orthodontic treatment need using IOTN AC index.
Conclusion
Orthodontic treatment need in children of ethnic minorities does not differ significantly from the vast majority of white children. However treatment need based on aesthetic index continues to vary in all ethnic groups from the professional aesthetic assessment
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Background
Orthodontic treatment is one of the most costly and challenging issues to NHS dentistry. In 2003, fees for orthodontic treatment accounted for more than a quarter of all child fees [1]. Whilst the focus has been often on strategies to improve access and increase budget of orthodontic care, evaluation of the criteria used for defining the need has received little attention. Most orthodontic indices have been primarily based on white populations; this may raise a concern since the demography of the United Kingdom has changed rapidly in the last few decades. The majority of ethnic minorities reside in the England region with London being the most diversified city; almost 30% of London inhabitants are of ethnic minorities [2].
The health needs of ethnic minorities may be different from the white population. In a comparative review of subgingival calculus formation, association between plaque formation and ethnicity has been reported [3]. These needs may be particularly different when they are related to aesthetics, where confounding factors such as cultural or societal values play a major role in shaping these needs. It has been shown that black people are more likely to have different orthodontic needs than other ethnicities; they are more likely to have class III occlusion, anterior open bites and mid-line diastema than their white or Asian counterparts [4,5]. Kiyak [6] showed that Pacific Asians had different dental beliefs and behaviours and different views about aesthetics compared to Caucasians.
Two of the properties of the ideal orthodontic index laid out by Shaw et al [7] were; 1- sensitive to the needs of the patients, 2- acceptable to both the public and the profession. Orthodontic care in the UK is currently provided on the definite need basis of the Index of Orthodontic Treatment Need (IOTN) devised by Brook and Shaw [8]. Under this category only severe cases are eligible for treatment under the scheme. IOTN, however, has two components; the Dental Health Component (DHC) and the Aesthetic Component (AC). The latter has gained increased popularity in recent years because a) the primary motive for seeking orthodontic treatment is improving appearance [9,10] and an aesthetic component would seem necessary to any diagnostic tool. The aesthetic component should therefore be accounted for when planning treatment using the DHC component, b) since patient satisfaction is the one of the main outcomes of treatment, a tool should be devised to be used by both patients and professionals to measure this outcome; the AC component has been shown to be capable of facilitating this task [10]. Moreover, a strong correlation between the AC component and psychosocial outcomes was reported by Bennett et al [11]. Mandall et al [12] also reported an association between child self esteem and IOTN AC but not with IOTN DHC. However, the AC is more subjective and less reliable than the DHC; studies which compared the need based on the two components demonstrated poor correlation [13-15]. Researchers suggested adjustment to the DHC defined need in order to balance for the aesthetic component.
The aims of the study were, first to explore the need for treatment in a multiethnic community. Secondly to assess whether the need for orthodontic treatment in ethnic minorities differs from the white population based on the dental health component and on the aesthetic component and, thirdly to test the agreement between normative and perceived need for orthodontic care across all ethnicities.
Methods
The study took place 2002/2003. All schools in the boroughs of Harrow and Hillingdon were included in the initial sampling. Fourteen out of the twenty seven schools in the two boroughs were selected using a one to one simple sampling technique. The required sample size for each ethnic group was based on our pilot study [14]. Children aged 12 to14 were included, the study population consisted of 3,500 children. Dental examination was carried out in accordance with BASCD criteria [16], DMFT was recorded together with IOTN DHC and AC, the examiner (PJ) was calibrated for both DMFT and IOTN indexes with high intra examiner reliability (for the Aesthetic Component of IOTN, the examiner achieved a weighted kappa score of 0.82 showing very good agreement, sensitivity of 100% and specificity of 84.2%). The questionnaire was developed by the Transcultural Oral Health Centre at an earlier stage [14]. This was then modified and tested in a second pilot.
Measurement of IOTN AC was recorded using a 10-point analogue scale with two pictures at each end of the scale representing IOTN AC scores of 1 and 10, this was used to avoid the bias reported by Burden and Pine [17] in recording children's perception of the IOTN AC. Normative IOTN AC and DHC were recorded according to the calibration criteria. Major ethnic groups in the UK described by the national census were used to identify the ethic background of the child. Ethnicity was therefore categorized as follows:
- White: English, Irish or any other Caucasian white.
- Black: African, Caribbean or black other.
- Asian: Indian, Sri Lankan, Bangladeshi and Pakistani.
- Chinese: Mainland Chinese, Korean, Taiwanese and Japanese.
- Mixed: Any mixed race.
Data was encoded and entered onto SPSS software, descriptive analysis was undertaken to report frequency distributions of IOTN scores. Logistic regression was undertaken to explore the ethnic variation with agreement/disagreement with different IOTN AC threshold levels. Kappa coefficient test was utilized to estimate the agreement between normative and subjective need for treatment.
Ethical approvals were obtained from the local ethic committee in the two boroughs. Consent letters were sent out to schools and parents.
Results
Out of the 3,500 children, 2,788 children were examined and completed the questionnaire representing a response rate of 80%. Distribution of sex was almost equal (48% males Vs 52% females). Over half of children (54%) were of white ethnic background. 12% Blacks, 25% Indians, 4% Chinese and 6% were of mixed race.
442 children (16%) were undergoing orthodontic treatment during the screening, when recording IOTN AC all children were included to explore the need based on aesthetic grounds irrespective of previous or current orthodontic treatment. However when DHC was used, only those who had not received or undergoing any orthodontic treatment were included in the analysis.
To ensure reliability of assessing normative need, 280 subjects of the 2,788 were re-examined. The Cohen's Kappa score would give an indication of the level of agreement between the first and second reading thus indicating intra-examiner reliability.
Kappa scores can either be measured from a 2 × 2 table, giving a simple Kappa score. Alternatively, it can be weighted to account for 'near miss' scores. Both methods were used to measure intra-examiner reliability. The simple Kappa score was 0.89 representing a very good agreement.
Using weighted kappa score, the same principle is used as for measuring simple Kappa but instead the weight is taken into account, Microsoft Excel® was used to calculate weighted Kappa values. For intra-examiner agreement in this study, weighted Kappa was found to be 0.79, representing substantial agreement.
Perceived need
Using the IOTN AC index children graded their teeth accordingly. Almost half of children (48%) scored their teeth as 2 or 3 on the index. Less than 2% had severe scores (8–10). Distribution of responses to self-grading of teeth is illustrated graphically in figure 1. Grouping the responses in three categories to estimate treatment need based on the IOTN AC index revealed that three quarters (75%) of the children perceived no need for treatment (AC = 1–3), whilst 23% perceived borderline need (AC = 4–7) and only 2% perceived definite need for treatment (AC = 8–10). Distribution of responses according to ethnicity is presented in table 1 where children from black ethnic minority had the least perceived need for treatment compared to other ethnicities. Variations were small in the definite need for treatment except for children from Chinese ethnic background who did not report any definite need for treatment.
Figure 1 Children's assessment of IOTN AC.
Table 1 Children's rated IOTN AC by treatment category and ethnicity
Treatment need IOTN AC score White Black Indian Chinese Mixed (others)
No need for treatment 1–4 1123
(74.9) 263
(80.2) 493
(72.1) 86
(74.8) 135
(83.3)
Moderate/Borderline need 5–7 338
(22.5) 59
(18.0) 179
(26.2) 29
(25.2) 24
(14.8)
Need for treatment 8–10 38
(2.5) 6
(1.8) 12
(1.8) 3 (1.9)
Total 1499 (100.0) 328
(100.0) 684
(100.0) 115
(100.0) 162
(100.0)
Normative need using IOTN AC
The examiner scored over two thirds (69%) of children's teeth as 2 or 3 on the IOTN AC index. No child was assessed as having grade 10 and less than 2% had grade 8 and 9. Results are illustrated in figure 2. Based on treatment need categories 87% of children were assessed as having no need for treatment, 11% had borderline need for treatment and 2% has definite need for treatment need. The main descriptive difference is clear at the 'no' and borderline need levels for treatment.
Figure 2 Dentist's assessment of IOTN AC.
Differences between ethnic groups were less obvious in the professional assessment; the majority of children from all ethnic backgrounds were assessed as having low need for treatment (AC 1–3). Results are summarised in table 2.
Table 2 Examiner's rated IOTN AC by treatment category and ethnicity
Treatment need IOTN AC score White Black Indian Chinese Mixed (others)
No need for treatment 1–4 1293
(86.3) 292
(89.0) 590
(86.3) 103
(89.6) 146
(90.1)
Moderate/Borderline need 5–7 177
(11.8) 33
(10.1) 77
(11.3) 12
(10.4) 16
(9.9)
Need for treatment 8–10 29
(1.9) 3
(0.9) 17
(2.5)
Total 1499
(100.0) 328
(100.0) 684
(100.0) 115
(100.0) 162
(100.0)
Normative need using DHC component
Using the dental health component, two thirds of children (68%) had no need for treatment, 17% had moderate need for treatment and 15% had definite need for treatment. Variations in need for treatment with ethnicity are presented in table 3. Children from black ethnicity had less need for treatment than did their white peers, whereas children of Chinese and Indian ethnicities had slightly more need for treatment. However these differences were not statistically significant when entered in a regression model using ethnicity as an explanatory variable.
Table 3 Normative need (IOTN DHC) and ethnicity
Treatment need IOTN DHC White Black Indian Chinese Mixed (others)
No need for treatment 1–2 878
(69.0) 213
(72.9) 365
(65.2) 54
(62.1) 94
(69.6)
Moderate/Borderline need 3 208
(16.4) 40
(18.6) 104
(18.6) 19
(21.8) 25
(18.5)
Need for treatment 4–5 186
(14.6) 39
(13.4) 91
(16.3) 14
(16.1) 16
(11.9)
Total 1272
(100.0) 292
(100.0) 560
(100.0) 87
(100.0) 135
(100.0)
Agreement between normative and perceived treatment need
Differences between children's and professionals' AC scores are illustrated in figure 3 which is a combination of figure 1 and 2. Variations are clear at the borderline level of need (AC = 4–7). In the logistic regression using the need definition of the scale as a cut-off point for comparison, the influence of ethnicity was not statistically significant in the perceived assessment of IOTN AC neither in normative assessment IOTN DHC (P > 0.05). Agreement between children's ratings and the dentist's rating is presented in table 4, at the low need level (AC 1–4) there was agreement in 80% of cases. This decreased to 50% at the borderline need level (AC 5–7). At the definite need level the number of cases was small so was the agreement (6%). Since the majority of responses of both children and the dentist are in the low need and borderline need level, the scale mid point was used (AC 5) to test the agreement/disagreement across different ethnicities. Cohen Kappa's test revealed poor overall agreement (K = 0.18). In the ethnic context; white ethnicity demonstrated a kappa score of 0.15, black, Indian, Chinese and mixed scored 0.27, 0.18, 0.43, and 0.48 respectively indicating poor agreement with the professional assessment.
Figure 3 Comparison between children's and dentist's ratings of IOTN AC.
Table 4 Agreement between the dentist and children on IOTN AC
* Low (1–4) Moderate (5–7) High (8–10) Total
Low (1–4) 1938 (80.0%) 147 15 2100
Moderate (5–7) 442 156 (49.5%) 31 629
High (8–10) 44 12 3 (6.1%) 59
Total 2424 315 49 2788
*Dentist's scores are displayed vertically in the table
Discussion
In this cross-sectional study, the proportion of children who were professionally assessed as having a clinically definite need for treatment is lower than previously reported in the child dental health survey for the UK [18]. The perceived need based on the AC component was very low and the overall rating of AC varied slightly between children of ethnic minorities. The only small difference was seen in black children where they perceived their teeth as more attractive than did their white counterparts but again these differences were not statistically significant. Ahmed et al [14] reported that children from black ethnic minorities were more likely to perceive less need for treatment compared to professional assessment. In contrast, Otuyemi et al [19] showed no difference in the perceptions of dental aesthetics between adult Nigerians and Americans.
In this study ethnicity did not influence orthodontic need for treatment based on clinical or aesthetic grounds. However children of Indian and Chinese ethnicities had a slightly higher clinical need for treatment. These results are mirrored in Mandall et al [15] study where they reported that ethnicity did not influence self grading of aesthetics and that Asian adolescents had more need for orthodontic treatment compared to Caucasians using the DHC component.
The disagreement between professional and children's grading of the AC is not unexpected, however this disagreement was not influenced by ethnicity, a result in line with and confirming findings from Mandall et al study [15].
Conclusion
Perception and prevalence of malocclusion in children of ethnic minorities is not different from the White ones. Perhaps their perceptions may have been influenced by the cultural and societal circumstances in their current place of living and these may be different from the perceptions held by peers living in their original countries.
Self-perception of aesthetics of malocclusion differed significantly from professional assessment; however this disagreement was not confined to ethnicity. The majority of disagreements between children and dentists were at the borderline level of need, perhaps a different threshold of need definition may resolve this discrepancy. It should be noted that disagreement is also influenced by the prevalence of malocclusion and previous experience, in this study the prevalence of severe malocclusion based on aesthetic grounds was low therefore overall agreement should be interpreted carefully.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MNK managed the study and analysed the data. RB is the principal investigator he wrote the paper with MNK. CF, PJ and SA coordinated with the schools and local health authorities, collected the data and contributed to writing of the discussion.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study was funded by a grant from Department of Health 112/RDC02055
==== Refs
Dental Review 2003–2004 2004 Dental Practice Board Publications
Census 2001 National Report for England and Wales 2003 Stationary Office, London
Roberts-Harry EA Clerehugh V Subgingival calculus: where are we now? A comparative review J Dent 2000 28 93 102 10666966 10.1016/S0300-5712(99)00056-1
Trottman A Elsbach HG Comparison Of Malocclusion In Pre-school Black And White Children Am J Orthod 1996 110 69 72
Brunelle JA Bhat M Lipton JA Prevalence and distribution of selected occlusal characteristics in the US population, 1988–1991 J Dent Res 1996 75 706 13 8594094
Kiyak HA Dental beliefs, behaviors and health status among Pacific Asians Caucasians Community Dent Oral Epidemiol 1981 9 10 4 6941871
Shaw WC O'Brien KD Richmond S Brook P Quality Control In Orthodontics: Risk/Benefit Considerations Br Dent J 1991 170 33 37 2001299 10.1038/sj.bdj.4807399
Brook PH Shaw WC The Development of an index of orthodontic treatment priority Eur J Orthod 1989 11 309 320 2792220
Gochman DS The measurement and development of dentally relevant motives J Public Health Dent 1975 35 160 164 1057022
Jacobson A Psychological aspects of dentofacial esthetics and orthognathic surgery Angle Orthod 1984 54 18 35 6584049
Bennett ME Tulloch JF Vig KW Phillips CL Measuring orthodontic treatment satisfaction: questionnaire development and preliminary validation J Public Health Dent 2001 61 155 60 11603319
Mandall NA Wright J Conboy FM O'Brien KD The relationship between normative orthodontic treatment need and measures of consumer perception Community Dent Health 2001 18 3 6 11421402
Birkeland K Boe OE Wisth PJ Orthodontic concern among 11-year-old children and their parents compared with orthodontic treatment need assessed by index of orthodontic treatment need Am J Orthodont 1996 110 197 205
Ahmed B Gilthorpe MS Bedi R Agreement between normative and perceived orthodontic need amongst deprived multiethnic school children in London Clin Orthod Res 2001 4 65 71 11553087 10.1034/j.1600-0544.2001.040202.x
Mandall NA McCord JF Blinkhorn AS Worthington HV O'Brien KD Perceived aesthetic impact of malocclusion and oral self-perceptions in 14–15-year-old Asian and Caucasian children in greater Manchester Eur J Orthod 2000 22 175 83 10822891 10.1093/ejo/22.2.175
Pitts(1) NB Evans(2) DJ Pine(3) C British Association for the Study of Community Dentistry (BASCD) Diagnostic Criteria for Caries Prevalence Surveys – 1996/97 Community Dent Health 1997 14 6 9 9114553
Burden DJ Pine CM Self-perception of malocclusion among adolescents Community Dent Health 1995 12 89 92 7648417
O'Brien M Children's dental health in the United Kingdom 1993 1994 London: HMSO
Otuyemi OD Ogunyinka A Dosumu O Cons NC Jenny J Kohout FJ Jakobsen J Perceptions of dental aesthetics in the United States and Nigeria Community Dent Oral Epidemiol 1998 26 418 20 9870542
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-961616229410.1186/1471-2458-5-96Research ArticleComparison of prevalence and severity of asthma among adolescents in the Caribbean islands of Trinidad and Tobago: results of a nationwide cross-sectional survey Monteil Michele A [email protected] Gina [email protected] Catherine [email protected] Gillian [email protected] Robin M [email protected] Department of Para-Clinical Sciences, Faculty of Medical Sciences, University of the West Indies, St. Augustine, Trinidad, West Indies2 Scarborough General Hospital, Tobago Regional Health Authority, Scarborough, Tobago, West Indies3 Department of Mathematics and Computer Science, Faculty of Sciences and Agriculture, University of the West Indies, St. Augustine, Trinidad, West2005 14 9 2005 5 96 96 6 1 2005 14 9 2005 Copyright © 2005 Monteil et al; licensee BioMed Central Ltd.2005Monteil 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
Asthma is a growing problem in the Caribbean but the prevalence in most islands is unknown and possible inter-island variation in prevalence has not been determined. A nationwide cross-sectional survey was conducted to compare the prevalence of asthma symptoms among high school students in the two islands of the Republic of Trinidad and Tobago.
Methods
Questionnaire and video instruments based on those developed by the International Study of Asthma & Allergy in Childhood (ISAAC) were used to assess asthma prevalence among 6394 children (age range, 11–19 years; mean age, 14.08 yrs) in the second and third years of 35 randomly selected high schools in Trinidad and Tobago. This cross sectional survey was conducted between September and December 2002.
Results
A total of 4988 questionnaires were available for analysis (3519 in Trinidad and 1469 in Tobago). Among respondents from the two islands, there were no significant differences in the prevalence of ever wheezing (24.1% and 24.3% for Trinidad and Tobago, respectively, RR 0.99, 95% CI, 0.90–1.08); wheezing in the previous 12 months (13.1% & 13.4%, RR 0.98, 95% CI 0.84–1.15); a previous or current diagnosis of asthma (12.8% & 13.5%, RR 0.95, 95% CI 0.82–1.12) and night cough in the past 12 months (35.4% & 38.3%, RR0.93, 95% CI 0.86–1.00). However, symptoms of severe asthma were significantly more common among students from Tobago and included having had more than one acute attack in the past year (13.4% & 15.8%, RR 0.85, 95% CI 0.73–1.00, p = 0.0004), night waking as a result of wheeze (7.4% & 10.9%, RR 0.68, 95% CI 0.56–0.83, p < 0.0001) and speech limitation in the past year (5.2% & 8.7%, RR 0.59, 95% CI 0.47–0.74, p < 0.001) Exercise-associated wheezing was also more frequent among Tobagonian adolescents (17.5% & 20.2%, RR 0.87, 95% CI 0.76 – 0.98, p = 0.04).
Conclusion
Self-reported wheeze is common among adolescents in Trinidad and Tobago. Variation in symptoms was found between the two territories; high school students from Tobago, the less industrialized of the two islands, reported more symptoms of severe asthma and exercise-induced wheeze. Difference in the ethnic composition rather than socio-economic factors may be contributing to the observed differences in symptom prevalence.
==== Body
Background
Asthma is a common clinical problem in the Caribbean [1,2] and its prevalence seems to have increased since the 1970s. In Barbados for example, between 1970 and 1990, the attendance at the Asthma Bay, an acute asthma management centre at the only public Accident & Emergency (A&E) department on the island increased from 36 to 360 per month despite an increase of only 10% in the island's population during that time [3]. Despite a growing regional problem, there is still very little known about the prevalence of the disease in most Caribbean territories and whether it varies in different islands. There is also little information available about possible environmental and genetic factors that may contribute to the development and exacerbation of asthma in the Caribbean.
The islands of the Republic of Trinidad and Tobago allow a comparison of prevalence and severity of asthma in two differing Caribbean environments. Trinidad & Tobago are located at the southern end of the Caribbean archipelago and are separated by 32 kilometres. Trinidad (4828 km2,) is the most industrialised of the Anglophone Caribbean territories and has petrochemical and gas-based industries [4]. In comparison, tourism is the main source of income for Tobago (300 km2). Despite the difference in industrialization, socio-economic parameters such as the level of employment are similar in the two territories; Trinidad and Tobago have employment rates of 62.9% and 66% respectively. The average monthly income for Trinidad ranges from 383USD from one rural county to 647USD in an urban centre. The average monthly income in Tobago falls within this range at 433USD (personal communication from Central Statistical Office (CSO) of Trinidad &Tobago, 2005).
The ethnic composition of the two islands differs substantially; Tobago has a predominantly African population which is similar to many of the other smaller Caribbean territories but Trinidad is multi-ethnic with citizens of African, Amerindian, Caucasian, Chinese, South Asian and Mixed ethnicity [5]. Trinidad and Tobago also exhibit geomorphologic differences. The official language in both islands is English.
A nationwide ISAAC-based questionnaire and video survey was conducted among over 6000 adolescents in the two islands to elucidate the prevalence of symptoms of asthma and to determine if differences in symptom prevalence or severity exist. If such differences exist, they may suggest specific environmental or genetic factors that would be explored by further research.
Methods
Study design and study instruments
For our nationwide questionnaire and video survey, we used a computerized database, created by the project team, of all public and private high schools in Trinidad and Tobago. The SPSS version 9.0 statistical program (SPSS Inc., Chicago, Illinois, United States of America) was used to generate a random selection of 20% of schools in Trinidad. Schools were approached in the order in which they were selected. Of the 25 Schools selected in Trinidad, 24 (96%) participated. In Tobago, there are only 11 high schools and all agreed to participate (100%).
The questionnaire and video instruments were based on those developed for global use by the International Study of Asthma and Allergies in Childhood [6]. To the "written" ISAAC questions we added a question related to a family history of asthma: Does your father or mother or brother(s) or sister(s) have asthma? Ethnicity of the respondents was assessed by asking them to state if they were one of the following; African (black), Caucasian (white), South Asian (Indian), Chinese, Mixed or Other. Information on participants' ethnicity was obtained since there have been reports elsewhere of racial differences in asthma prevalence and severity and we wished to determine if such differences exist in our multi-ethnic population. The questionnaires are shown in Appendix 1 [see Additional file 1].
Questionnaires for further analysis were obtained from 4988 students in the selected schools. The students were in either Year 2 or Year 3 of secondary school (high school). Children in Trinidad and Tobago typically begin secondary school at age 11–12 years so that students are generally 12–13 years of age in Year 2 and 13–14 years old in Year 3.
All students in these classes were eligible for participation and were provided with information sheets to take home for their parents. Parents were asked to send a note stating that they did not want their child to participate otherwise all students were included in the survey when research staff visited schools. Some children chose not to participate of their own accord. All schools on the two islands were visited by the same two members of the research team. Researchers distributed questionnaires, read through each question slowly and allowed students to complete the questions. Then a 7-minute video showing asthmatics in distress was shown and the video-associated questionnaires ("video" questionnaire) were administered as described above. Forms were collected from students after completion. The research group, because of funding limitations, was unable to determine reasons why students chose not to participate and also if there were differences between students who responded and those who chose not to.
The study was approved by the Ethics Committee of the Faculty of Medical Sciences, University of the West Indies. Permission to visit schools was obtained from the Ministry of Education.
Responses from completed questionnaires were numbered consecutively and entered into computerized databases using Access software (Microsoft Professional 2000, Microsoft Corporation, Redmond, Washington, United States of America)
Outcome measures
The primary outcome measure was the prevalence of wheeze in secondary school-aged children in Trinidad & Tobago in the 12 months prior to the survey. Secondary outcome measures included the prevalence of symptoms suggestive of severe asthma, exercise induced wheeze and a family history of asthma.
Statistical analysis
In this study we compared the prevalence of wheeze and symptoms suggestive of severe asthma in high school students in Trinidad and Tobago. Data were analyzed using SPSS (version 9.0) statistical package. The chi-square test (χ2) was used to compare the prevalence of symptoms while the significance of relative risks (RR) was assessed with 95% confidence intervals (CIs). Correlation between responses to the "written" and "video" questionnaires was determined by comparing the degree of agreement of positive and negative responses to the following questions: the first two questions of the "written" questionnaire with the first two responses of the "video" questionnaire in relation to Scene 1 on the video; the responses to the questions on wheeze after exercise in the past 12 months (question 7 in "written" questionnaire and question 2 in relation to Scene 2 on the video); the responses to the questions on night cough and night waking in the past 12 months (questions 4 and 8 in the "written" questionnaire and questions 2 in relation to scenes 3 and 4 on the video) [see Additional file 1].
Results
Response rates
A total of 3519 completed questionnaires were retrieved from secondary school children in Trinidad and from 1469 pupils in Tobago. Response rates were 73.3% and 92% for Trinidad and Tobago respectively. These figures correspond to approximately 9% and 92% of all Year 2 and 3 students in Trinidad and Tobago, respectively. There were no negative responses from parents. Student participation at many schools depended on the willingness of students to participate when researchers visited each school. The locations of all participating schools are shown in Figure 1.
Figure 1 Maps of Trinidad & Tobago showing locations of schools (red dots) that participated in the survey.
Demographic analysis of participants
Students in the study ranged in age from 11–19 years with average ages of 13.8 and 14.4 years for Trinidad and Tobago respectively. Female participants outnumbered males in both islands (Table 1). In the Trinidadian sample, 36% were South Asian, 35% Mixed, 23.6% African with less than 5% of Chinese, Caucasian or Other ethnicity and 1% were non-responders. On Tobago, there was a predominance of African students (68.5%) with 26% Mixed, 1.4% South Asian and 2% self-reported as being of Chinese, Caucasian or Other race. 2% of Tobagonian students did not respond.
Table 1 Summary of the demographic features of study participants
Trinidad-Secondary School Students Tobago-Secondary School Students
Mean Age, years (range) 13.77 (11–19) 14.39 (11–18)
% students of 13–14 years of age 77.12 70.0
Male: Female Ratio 1: 1.47 1.1.23
Ethnic Composition (%)
South Asian 36 1.4
Mixed 34.8 26
African 23.6 68.5
Caucasian 2.0 0.4
Chinese 0.5 0.2
Other 2.0 1.4
Prevalence of wheeze
848 of 3519 respondents (24.1%) in Trinidad and 357 of 1469 (24.3%) in Tobago reported wheezing in the past (RR 0.99, 95% CI 0.90 – 1.08). Prevalence of wheeze in the previous 12 months was also similar in the two territories; 13.1 % and 13.4% for Trinidad and Tobago respectively, (RR 0.98, 95% CI, 0.84 – 1.15) (Table 2). Four hundred and fifty (12.8%) Trinidadian students and 198 Tobagonian students (13.5%) reported a previous or present diagnosis of asthma (RR 0.95, 95% CI 0.82–1.12). Night cough was the commonest symptom reported by students from both islands. It occurred in 1246 (35.4%) Trinidadian and 563 (38.3%) Tobagonian pupils (RR 0.93, 95% CI 0.86 – 1.00). Exercise-induced wheeze was reported more frequently by students from Tobago (RR 0.87, 95% CI 0.76–0.98, p = 0.04)
Table 2 Prevalence and severity of asthma and asthma-associated symptoms among adolescents in Trinidad and Tobago
Prevalence, %
Trinidad, n = 3519 Tobago, n = 1469 Relative Risk**, (95% CI)
% Participation 73.3 92
12 month prevalence
wheeze 13.1 13.4 0.98 (0.84–1.15)
No. of attacks in past yr
1–3 10.5 10.9 0.97 (0.81–1.15)
4–12 1.9 3.0 0.65 (0.44–0.94)
>12 0.9 1.8 0.51 (0.31–0.85)
Sleep disturbance from wheeze
≤1 per week 4.7 5.8 0.81 (0.63–1.05)
>1 per week 2.7 5.1 0.53 (0.39–0.71)
Speech limitation 5.2 8.7 0.59 (0.47–0.74)
Night cough in past year 35.4 38.3 0.93 (0.86–1.00)
Exercise-induced wheeze 17.5 20.2 0.87 (0.76–0.98)
Lifetime prevalence
Wheeze 24.1 24.3 0.99 (0.90–1.08)
Asthma 12.8 13.5 0.95 (0.82–1.12)
Family History of Asthma 23.0 13.4 1.72 (1.49–1.98)
** Relative Risk of symptom prevalence between Trinidad & Tobago. Tobago is the referent island
Symptoms suggestive of severe asthma were reported more frequently by students from Tobago: 4 or more attacks of wheeze in the past 12 months (99 (2.8%) of 3519 in Trinidad and 71(4.8%) of 1469 in Tobago, RR 0.58, 95% CI 0.43 – 0.79, p = 0.0005); sleep disturbance from wheeze 1 or more times per week in the past year (261 (7.4%) Trinidadian students versus 160 (10.9%) Tobagonian pupils, RR 0.68, 95% CI 0.57 – 0.82, p < 0.0001); speech limitation in the past year (183 (5.2%) Trinidadians students versus 128 (8.7%) students from Tobago, RR 0.60, 95% CI 0.48 – 0.74, p < 0.0001).
Prevalence of wheeze from video-related questionnaire
A total of 4943 completed video-related questionnaires were available for further analysis, 3489 from Trinidad and 1454 from Tobago. Fewer students reported symptoms of wheeze on the video-related questionnaire: 395 (11.32%) Trinidadian and 250 (17.2%) Tobagonian students reported wheezing in the past and 262 (7.5%) Trinidadian and 179 (12.3%) Tobagonian pupils wheezed in the past 12 months. The prevalence of all symptoms on the video-related questionnaire was significantly higher among Tobagonian students than those from Trinidad (Table 3). There was good agreement between the written and video questionnaire responses in both islands; in Trinidad, there was 64% agreement for positive responses and 78% agreement for negative responses and in Tobago, the corresponding figures were 64% and 76% respectively.
Table 3 Comparison of responses to asthma video questionnaire by secondary school students in Trinidad and Tobago
13–14 yr olds Trinidad % (no.) 13–14 yr olds Tobago % (no.) Relative Risk** (95% CI) P value
Wheeze ever 11.32 (395) 17.2 (250) 0.66 (0.57–0.76) p < 0.0001
Wheeze, 12 mths 7.5 (262) 12.3 (179) 0.61 (0.51–0.73) p < 0.0001
Wheeze, 1 mth 4.7 (163) 8.7 (126) 0.54 (0.43–0.67) p < 0.0001
Exercise wheeze ever 21.1 (737) 27.6 (401) 0.76 (0.69–0.85) p < 0.0001
Exercise wheeze, 12 mt 14.7 (514) 20.2 (293) 0.73 (0.64–0.83) p < 0.0001
Exercise wheeze, 1 mth 9.1 (316) 14.3 (208) 0.63 (0.54–0.74) p < 0.0001
Night wheeze ever 7.6 (266) 11.2 (162) 0.68 (0.57–0.82) p < 0.0001
Night wheeze, 12 mth 3.9 (136) 7.4 (108) 0.52 (0.41–0.67) p < 0.0001
Night wheeze, 1 mth 2.3 (81) 5.8 (84) 0.40 (0.30–0.54) p < 0.0001
Night cough ever 24.9 (870) 31.8 (461) 0.78 (0.71–0.86) p < 0.0001
Night cough, 12 mth 16.0 (558) 21.8 (316) 0.73 (0.65–0.83) p < 0.0001
Night cough, 1 mth 7.3 (256) 12.9 (187) 0.57 (0.48–0.68) p < 0.0001
Severe wheeze ever 8.9 (312) 12.6 (183) 0.71 (0.60–0.84) p = 0.0001
Severe wheeze 12 mth 5.4 (189) 7.9 (114) 0.69 (0.55–0.86) p = 0.0014
Severe wheeze 1 mth 3.1 (109) 6.0 (87) 0.52 (0.40–0.69) p < 0.0001
**Relative Risk of symptom prevalence between Trinidad & Tobago. Tobago is the referent island
Gender difference in prevalence of asthma
In both islands wheeze and a diagnosis of asthma were more common among girls than boys (Table 4). However, this gender difference only achieved statistical significance in the Trinidadian population.
Table 4 Prevalence of asthma and wheeze by gender
Trinidad, % Tobago, %
Asthma Ever
Boys 5.4 6.7
Girls 7.4 6.7
*RR 0.73 (0.56–0.95) *RR 1.0 (0.68–1.47)
Wheeze Ever
Boys 10.2 11.6
Girls 13.9 12.5
*RR 0.74 (0.61–0.89) *RR 0.98 (0.70–1.22)
Wheeze in past yr
Boys 5.3 5.7
Girls 7.8 7.7
*RR 0.68 (0.53–0.89) *RR 0.74 (0.50–1.10)
* Relative Risk of symptom between boys and girls (referent) in the individual islands
Ethnic difference in prevalence of asthma
The island of Trinidad is well-known for its multi-ethnic composition; Africans (39.6%), South Asians (40.3%), Mixed (18.4%) and the rest (1.6%) [5]. An analysis of video responses by self-reported ethnicity from among the cohort of Trinidadian students (Table 5) showed that symptoms were significantly less common among South Asian adolescents compared with those of African or Mixed ancestry.
Table 5 Comparison of responses to asthma video questionnaire by secondary school students of different ethnic groups in Trinidad
13–14 yr olds in Trinidad of African ethnicity % (no.) 13–14 yr olds in Trinidad of South Asian ethnicity % (no.) 13–14 yr olds in Trinidad of Mixed ethnicity % (no.)
Wheeze ever 12 (97) 7.8 (98)* ^ 14.5 (175)
Exercise wheeze ever 23 (185) 18.5 (233)**! 23.6 (284)
Night wheeze ever 9.8 (78) 4.5 (59)#! 9.1 (109)
Night cough ever 31.1 (247) 16.5 (207)#! 30.3 (360)
Severe wheeze ever 8.8 (70) 6.6 (83)! 11.5 (137)
Comparison of African and South Asian, * = p < 0.01; ** ; ** = p < 0.05; # = p < 0.0001
Comparison of Mixed and South Asian, ^ = p < 0.0001; ! = p < 0.001
Discussion
We report on the results of an ISAAC based questionnaire and video survey conducted in the twin island republic of Trinidad and Tobago involving almost 5000 adolescents. As far as these authors are aware, this is the largest survey of its kind conducted in the Anglophone Caribbean. Wheeze in the past was reported by almost a quarter of participants on both islands while wheeze in the past 12 months was reported by about 13% of students in both territories. Symptoms suggestive of severe asthma occurred more frequently in students from Tobago. There was good correlation between the questionnaire and video associated responses in both territories. Night cough was the commonest reported symptom and occurred in over 35% of respondents. Symptoms were reported more commonly by female participants and in the Trinidadian cohort, South Asian students reported fewer symptoms on the video questionnaire.
Wheeze in the past and wheeze in the last 12 months occurred less frequently among adolescents in Trinidad and Tobago compared with previous reports from Barbados; 24.2% and 13.3% respectively for T&T compared with 30.1% and 24.2% for Barbados [1]. The prevalence of wheeze in the last 12 months in Trinidad and Tobago is also lower than that reported by many Central and South American countries [7]. The higher prevalence of wheeze reported from Barbados may reflect an increased awareness of asthma symptoms in that island where there have been very successful public education programmes about the disease for several years [8]. The observed difference in prevalence between the two countries is also in keeping with previous reports of higher prevalence with higher per capita income of a country [9]; GDP per capita of Barbados is 16,200USD compared with 9,600 for Trinidad and Tobago [10].
The frequency of occurrence of symptoms of night cough in the past 12 months (35.4% & 38.3% in Trinidad and Tobago, respectively) far exceeded the prevalence of wheeze. The prevalence of night cough was more akin to that reported for rhinitis symptoms in the last year (33.4% & 30.9% in Trinidad and Tobago respectively) which have been reported elsewhere [11] and raises the possibility that in this population night cough could be due to untreated rhinitis rather than asthma.
Symptoms of severe asthma such as sleep disturbance more than once per week (2.7% in Trinidad & 5.1% in Tobago), speech limitation (5.2% & 8.7% in Trinidad and Tobago respectively) and 4 or more attacks of asthma in the past year (2.8% in Trinidad & 4.8% in Tobago) all occurred more commonly among students from Tobago. Additionally, more students from Tobago responded positively on the video questionnaire which asks for information about more severe asthma [7]. The ISAAC video shows 5 scenarios with young asthmatics at rest but in mild respiratory distress; with exercise induced wheezy breathing; with nocturnal cough; with nocturnal dyspnoea and with severe respiratory distress and wheezing.
The increased prevalence of severe asthma symptoms in Tobagonian adolescents compared to Trinidadian students is unexpected since Tobago has less industrial development and is more reliant on tourism and fishing than Trinidad where there are petrochemical parks and more manufacturing industries. Uneven availability of healthcare may have been one factor which contributed to differences in public awareness, under-diagnosis and management of asthma between the two islands. However, we have calculated similar doctor/patient ratios for the two islands (9.26 doctors/10,000 population in Trinidad and 9.95 doctors/10,000 population in Tobago). These figures are based on manpower figures for 2003/2004 from the Ministry of Health and Tobago Regional Health Authority (personal communication) and population data from the 2000 census [5].
Economic parameters as employment rates and monthly income are similar in Trinidad and Tobago suggesting that such factors may not be risk factors for the higher rate of severe asthma symptoms reported by Tobagonian students. In contrast, student ethnicity could be a contributory factor. In Trinidad, African and Mixed adolescents responded positively more often to video-related questions that did students of South Asian race. Since the video questionnaire seeks information about more severe symptoms, this result suggests that children of African and Mixed race may be experiencing more severe symptoms. The predominant ethnic groups in the Tobagonian cohort were African and Mixed (94.5%) so that race may be a factor in the higher level of positive responses to severe symptoms observed in Tobagonian students. Our findings are in keeping with other reports of race as an important association with severe asthma. Fox Ray and her colleagues [12] in their study of racial and income factors associated with hospital admissions for paediatric and adult asthmatics in California found black patients had a four-fold higher admission rate than other ethnic groups even after controlling for income.
Environmental factors such as environmental tobacco smoking (ETS) furry pets and use of foam pillows [13] have been associated with current wheeze and severe asthma in adolescents. Our survey of asthma did not ask about these environmental triggers. In Trinidad and Tobago, foam pillows are commonly used and about 25% of the adult male population smokes [14] so that these factors could be triggers of asthma among our adolescent population and should be explored in future research. We have previously noted the increased prevalence of asthma symptoms among 6–7 year olds who are exposed to ETS [15]. Furry animals such as cats, rabbits, gerbils and hamsters are not popular in our islands. The most popular pets are dogs which tend to be kept outdoors. We therefore do not anticipate pet exposure to be a risk factor for asthma in our context.
In our study, female students experienced more symptoms of wheeze. Gender difference in symptom prevalence has been reported in some but not all ISAAC surveys among adolescents [16]. We also noted that the difference in asthma symptoms between boys and girls to be significant in Trinidad only. We have no explanation for this but hope to explore this in future research.
Conclusion
Results from a two island ISAAC questionnaire and video survey among almost 5000 adolescents in Trinidad and Tobago have shown that wheeze in the past 12 months is a common symptoms in the population. Variation in the prevalence of more severe symptoms has been noted between the two islands with the surprising results that students from the less industrialised island of Tobago have a higher prevalence of severe symptoms. The observed difference may be related to genetic factors such as race but environmental and cultural contributory factors cannot be ruled out and merit further research.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MAM designed and coordinated study and wrote this paper
GJ and CC conducted all field work in both islands
GW coordinated the study in Tobago
RMA provided assistance with the statistical analysis
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional file 1
Appendix 1: questionnaires on asthma symptoms used in nationwide survey. This file contains the modified ISAAC questionnaires used in the nationwide survey of asthma and allergic symptoms which we conducted in Trinidad and Tobago
Click here for file
Acknowledgements
This project was commissioned by the Environmental Management Authority of Trinidad and Tobago and was funded through a Pan American Health Organization (PAHO) grant to Michele Monteil. The authors wish to thank the following persons for their assistance with this project: Owen Cuffie, project chauffeur; Avril Siung Chang, PAHO Office, Trinidad and Tobago; David MacIntosh and Wayne Rajkumar, Environmental Management Authority; the Ministry of Education of Trinidad and Tobago; the Tobago House of Assembly, Education Desk; the Health Education Unit, Tobago; and the principals and students of all the participating schools
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Howitt ME Roach TC Naidu R Prevalence of asthma and wheezing illnesses in Barbadian school children: The Barbados National Asthma & Allergy Study [Abstract] West Indian Med J 1998 47 22 23 10368619
Tam Tam HB Deva Tata M Ganganaidu K Aiyaroo K Prevalence of asthma related symptoms in school children in Port-of-Spain, Trinidad [Abstract] West Indian Med J 1998 47 22 10368619
Naidu RP Use of the log book in a quality assurance exercise in the Hospital Emergency Department in Barbados [Abstract] West Indian Med J 1990 39 44
PROCICARIBE-The Caribbean Agricultural Science and Technology Network System [Web site] Accessed October 28th 2004
Trinidad and Tobago Central Statistical Office Central Statistical Office [Web site] Accessed 5th October 2004
ISAAC Steering Committee and ISAAC Phase Three Study Group ISAAC Phase three manual 2000 Auckland; ISAAC
International Study of Asthma and Allergies in Childhood Steering Committee Worldwide Variation in the prevalence of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC Lancet 1998 351 1225 1232 9643741 10.1016/S0140-6736(97)07302-9
Global Initiative For Asthma Newsletter – GINA Asthma Association Directory [Web site] Accessed December 2004
Stewart AW Mitchell EA Pearce N Strachan DP Weilandon SK ISAAC Steering Committee International Study for Asthma and Allergy in Childhood. The relationship of per capita gross national product to the prevalence of symptoms of asthma and other atopic diseases in children (ISAAC) Int J Epidemiol 2001 30 173 9 11171881 10.1093/ije/30.1.173
The World Factbook [Web site] Accessed October 2004
Monteil MA Joseph G Changkit C Siung Chang A Wheeler G McIntosh D Antoine RM Rajkumar W Prevalence of Common Allergic Disorders among Primary & Secondary School-Aged Children in Trinidad & Tobago – The International Study of Asthma & Allergies in Childhood Study (T&T). [Abstract] Caribbean Med J 2003 65 36 37
Fox Ray N Tharner M Fadillioglu B Gergen PJ Race, Income, Urbanicity and Asthma Hospitalization in California – a small area analysis Chest 1998 113 1277 84 9596306
Burr ML Anderson HR Austin JB Harkins LS Kaur B Strachan DP Warner JO Respiratory symptoms and home environment in children: a national survey Thorax 1999 54 27 32 10343627
World Health Organization The tobacco atlas 2002 Accessed October 2004
Monteil MA Joseph G Chang Kit C Wheeler G Antoine RM Smoking at home is strongly associated with symptoms of asthma and rhinitis in children of primary school age in Trinidad and Tobago Rev Panam Salud Publica 2004 16 193 8 15507187
Habbick BF Pizzichini MM Taylor B Rennie D Senthilselvan A Sears MR Prevalence of asthma, rhinitis and eczema among children in 2 Canadian cities: the International Study of Asthma and Allergies in Childhood CMAJ 160 1824 8 1999 Jun 29 10405666
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-971617658310.1186/1471-2458-5-97Research ArticleA population-based study of asthma, quality of life, and occupation among elderly Hispanic and non-Hispanic whites: a cross-sectional investigation Arif Ahmed A [email protected] James E [email protected] George L [email protected] Texas Tech University Health Sciences Center, Department of Family & Community Medicine, Division of Public Health, Lubbock, TX, USA2 The University of Texas School of Public Health- Houston, Division of Environmental and Occupational Health Sciences, Houston, TX, USA2005 21 9 2005 5 97 97 5 7 2005 21 9 2005 Copyright © 2005 Arif et al; licensee BioMed Central Ltd.2005Arif 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 U.S. population is aging and is expected to double by the year 2030. The current study evaluated the prevalence of asthma and its correlates in the elderly Hispanic and non-Hispanic white population.
Methods
Data from a sample of 3021 Hispanics and non-Hispanic White subjects, 65 years and older, interviewed as part of an ongoing cross-sectional study of the elderly in west Texas, were analyzed. The outcome variable was categorized into: no asthma (reference category), current asthma, and probable asthma. Polytomous logistic regression analysis was used to assess the relationship between the outcome variable and various socio-demographic measures, self-rated health, asthma symptoms, quality of life measures (SF-12), and various occupations.
Results
The estimated prevalence of current asthma and probable asthma were 6.3% (95%CI: 5.3–7.2) and 9.0% (95%CI: 7.8–10.1) respectively. The majority of subjects with current asthma (Mean SF-12 score 35.8, 95%CI: 34.2–37.4) or probable asthma (35.3, 34.0–36.6) had significantly worse physical health-related quality of life as compared to subjects without asthma (42.6, 42.1–43.1). In multiple logistic regression analyses, women had a 1.64 times greater odds of current asthma (95%CI: 1.12–2.38) as compared to men. Hay fever was a strong predictor of both current and probable asthma. The odds of current asthma were 1.78 times (95%CI: 1.24–2.55) greater among past smokers; whereas the odds of probable asthma were 2.73 times (95%CI: 1.77–4.21) greater among current smokers as compared to non-smokers. Similarly fair/poor self rated health and complaints of severe pain were independently associated with current and probable asthma. The odds of current and probable asthma were almost two fold greater for obesity. When stratified by gender, the odds were significantly greater among females (p-value for interaction term = 0.038). The odds of current asthma were significantly greater for farm-related occupations (adjusted OR = 2.09, 95%CI: 1.00–4.39); whereas the odds were significantly lower among those who reported teaching as their longest held occupation (adjusted OR = 0.36, 95%CI = 0.18–0.74).
Conclusion
This study found that asthma is a common medical condition in the elderly and it significantly impacts quality of life and general health status. Results support adopting an integrated approach in identifying and controlling asthma in this population.
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Background
The U.S. population is aging and is expected to double by the year 2030, with the elderly comprising up to 20 percent of the total population. The population age 85 years and older will reach 21 million by the year 2050 [1]. Additionally, baby boomers will also reach 65 years of age in less than a decade. Therefore, epidemiologic studies of aging and age-associated diseases have national relevance.
Despite the worsening national trends for asthma for the past 25 years, bronchial asthma in the elderly has not received as much attention as asthma among children and adults. Many national and international studies exclude elderly when studying asthma, partly because asthma is difficult to distinguish from chronic obstructive pulmonary disease and congestive heart failure in older age [2,3]. However, recent studies have indicated that asthma is not an uncommon condition among the elderly. In the U.S., prevalence of asthma among the elderly range between 4% and 10% [4-7]. According to the Centers for Disease Control and Prevention (CDC) self-reported asthma rates in the elderly U.S. population increased sharply from 31 per 1000 in 1980 to 45 per 1000 in 1994 [8].
Long-term exposure to occupational agents at the workplace may result in poor quality of life later in life; however, the precise relationship between different occupations and asthma has not been studied previously in the elderly. According to state projections, by the year 2025 Texas will have the third largest population of individuals aged 65 and older after California and Florida [9]. Since morbidity due to asthma is on the rise, understanding factors associated with asthma and its association with the quality of life of older individuals is important. In this study the prevalence of asthma and asthma symptoms and their relationship with occupation and health related quality of life were estimated among older individuals in a largely sparsely settled region of west Texas.
Methods
The study data were collected as part of a large ongoing telephone-based cross-sectional study of individuals 65 years and older residing in 108 counties that comprise west Texas. A detailed description of the survey methods has been previously described [10]. Three waves of the surveys have been completed. The original sample comprised of 5006 subjects. The focus of wave-3 of the survey was respiratory conditions and symptoms and their effects on the older population. The cooperation rate for the wave-3 survey (completed interviews/ (completed interviews+ refusals)) was 90.4%; the response rate (completed interviews/ (completed interviews + refusals + eligible non contact)) was 86.7% [11,12]. The analysis for the present study was limited to the third wave of the survey, conducted from October 2001 through December 2001. Of the 3392 subjects interviewed during this third wave, 237 reported a prior history of emphysema, as determined by an affirmative response to the question, "Have you ever been diagnosed by a physician to have emphysema?" and were excluded from the analysis, leaving a sample of size 3155. Of these, 3021 were non-Hispanic whites or Hispanics and were included in the final analysis. During wave 3 of the survey, subjects were asked questions on general demographics, presence of asthma, asthma symptoms, allergies, smoking habits, housing characteristics, family history of asthma and allergies, chronic bronchitis and emphysema (collectively referred to as "COPD"), health-related quality of life (SF-12), and asthma-specific quality of life (mini Asthma QoL).
Asthma-related questionnaire items in this study were derived mainly from the International Union Against Tuberculosis and Lung Disease (IUATLD) bronchial symptom questionnaire [13] which has been previously validated in several countries. In addition, a cluster of five previously validated questions on asthma symptoms, collectively referred to as the Discriminative Function Predictor (DFP) were included in the final questionnaire.
Dependent variable
Our main outcome was a three category asthma variable coded as no asthma (reference category), current asthma, and probable asthma. Current asthma was defined as those responded in affirmative to questions, "have you ever been diagnosed by a physician to have asthma?" and "Do you still have asthma?" Diagnosis of asthma made by a health care provider still remains the most common approach used to define asthma in epidemiological studies [14]. The approach used in defining current asthma is similar to that used regularly in the U.S. National Health Interview Survey (NHIS) [15]. It is on this basis that the NHIS establishes its national prevalence estimates for current asthma. Probable Asthmawas defined using the weighted 5-item asthma symptoms questions, collectively referred to as Discriminant function predictor (DFP) [13]. The items included in DFP were weighted using the following logit equation: Logit P(X) = (-2.92) + 1.42(W) +1.39(SOB) + 1.00(TRB_C)+ 1.51(TRB_N) +2.37 (CT_D) where W = wheezing in the past 12 months; SOB = nocturnal shortness of breath in the past 12 months; TRB_C = continuous trouble with breathing; TRB_N = breathing is never quite right; CT_D = chest tightness around dust, animals, or feathers. To construct the variable "probable asthma" we used logit coefficients to generate logit scores. The default cut-off value of p > 0.5 was used to classify subjects as having probable asthma. Based on these criteria a total of 207 subjects were classified as having current asthma and a total of 265 subjects were classified as having probable asthma; these two groups did not overlap. A total of 2,549 subjects were classified as having neither current nor probable asthma.
Occupations
Each study subject was asked about their longest held occupation. This question was derived from the National Health and Nutrition Examination Survey III (item HAS17R) and asked from each study participant: "Thinking of all the paid jobs or businesses you ever had, what kind of work were you doing the longest?"[16]. Occupations were coded using the1980 U.S. Bureau of Census Occupational Classification Codes [17]. Those who reported never having worked (n = 312) and those who employed in the Armed Forces (n = 61) were excluded from the analysis. Based on prior studies by the authors [18], together with a review of literature, the coded occupations were grouped into seven categories: administrative/secretarial, health-related, teaching, service-related, farm- related, precision production, and other occupations.
Health-related Quality of Life (QoL)
The Medical Outcomes Study Short Form-12 (SF-12) health-related quality of life instrument was administered to all study participants. The SF-12, an abbreviated version of the SF-36, is commonly included in population-based studies to assess perceived health status [19], and its use has been validated in studies of older persons [20] and in clinical and community settings [21]. Scores on the 12 items were used to create two separate summary scores: a physical component score (PCS) and a mental component score (MCS). Scores ranged from 0 (the worst possible health) to 100 (the best possible health). In addition, the mini Asthma Quality of Life (mini-Asthma QoL) questionnaire was administered to those study participants who met the case definition for current asthma (n = 207). Mini-Asthma QoL measures functional impairments that are most troublesome to subjects with asthma during the 2 weeks prior to responding to the survey, and has four domains: 1) symptoms (5 items); 2) activity limitation (4 items); 3) emotional function (3 items); and 4) environmental stimuli (3 items). All responses were recorded on a 7-point Likert scale (from 1 = maximum impairment to 7 = no impairment). Responses to both the SF-12 scale and mini-Asthma QoL were scored according to published guidelines [21,22].
Other measures
The following covariates were also included in the analysis: 1) age (four categories); 2) sex (male, female); 3) education level (four categories); 4) income level (four categories); 5) geographic location (urban, rural); 6) history of hay fever; 7) pet ownership (three categories); 8) smoking status (non-smoker, current smoker, and past smoker): this variable was defined using the two questions: have you smoked at least 100 cigarettes during your entire life? Those who replied "yes" were asked Do you smoke cigarettes now?; those who responded in affirmative to both questions were classified as current smoker, those who smoked cigarettes in the past but no longer smoke cigarettes were classified as past smoker, and those who stated that they never smoke at least 100 cigarettes in their entire life were classified as non-smoker; 9) environmental tobacco smoke was defined based on responses to the question "other than the [respondent] how many people in home smoke?" 10) self-rated health was assessed using the question: "in general, would you say your health is excellent, very good, good, fair, or poor?" The responses were dichotomized into excellent/good and fair/poor; 11) complaint of pain: respondents were asked how often they were troubled with pain and how bad was their pain most of the time. The responses were grouped into three categories: no pain, mild pain, and severe pain; 12) body mass index (BMI): The BMI was defined as the weight in kilograms divided by the height in metres squared (kg/m2). This variable was computed based on self-reported weight and height and categorized into: normal weight (BMI <25), overweight (BMI 25–29.9), and obese (BMI = 30). Missing values were coded as a separate category; and 13) health insurance status. Nocturnal symptoms of asthma were defined using the question (asked separately for each symptom): "At any time in the last 12 months, have you been awakened at night by an attack of: 1) wheezing, 2) chest tightness, 3) shortness of breath, 4) cough."
To compare our study results with the prior published studies of asthma in the elderly, we performed a comprehensive MEDLINE search for English language articles published between 1966 and April 2005, using keyword terms "asthma" "elderly", "Health surveys or prevalence", and "Epidemiology". A total of 13 population or community-based studies were identified and data on type of the study, sample size, response rate, definition of asthma, and prevalence estimates of asthma were abstracted and summarized (Table 6). Only those studies which enrolled subjects aged 65 years and older, with clearly defined asthma as one of the outcome variables, and published prevalence estimates of asthma, were included in the summary table.
Statistical analysis
Comparison of the sample data to the U.S. Census 2000 data for west Texas suggested that the sample slightly underestimated the proportion of Hispanics and overestimated women. Therefore, data were weighted using post-stratification. The post-stratification adjustment cells were made up of age (65–69, 70–74, 75–79, and 80+), sex (Male, Female) and ethnicity (Hispanics, non-Hispanic White) categories. First, the census data (for 108 west Texas counties) and the wave-3 sample were stratified by age, sex, and ethnicity; then, an adjustment factor was computed by dividing the census cell proportion by the sample cell proportion. Finally, sampling weights were computed using the following formula [23]: Final Weight = (Total Number in Census Population/Total # in Sample) * Adjustment Factor
Weighted prevalence estimates and their corresponding 95% confidence intervals were computed. Since the outcome variable was categorical, polytomous logistic regression analyses were used to compute the odds ratios and their corresponding 95% confidence intervals. In polytomous logistic regression, the odds of current and probable asthma were simultaneously compared to no asthma, the common reference category. Odds ratios were adjusted for age, sex, race/ethnicity, smoking status, and history of hay fever. STATA statistical software version 9.0 (Stata Corp, College Station, TX), which incorporated sampling weights, was used for all the analyses.
Results
The socio-demographic sample characteristics of the study are presented in Table 1. The mean age of the study participant was 75.5 years (SD = 6.4). Of the 3021 participants, 878 were male and 2143 were female. Approximately 19% were obese (BMI = 30). The prevalence patterns of current and probable asthma by selected characteristics are presented in Table 2. The overall weighted prevalence of current asthma was 6.3% (95%CI: 5.3–7.2), whereas an additional 9.0% (95%CI: 7.8–10.1) of the respondents had probable asthma. Hispanic Americans reported a lower prevalence of current asthma (4.0%, 95%CI: 1.9–6.1) as compared to non-Hispanic whites (7.1%, 95%CI: 6.1–8.1). No significant race/ethnic differences were observed for probable asthma (Table 2). The prevalence estimates of current and probable asthma were slightly higher among females as compared to males. More than half of the sample were non-smokers (Table 1); only 8.2% reported currently smoking cigarettes and the prevalence of probable asthma was significantly higher in this group (16.8%, 95%CI: 11.8–21.8) as compared to non-smokers and ex-smokers (Table 2). Current smokers (33.7%, 95%CI: 27.3–40.1) and ex-smokers (24.2%, 95%CI: 21.2–27.2) also had significantly higher prevalence of wheezing as compared to non-smokers (15.1%, 95%CI: 13.1–17.1). The prevalence of nocturnal symptoms was significantly higher among those with current asthma, as compared to those with no asthma, and ranged from as low as 21.9% (95%CI: 15.2–28.6) for nocturnal wheezing to as high as 48.0% (95%CI: 40.4–55.6) for nocturnal cough (Figure 1). The prevalence of current asthma was highest among those who reported farm-related occupations as their longest held job (9.5%, 95%CI: 3.6–15.4), whereas the prevalence of probable asthma was highest among those who reported service-related occupations (12.8%, 95%CI: 8.4–17.1) as their longest held occupation (Table 2). When data was separated by gender, the prevalence of probable asthma was slightly higher among women (13.0%, 95%CI: 8.1–17.8) as compared to men (11.8%, 95%CI: 1.6–22.0) in this occupation category. Approximately 25% of Hispanics (95%CI: 19.4–29.6), as compared to 9.5% (95%CI: 8.3–10.7) of non-Hispanic whites, reported service-related occupation as their longest held occupation.
Table 1 Demographic, social, and health characteristics of the study sample
Characteristics Unweighted n (n = 3021)a Weighted %
Age-
65–69 700 29.0
70–74 938 25.8
75–79 656 19.7
80 and over 727 25.9
Sex
Male 878 40.5
Female 2143 59.5
Race/Ethnicity
NH-White 2671 73.6
Hispanic 350 26.4
Education
< HS 732 33.5
HS/GED 1001 29.1
Some College 698 19.8
College 581 17.7
Household Annual Income
≤ 20 k 1091 38.5
20–40 k 820 24.6
≥ 40 k 530 16.8
Missing 580 20.1
Geographic Location
Urban 1643 57.8
Rural 1378 42.2
Hay Fever
No 2283 77.8
Yes 718 22.2
Pet Ownership
Don't Have a Pet 1945 64.5
Dog/Cat 954 30.8
Other pets 120 4.6
Smoking Status
Non-Smoker 1657 54.1
Current Smokers 267 8.2
Past Smokers 1082 37.7
Environmental tobacco smoke
No 2704 88.3
Yes 314 11.7
Self-rated Health
Excellent/Good 2060 63.6
Fair/Poor 950 36.4
Complaint of Pain
No Pain 1747 59.1
Mild Pain 518 16.7
Severe Pain 747 24.1
Body Mass Index (BMI)c
Normal Weight 1225 36.3
Overweight 1100 37.5
Obese 547 18.8
Health Insurance
No 81 3.8
Yes 2937 96.2
Occupations
Administrative 279 10.0
Health Related 170 5.3
Teaching 275 9.3
Secretarial 602 18.5
Service Related 321 13.2
Farm Related 157 7.8
Precision Production 237 11.8
Other Occupations 574 24.1
a n may not total to 3021 in some variables due to missing values.
Table 2 Weighted prevalence estimates (95% confidence interval) of current and probable asthma by selected characteristics.
Characteristics Current Asthmaa %
(95%CI) Probable Asthmaa %
(95%CI)
Overall 6.3 (5.3–7.2) 9.0 (7.8–10.1)
Age-
65–69 7.4 (5.3–9.5) 9.8 (7.3–12.3)
70–74 5.5 (4.0–6.9) 7.8 (5.8–9.8)
75–79 6.6 (4.5–8.7) 9.3 (6.6–12.0)
80 and over 5.6 (3.8–7.3) 8.9 (6.6–11.1)
Sex
Male 5.2 (3.6–6.7) 7.8 (5.8–9.8)
Female 7.0 (5.9–8.2) 9.7 (8.3–11.2)
Race/Ethnicity
NH-White 7.1 (6.1–8.1) 8.4 (7.3–9.5)
Hispanic 4.0 (1.9–6.1) 10.5 (7.2–13.8)
Education
< HS 5.1 (3.3–6.9) 11.6 (9.0–14.3)
HS/GED 7.5 (5.8–9.1) 6.7 (5.1–8.4)
Some College 7.9 (5.9–10.0) 8.8 (6.4–11.2)
College 4.8 (2.9–6.6) 7.7 (5.3–10.1)
Household Annual Income
≤ 20 k 7.3 (5.6–8.9) 11.2 (8.9–13.3)
20–40 k 6.6 (4.9–8.3) 5.9 (4.2–7.6)
≥ 40 k 4.8 (2.8–6.8) 6.8 (4.4–9.1)
Missing 5.2 (3.2–7.2) 11.1 (8.0–14.2)
Geographic Location
Urban 6.2 (5.0–7.4) 8.7 (7.0–10.3)
Rural 6.3 (4.9–7.7) 9.3 (7.6–11.0)
Hay Fever
No 4.3 (3.4–5.2) 7.5 (6.2–8.7)
Yes 13.3 (10.7–15.8) 13.9 (11.0–16.9)
Pet Ownership
Don't Have a Pet 6.3 (5.1–7.6) 9.1 (7.6–10.6)
Dog/Cat 6.4 (4.9–8.0) 8.4 (6.4–10.4)
Other pets 3.8 (1.1–6.5) 10.6 (4.8–16.4)
Smoking Status
Non-Smoker 5.5 (4.4–6.6) 7.9 (6.4–9.5)
Current Smokers 3.4 (1.5–5.3) 16.8 (11.8–21.8)
Past Smokers 8.0 (6.2–9.8) 8.6 (6.7–10.6)
Environmental tobacco smoke
No 6.3 (5.4–7.3) 8.6 (7.3–9.8)
Yes 5.8 (2.8–8.7) 11.4 (7.5–15.3)
Self-rated Health
Excellent/Good 4.9 (3.9–5.9) 5.7 (4.6–6.8)
Fair/Poor 8.7 (6.8–10.6) 14.7 (12.1–17.3)
Complaint of Pain
No Pain 4.6 (3.6–5.7) 5.3 (4.1–6.5)
Mild Pain 7.2 (4.5–9.8) 8.8 (6.2–11.4)
Severe Pain 9.7 (7.4–11.9) 18.0 (14.6–21.3)
Body Mass Index (BMI)
Normal Weight (BMI <25) 5.0 (3.8–6.2) 7.2 (5.6–8.9)
Overweight (BMI 25–29.9) 6.5 (4.9–8.1) 8.4 (6.6–10.2)
Obese (BMI ≥ 30) 8.6 (6.2–11.0) 12.9 (9.7–16.2)
Health Insurance
No 8.5 (1.4–15.7) 12.8 (4.0–21.7)
Yes 6.2 (5.3–7.1) 8.8 (7.6–10.0)
Occupations
Administrative 8.3 (6.4–10.2) 9.3 (7.2–11.5)
Health Related 7.6 (3.5–11.7) 8.4 (3.0–13.8)
Teaching 3.1 (1.0–5.1) 9.7 (5.5–13.9)
Secretarial 8.7 (6.3–11.1) 10.3 (7.5–13.2)
Service Related 4.7 (2.2–7.1) 12.8 (8.4–17.1)
Farm Related 9.5 (3.6–15.4) 6.8 (2.1–11.5)
Precision Production 5.5 (2.5–8.5) 10.1 (5.5–14.6)
Other Occupations 6.3 (4.3–8.2) 6.8 (4.6–9.0)
The outcome is a three category variable: no asthma (reference), current asthma, probable asthma.
a The prevalence estimates were obtained by cross tabulating individual characteristics with the three category outcome variable (No Asthma, Current Asthma, and Probable asthma).
Figure 1 Prevalence of nocturnal symptoms among subjects with current asthma compared to no asthma.
The majority of subjects with current or probable asthma rated their health as fair or poor (Table 2) and had significantly worse physical health-related quality of life, as determined by lower scores on physical component part of SF-12 scale, compared to subjects without asthma (Table 3). Among the subsample of 207 subjects with current asthma who were administered the mini-Asthma QoL, significant impairment (Mean Score 4.6, 95%CI: 4.4–4.9) was observed only for the environmental stimuli domain (Table 4).
Table 3 SF-12 scores among subjects with and without Asthma
Asthma Status Meanb (95%CI)
Physical Component Score (PCS12)a
No Asthma 42.6 (42.1, 43.1)
Current Asthma 35.8 (34.2, 37.4)
Probable Asthma 35.3 (34.0, 36.6)
Mental Component Score (MCS12)a
No Asthma 53.4 (52.9, 53.8)
Current Asthma 52.9 (51.5, 54.3)
Probable Asthma 49.7 (48.2, 51.5)
a A lower score reflects a poorer quality of life.
b Weighted mean and 95% Confidence Interval.
Table 4 Mini-Asthma Quality of Life scores among subjects with Current Asthma
Mini-Asthma quality of life scorea Meanb (95%CI)
Overall 5.4 (5.2, 5.6)
Symptoms domain 5.4 (5.3, 5.6)
Activity limitation 5.6 (5.4, 5.9)
Emotional function 5.7 (5.4, 5.9)
Environmental stimuli 4.6 (4.4, 4.9)
a A lower score reflects a poorer quality of life.
b Weighted mean and 95% Confidence Interval.
The estimated crude and adjusted odds ratios of association for current and probable asthma with selected variables are presented in table 5. In the polytomous multiple logistic regression analysis, the adjusted odds of current asthma and probable asthma among women were 1.64 times (95%CI: 1.12–2.38) and 1.41 (95%CI: 1.00–2.01) times greater, respectively, as compared to men. Hispanics had significantly lower odds of current asthma as compared to non-Hispanic whites in the univariate analysis only (OR = 0.57, 95%CI 0.32–0.99). Hay fever was a strong predictor for both current and probable asthma. A past history of smoking was associated with 1.78 times greater odds of current asthma (95%CI: 1.24–2.55); however, for probable asthma, an increased odds of association was observed among current smokers only (adjusted OR 2.73, 95%CI: 1.77–4.21). Approximately one-fourth of respondents reported severe pain that prevented them from performing every day activities (Table 1). The self-reported severe pain was associated with more than twice the odds of having current asthma (adjusted OR = 2.35, 95%CI: 1.64–3.36) and more than four times the odds of probable asthma (adjusted OR = 4.23, 95%CI: 2.99–5.99) when compared to those without pain. Those who reported being in fair or poor health also had more than twice the odds of current and probable asthma as compared to those who reported their health as excellent or good. Similarly, the adjusted odds of current and probable asthma were 1.98 times and 2.12 times greater among obese individuals, respectively, as compared to normal weight individuals (Table 5). A significant interaction was found between female gender and obesity (BMI = 30) for current asthma only (adjusted OR = 2.85, 95%CI: 1.06–7.66).
Table 5 Polytomous multiple logistic regression analysis of factors associated with current and probable asthma.
Current Asthma Probable Asthma
Characteristics Crude OR
(95%CI) Adjusted ORa
(95%CI) Crude OR
(95%CI) Adjusted ORa
(95%CI)
Age
65–69 1.00 1.00 1.00 1.00
70–74 0.70
(0.46–1.01) 0.68
(0.45–1.05) 0.76
(0.51–1.13) 0.80
(0.53–1.20)
75–79 0.88
(0.56–1.38) 0.86
(0.54–1.38) 0.93
(0.61–1.43) 1.05
(0.67–1.64)
80+ 0.73
(0.47–1.14) 0.70
(0.44–1.11) 0.87
(0.58–1.30) 1.07
(0.71–1.61)
Sex
Male 1.00 1.00 1.00 1.00
Female 1.43
(1.00–2.04) 1.64
(1.12–2.38) 1.30
(0.94–1.80) 1.41
(1.00–2.01)
Race/Ethnicity
Non-Hispanic White 1.00 1.00 1.00 1.00
Hispanics 0.57
(0.32–0.99) 0.69
(0.39–1.24) 1.23
(0.84–1.81) 1.48
(0.99–2.21)
Education Level
College Graduate 1.00 1.00 1.00 1.00
< HS 1.12
(0.65–1.94) 1.86
(1.05–3.30) 1.58
(1.04–2.42) 1.64
(1.02–2.64)
HS/GED 1.60
(0.99–2.56) 1.68
(1.03–2.73) 0.88
(0.58–1.36) 0.84
(0.54–1.29)
Some College 1.75
(1.07–2.87) 1.76
(1.06–2.93) 1.20
(0.77–1.88) 1.07
(0.68–1.68)
Household Annual Incomeb
≥ 40 k 1.00 1.00 1.00 1.00
≤ 20 k 1.64
(0.99–2.71) 2.43
(1.38–4.28) 1.77
(1.14–2.76) 1.85
(1.17–2.92)
20–40 k 1.38
(0.82–2.32) 1.42
(0.83–2.43) 0.88
(0.54–1.43) 0.90
(0.55–1.48)
Geographic Location
Rural 1.00 1.00 1.00 1.00
Urban 0.97
(0.71–1.34) 1.02
(0.72–1.43) 0.92
(0.69–1.23) 0.84
(0.63–1.13)
Hay Fever
No 1.00 1.00 1.00 1.00
Yes 3.74
(2.71–5.16) 3.62
(2.65–4.95) 2.26
(1.66–3.08) 2.46
(1.80–3.37)
Pet Ownership
Do not own a pet 1.00 1.00 1.00 1.00
Own a Dog or a Cat 1.01
(0.72–1.40) 0.92
(0.65–1.30) 0.92
(0.67–1.27) 0.84
(0.61–1.17)
Own some other Pet 0.59
(0.27–1.28) 0.53
(0.23–1.25) 1.16
(0.61–2.19) 0.99
(0.49–1.98)
Smoking Status
Non-Smoker 1.00 1.00 1.00 1.00
Current Smoker 0.67
(0.36–1.24) 0.73
(0.39–1.35) 2.30
(1.51–3.50) 2.73
(1.77–4.21)
Past Smoker 1.51
(1.08–2.10) 1.78
(1.24–2.55) 1.13
(0.82–1.57) 1.36
(0.95–1.96)
Environmental tobacco smoke
No 1.00 1.00 1.00 1.00
Yes 0.94
(0.53–1.65) 1.03
(0.58–1.85) 1.37
(0.90–2.08) 1.21
(0.77–1.89)
Self-rated Health
Excellent/Good 1.00 1.00 1.00 1.00
Fair/Poor 2.08
(1.51–2.86) 2.71
(1.96–3.76) 3.03
(2.25–4.06) 3.41
(2.48–4.68)
Complaint of Pain
No Pain 1.00 1.00 1.00 1.00
Mild Pain 1.65
(1.04–2.62) 1.61
(1.00–2.62) 1.77
(1.18–2.65) 1.83
(1.22–2.75)
Severe Pain 2.60
(1.84–3.68) 2.35
(1.64–3.36) 4.20
(3.01–5.87) 4.23
(2.99–5.99)
Body Mass Index (BMI)
Normal Weight (BMI <25) 1.00 1.00 1.00 1.00
Overweight (BMI 25–29.9) 1.34
(0.92–1.94) 1.34
(0.91–1.95) 1.20
(0.84–1.69) 1.28
(0.89–1.86)
Obese (BMI ≥ 30) 1.94
(1.29–2.88) 1.98
(1.30–3.01) 2.00
(1.36–2.94) 2.12
(1.41–3.12)
Health Insurance
No insurance 1.00 1.00 1.00 1.00
Have insurance 0.67
(0.26–1.71) 0.48
(0.17–1.33) 0.64
(0.28–1.43) 0.64
(0.28–1.47)
Occupationc
Administrative/Secretarial 1.46
(1.04–2.03) 1.18
(0.84–1.65) 1.08
(0.79–1.49) 1.01
(0.73–1.40)
Health Related 1.17
(0.63–2.17) 1.01
(0.53–1.93) 0.93
(0.45–1.91) 0.91
(0.44–1.89)
Teaching 0.43
(0.21–0.87) 0.36
(0.18–0.74) 1.04
(0.63–1.73) 1.14
(0.68–1.88)
Service Related 0.70
(0.39–1.25) 0.78
(0.42–1.44) 1.54
(1.00–2.37) 1.47
(0.93–2.30)
Farm Related 1.51
(0.74–3.08) 2.09
(1.00–4.39) 0.74
(0.34–1.59) 0.84
(0.39–1.77)
Precision Production 0.83
(0.45–1.51) 1.11
(0.56–2.17) 1.13
(0.66–1.93) 1.28
(0.73–2.22)
Other Occupations 0.91
(0.62–1.33) 0.94
(0.63–1.41) 0.67
(0.45–0.99) 0.65
(0.43–0.99)
The outcome is a three category variable: no asthma (reference), current asthma, probable asthma.
a Adjusted for age, sex, race/ethnicity, smoking status, and history of hay fever.
b Missing information was coded as a separate category.
c Occupations were coded using dummy variable approach and each occupation was regressed separately.
A significant positive association between current asthma and farm-related occupation was found in this study (adjusted OR = 2.09, 95%CI: 1.00–4.39). The odds of current asthma were significantly lower among those who reported teaching as their longest held occupation (adjusted OR = 0.36, 95%CI = 0.18–0.74) (Table 5). Those in the service-related occupations had 1.47 times greater odds of probable asthma but the results were only significant in the univariate analysis (unadjusted OR = 1.54, 95%CI: 1.00–2.37).
Discussion
Asthma is a frequently overlooked and misdiagnosed medical condition in older patients. Morbidity due to asthma, if not properly diagnosed and managed, can have serious debilitating effects for older individuals. This large population based survey was an attempt to estimate the prevalence of asthma and its correlates in this population in the west Texas region.
This study found the prevalence of current asthma of 6.3% (95%CI: 5.3–7.2) and an additional 9.0% (95%CI: 7.8–10.1) had probable asthma (symptoms based definition-DFP). In our earlier analysis of NHANES III data, using a similar case definition as reported in this study, we reported a prevalence of current asthma of 3.6% (95%CI 2.9–4.2) in the U.S. population aged 60 and above [24]. The review of previously published population- based studies in the elderly suggests a wide variation in the prevalence of asthma (Table 6). The U.S. studies, on average, have reported a lower prevalence of asthma [4-7] as compared to European studies [25-30]. The four previous studies from the U.S., included in the summary table, found a median prevalence of asthma of 4.7% (range 3.9% to 10%); whereas the median prevalence from the six European studies was 7% (range 6% to 8.4%) (Table 6). The three studies from the Asia-Pacific region [31-33] reported a median prevalence of asthma of 5.5% (range 3.9–10.5). The wide variation in reported prevalence estimates could in part be due to use of different case definitions of asthma or different geographical region which complicates comparison among studies.
Table 6 Summary table of asthma studies in the elderly
Reference (Year) Country Type of Study Age Group Final Sample Size (Response Rate-%) Outcome Prevalence (95%CI)
Hardie et al [25] (2005) Norway Cross-Sectional Survey 70 years and older 1649 (56%) Current Asthma All: 8.0% (6.5–9.5)
Men: 8.1% (6.0–10.2)
Women: 8.0% (5.9–10.0)
Wheezing All: 7.7 (6.3–9.1)
Men: 10.3 (8.0–12.5)
Women: 6.2 (4.4–8.0)
Malik et al [7] (2004) USA Community-based Cross-Sectional Survey > 60 years 380 Doctor Diagnosed Asthma 10% (9.0–16.0)
Mishra [31] (2003) India Interview administered Cross-sectional survey 60 years and older 38,582 (98% overall) [Self-reported] Asthma Range: 8.5%–12.4% (various age groups)
Men: 9.5%–14.0%
Women: 7.5%–10.5%
Choy et al. [32] (2002) China Cross-sectional survey 70 yrs and older 179 (72%) [Clinical] Asthma 3.9% (1.6–7.9)
[Symptom-based] Asthma 5.0% (2.3–9.3)
Saks et. al. [33] (2001) Estonia Interview administered survey ≥ 65 years of age 811 (81.1%) Doctor Diagnosed Asthma 5.5% (3.9%–7.2%)
Romero et al. [4] (2001) USA community-based cross-sectional survey ≥ 65 years of age 883 (53%) [Self reported] Asthma NHWM*: 9.3%
NHWF* : 7.6%
HM*: 4.2%
HF*: 6.3%
Enright et. al. [6] (1999) USA Prospective study-Cardiovascular Health Study ≥ 65 years of age 4581 Definite Asthma 3.9%
Probable Asthma 4.1%
Parameswaran et. al. [26] (1998) U.K. Cross-Sectional Survey > 65 years of age 1362 (68%) Asthma 7.0%
Nejjari et al. [27] (1996) France Cross-sectional based on PAQUID Cohort ≥ 65 years of age 2355 (97.9%) Asthma Overall: 6.1%
Men: 7.4% (5.7–9.0)
Women: 5.2% (4.1–6.4)
Current Asthma Overall: 2.5%
Men: 2.9%
Women: 2.2%
Isoaho et al. [28] (1994) Finland Cross-sectional survey ≥ 65 years of age 1196 (93%) Self reported Asthma Men: 7.0%
Women: 8.6%
Current Asthma Men: 2.9%
Women: 3.8%
Burrows et al. [5] (1991) USA Cross-sectional as part of a longitudinal study ≥ 65 years of age 804 [Self reported] Asthma Men: 3.8%
Women: 7.1%
Active Asthma Overall: 7.5%
Men: 7.9%
Women: 7.1%
Horsley et al. [29] (1991) U.K. Postal Cross Sectional Survey ≥ 65 years of age 1803 (96.2%) Asthma Overall: 8.4%
Men: 9.6%
Women: 7.2%
Current Asthma Overall: 4.2%
Men: 4.9%
Women:3.6%
Wheezing Overall: 24.2%
Men: 29.2%
Women: 19.7%
Burr et al. [30] (1979) U.K. Random Cross-sectional survey 70 yrs and older 418 (86.2%) Current Asthma Overall: 2.9%
Men: 5.1%
Women: 1.8%
[Ever] Asthma 6.5%
* NHWM = non-Hispanic white male, NHWF = non-Hispanic white female, HM = Hispanic male, HF = Hispanic female.
Some of the previously well recognized correlates of asthma, such as female gender, low socioeconomic status (as measured by education and income) and hay fever were also identified in this study [24,34]. In addition, smoking, poor health-related QoL, obesity, and certain occupational groups were associated with current or probable asthma.
Associations between smoking and asthma remain a subject of debate. In this study the prevalence of probable asthma was approximately 17% among current smokers; ex-smokers had a higher odds of current asthma (adjusted OR = 1.78, 95%CI: 1.24–2.55) whereas current smokers were more likely to have probable asthma (adjusted OR = 2.73, 95%CI: 1.77–4.21). In a recent study, Hardie et. al., [25] reported a greater than two-fold increased odds of current asthma among ex-smokers age 70 years and older. Similarly, a recent incident case-control study reported an increase risk of asthma among ex-smokers [35]. Prior population-based surveys, focusing on younger adults, have largely failed to find such an association. In the NHANES III analysis, a positive association of current smoking was found with the presence of wheezing, but not with current asthma, suggesting possible confounding or misclassification with non-asthma causes of wheezing, such as emphysema or chronic bronchitis [24]. Similarly, results from the European Community Respiratory Health Survey (ECRHS) also found no association of asthma with either a current or past history of smoking [34]. Since ECRHS is a study of young adults, it is possible that, being of lesser duration, exposure to tobacco smoke has not yet had time to cause serious damage to airways that may contribute to the appearance of asthma. An alternative explanation could be that the general decline in prevalence of smoking in most developed countries partly explains the lack of association observed in the younger population. The results of this study suggest that despite quitting smoking, the airway damage is not completely reversible. However, further studies are needed in older populations to assess the long term impact of smoking on asthma.
Self-rated health is considered a valid measure of person's health [36,37] and has been shown to relate directly to quality of life [38]. The SF-12 has been used previously to measure health outcomes for persons suffering from asthma [39] and COPD [40]. Use of both generic and asthma specific QoL measures are recommended to assess the impact of asthma on patient's daily life [41]. In a large community survey of elderly, Enright and colleagues [6] reported that subjects with asthma had significantly lower QoL and higher degree of impairment of activities of daily living. They were more likely to report symptoms of depression and poor general health. Similarly, Nejjari and colleagues [42] in a population based case-control study reported that older subjects with asthma were more likely to report lower QoL than controls. Breathlessness was reported as a major cause of lower QoL. In this study more than one-third of participants rated their health as fair or poor. Among those with current and probable asthma this percentage increased to approximately 50% and 60%, respectively. Since such a large proportion of subjects with probable asthma (i.e., without a clinical diagnosis of asthma) complained of poor health, it is possible they represent a group with as yet undiagnosed (and, hence, untreated) asthma. In addition, both current and probable asthma were associated with severe pain, poor physical health related quality of life and poor performance on the mini-Asthma QoL environmental domain subscale, all of which add consistency to this impression.
Although several recent studies are finding an association, the relationship between asthma and obesity remains controversial or, at best, unexplained. This association has been observed in children and adults, [24,43] as well as among nurses [44] and other health care workers (author's unpublished data). In this study we report a positive association between current asthma and obesity in the elderly, which was only significant among females (adjusted OR = 2.74, 95%CI 1.74–4.33; p value for interaction term = 0.038). The interaction term was not statistically significant for probable asthma. These findings are consistent with those of other population-based studies [44-47]. Beckett et al., [46] in a prospective study of 4547 African-American and White men and women, found a significant association between incident asthma and body mass index in females only. Camargo and colleagues, in a prospective study of registered nurses, found an association between body mass index and incident cases of asthma [44]. Chen et al., [47] in a large longitudinal study of the Canadian population reported a significant association between obesity and development of asthma among women. However, these and our results contrast somewhat to recently reported results from an incident case-control study on Swedish adults which reported an odds ratio of 3.0 and 3.3 in both females and males respectively [35]. The authors included 309 cases of incident asthma of which 202 (65%) were women. Although the authors enrolled an equal number of controls, they did not provide information on the gender distribution of this comparison group. Moreover, they did not adjust their results for known confounders including smoking and hay fever, which in part could explain the discrepant findings. Although a biological mechanism to explain such an association remains elusive, the strong evidence observed across all age groups, among different occupational groups, and from studies of all types (cross-sectional surveys, case-control studies and prospective studies) suggests a possible causal relationship between obesity and asthma.
In this study two occupational groups were significantly associated with current asthma. Those who reported teaching as their longest held occupation were 0.36 times less likely to have current asthma. This is in contrast to other reports that found higher rates of asthma among teachers including our own studies in other populations [18,48]. Kraut et. al.,[48] reported elevated odds ratios for "other teaching and related occupations" (OR 2.54, 95% CI 1.18–5.44); Whelan et. al., [49] reported higher prevalences of work-related upper respiratory symptoms and wheezing among teachers, but not asthma. Differences in the study population could in part explain the discrepant findings. Alternately, the lower odds observed among teachers in this study could reflect a cohort effect. Following the energy crisis of the 1970s, schools were made more airtight. This resulted in school buildings with poor ventilation and excess moisture, and the subsequent risk of exposure to multiple antigens, including mold and other indoor air contaminants [50,51]. It is plausible that teachers in this group may have worked in this profession before changes were made to school building codes, and may not have been exposed to the poor indoor air quality and other environmental conditions that are being reported by the younger working population.
Farm-related occupations have previously been reported to be associated with asthma among adults, as is in this study. In the present study, subjects with farm-related occupations had twice the odds of current asthma. When the data were stratified by gender, the association was primarily seen in males (adjusted OR = 2.51, 95%CI: 1.02–6.21). There was no difference in the prevalence of hay fever among those with or without farming occupations, raising the possibility that the increased prevalence of current asthma in this population is of non-allergic origin. This is consistent with recently reported findings in Norwegian farmers with current asthma that was of non-atopic origin [52]. In our earlier analyses of NHANES III data, [18] a greater than four-fold odds of work-related asthma (OR = 4.22, 95%CI: 1.76–10.10) was observed among those with farm-related occupations. In the French PAQUID cohort, retired farm workers (aged 65 and older) had more than five times the odds (OR = 5.35, 95%CI: 1.33–21.50) of current asthma [27]. Similarly, Kogevinas et. al.,[53] reported an odds ratio of 2.62 (95% CI 1.29–5.35) among farmers who participated in the ECRHS.
The service-related occupation group had significantly higher odds of probable asthma in the unadjusted analyses only. The three major groups that made up this occupational category were: food-related, housekeepers/janitors, and hairdressers. All of these occupations, which involve use of chemicals and substances that are respiratory irritants, have previously been associated with increase risk of asthma [18,54].
There were some limitations of this study. Since the study was cross-sectional in nature, cause and effect relationship cannot be established. There were 41 subjects who reported having both current asthma and chronic bronchitis; inclusion of these subjects caused a slight overestimation of current asthma prevalence. Respondents with chronic bronchitis were not excluded from the analysis because symptoms of asthma and chronic bronchitis can overlap, especially in the old age. Smoking confounds asthma and subjects with asthma tend to become incomparable with regard to smoking habits than those without asthma. It was difficult to separate these associations in a cross-sectional survey. Another limitation of the study is possible misclassification of current asthma status. Study respondents whose asthma was in control or in remission at the time of study may have responded as not having asthma and hence been classified as being non-asthmatic; however, if their asthma was not under control, they may have responded affirmatively to questions on asthma diagnosis, being thus classified as having current asthma. The survey sample attrition over time is also a potential concern. However, no evidence for differential survival was found in the study. With advancing age, quality of life in asthmatics can be compromised due to the concurrent presence of other chronic medical conditions, which could also partly explain the poor physical QoL observed in this study. However, our results are consistent with earlier findings where both the moderate or severe persistent asthma was associated with poor QoL among the elderly [55]. Finally, no reference values are available for mini Asthma QoL in the general elderly population; this fact, in addition to absence of indoor monitoring data, makes the interpretation of low scores on the environmental domain subscale of the Asthma QoL (reflecting poor QoL) difficult.
Conclusion
This study found that asthma is a common medical condition among the elderly. Several factors including female gender, low socio-economic status, hay fever, obesity, and smoking status were associated with current or probable asthma. The majority of subjects with current or probable asthma rated their health as fair or poor and their quality of life was compromised. Male farmers had higher odds of current asthma; whereas lower odds of current asthma, possibly due to a cohort effect, were observed among those who were in a teaching occupation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
AA carried out the study as part of funding from the National Institute of Aging, performed statistical analyses and drafted the manuscript. JER participated in the design of the study and draft of the manuscript. GD participated in the analysis and interpretation of occupational section of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This research is supported in part by grant 90AM2378 from the Administration on Aging and 1R03AG020986-01 by the National Institute of Aging (NIH). The authors acknowledge technical and editorial support provided by Eun Sul Lee, Ty Borders, and Tom Xu.
==== Refs
U.S. Federal Interagency Forum on Aging-Related Statistics Older Americans 2004: Key indicators of Well-Being. Federal Interagency Forum on Aging-Related Statistics 2004 Washington D.C: U.S. Government Printing Office
Lee HY Stretton T Asthma in elderly Br Med J 1973 J4 93 95
Slavin RG The Elderly Asthmatic Patient Allergy & Asthma Proc 2004 25 371 373 15709446
Romero LJ Lindeman RD Liang HC Koehler KM Baumgartner RN Garry PJ Prevalence of self-reported illnesses in elderly Hispanic and non-Hispanic Whites in New Mexico Ethnicity & Disease 2001 11 263 272 11456001
Burrows B Barbee RA Cline MG Knudson RJ Lebowitz MD Characteristics of asthma among elderly adults in a sample of the general population Chest 1991 100 935 942 1914608
Enright PL McClelland RL Newman AB Gottlieb DJ Lebowitz MD Underdiagnosis and undertreatment of asthma in the elderly. Cardiovascular Health Study Research Group Chest 1999 116 603 613 10492260 10.1378/chest.116.3.603
Malik A Saltoun CA Yarnold PR Grammer LC Prevalence of obstructive airways disease in the disadvantaged elderly of Chicago Allergy & Asthma Proceedings 2004 25 169 173 15317321
Mannino DM Homa DM Pertowski CA Ashizawa A Nixon LL Johnson CA Ball LB Jack E Kang DS Surveillance for asthma–United States, 1960–1995 Mor Mortal Wkly Rep CDC Surveill Summ 1998 47 1 27
U.S. Bureau of Census State Population Projections: Detailed State Projections by Single Year of Age, Sex, Race, and Hispanic Origin: 1995 to 2025 Accessed Feb 12, 2005
Borders TF Aday LA Xu KT Factors associated with health-related quality of life among an older population in a largely rural western region J Rural Health 2004 20 67 75 14964929
American Association for Public Opinion Research Standard definitions: final dispositions of case codes and outcome rates for surveys 2000 Ann Arbor, MI: American Association for Public Opinion Research
Council of American Survey Research Organizations On the definition of response rates: a special report of the CASRO Task Force on Completion Rates 1982 Port Jefferson, NY: Council of American Survey Research Organizations
Burney PG Chinn S Britton JR Tattersfield AE Papacosta AO What symptoms predict the bronchial response to histamine? Evaluation in a community survey of the bronchial symptoms questionnaire (1984) of the International Union against Tuberculosis and Lung Disease International Journal of Epidemiology 1989 18 165 173 2656559
Pekkanen J Pearce N Defining asthma in epidemiological studies Eur Respir J 1999 14 951 957 10573248 10.1034/j.1399-3003.1999.14d37.x
Centers for Disease Control and Prevention Asthma prevalence and control characteristics by race/ethnicity – United States, 2002 MMWR 2002 53 145 148
National Center for Health Statistics (NCHS) Plan and operation of the Third National Health and Nutrition Examination Survey, 1988–94 Vital and health statistics, series 1: programs and collection procedures, no 32, DHHS publication no (PHS) 94-1308 (GPO no 017-022-01260-0) 1994 Hyattsville, MD: National Center for Health Statistics
U.S. Bureau of the Census 1980 Census of Population: Classified Index of Industries and Occupations PHC80-R4 1982 Washington, DC
Arif AA Delclos GL Whitehead LW Tortolero SR Lee ES Occupational exposures associated with work-related asthma and work-related wheezing among U.S. workers Am J Indust Med 2003 44 368 376 10.1002/ajim.10291
Ware JE Kosinski M Keller SD A 12-Item Short-Form Health Survey: Construction of Scales and Preliminary Tests of Reliability and Validity Medical Care 1996 34 220 233 8628042 10.1097/00005650-199603000-00003
Resnigh B Nahm ES Reliability and validity testing of the revised 12-item Short Form Health Survey in older adults J Nurs Management 2001 9 151 161
QualityMetric Inc. SF-12 Health Survey 2002
Juniper EF Guyatt GH Cox FM Ferrie PJ King DR Development and validation of the Mini Asthma Quality of Life Questionnaire Eur Respir J 1999 14 32 38 10489826 10.1034/j.1399-3003.1999.14a08.x
Lee ES Forthofer RN Lorimer RJ Analyzing complex survey data 1989 07–071 Newbury Park, CA: Sage
Arif AA Delclos GL Lee ES Tortolero SR Whitehead LW Prevalence and risk factors of asthma and wheezing among US adults: an analysis of the NHANES III data Eur Respir J 2003 21 827 833 12765429
Hardie JA Vollmer WM Buist AS Bakke P Morkve O Respiratory symptoms and obstructive pulmonary disease in a population aged over 70 years Respir Med 2005 99 186 195 15715185 10.1016/j.rmed.2004.06.006
Parameswaran K Hildreth AJ Chadha D Keaney NP Taylor IK Bansal SK Asthma in the elderly: underperceived, underdiagnosed and undertreated; a community survey Respir Med 1998 92 573 577 9692125 10.1016/S0954-6111(98)90311-0
Nejjari C Tessier JF Letenneur L Dartigues JF Barberger-Gateau P Salamon R Prevalence of self-reported asthma symptoms in a French elderly sample Respir Med 1996 90 401 408 8796232 10.1016/S0954-6111(96)90113-4
Isoaho R Puolijoki H Huhti E Kivela SL Tala E Prevalence of asthma in elderly Finns J Clin Epidemiol 1994 47 1109 1118 7722544 10.1016/0895-4356(94)90097-3
Horsley JR Sterling IJ Waters WE Howell JB Respiratory symptoms among elderly people in the New Forest area as assessed by postal questionnaire Age & Ageing 1991 20 325 31 1755387
Burr ML Charles TJ Roy K Seaton A Asthma in the elderly: an epidemiological survey BMJ 1979 21 1041 1044 312675
Mishra V Effect of Indoor Air Pollution from Biomass Combustion on Prevalence of Asthma in the Elderly Environ Health Perspect 2003 111 71 77 12515681
Choy DK Hui DS Li ST Ko FW Ho S Woo J Lai CK Prevalence of wheeze, bronchial hyper-responsiveness and asthma in the elderly Chinese Clinical & Experimental Allergy 2002 32 702 707 11994093 10.1046/j.1365-2222.2002.01395.x
Saks K Kolk H Allev R Soots A Koiv K Paju I Jaanson K Schneider G Health status of the older population in Estonia Croat Med J 2001 42 663 668 11740851
Janson CJ Anto P Burney S Chinn R de Marco J Heinrich D Jarvis N Kuenzli B Leynaert C Luczynska F Neukirch C Svanes J Sunyer M The European Community Respiratory Health Survey: what are the main results so far? on behalf of the European Community Respiratory Health Survey II Eur Respir J 2001 18 598 611 11589359 10.1183/09031936.01.00205801
Ronmark E Andersson C Nystrom L Forsberg B Jarvholm B Lundback B Obesity increases the risk of incident asthma among adults Eur Resp J 2005 25 282 288 10.1183/09031936.05.00054304
Finch BK Hummer RA Reindl M Vega WA Validity of self-rated health among Latino(a)s Am J Epidemiol 2002 155 755 759 11943694 10.1093/aje/155.8.755
Burstrom B Fredlund P Self rated health: Is it as good a predictor of subsequent mortality among adults in lower as well as in higher social classes J Epidemiol Community Health 2001 11 836 40 11604441 10.1136/jech.55.11.836
Rohrer JE Arif AA Pierce JR Blackburn C Unsafe Neighborhoods, Social Group Activity, and Self-Rated Health J Public Health Manag Pract 2004 10 124 129 14967979
Ford ES Mannino DM Redd SC Moriarty DG Mokdad AH Determinants of quality of life among people with asthma: findings from the Behavioral Risk Factor Surveillance System J Asthma 2004 41 327 36 15260466 10.1081/JAS-120026090
Miravitlles M Ferrer M Pont A Zalacain R Alvarez-Sala JL Masa F Verea H Murio C Ros F Vidal R IMPAC Study Group Effect of exacerbations on quality of life in patients with chronic obstructive pulmonary disease: a 2 year follow up study Thorax 2004 59 387 95 15115864 10.1136/thx.2003.008730
Richards JM JrHemstreet MP Measures of life quality, role performance, and functional status in asthma research Am J Respir Crit Care Med 1994 149 S31 9 8298767
Nejjari C Tessier JF Barberger-Gateau P Jacqmin H Dartigues JF Salamon R Functional status of elderly people treated for asthma-related symptoms: a population based case-control study Eur Respir J 1994 7 1077 1083 7925876
Arif AA Borders T Rohrer J Xu T Prevalence and risk factors of pediatric asthma in a largely rural U.S. population J Paediatr Child Health 2004 40 189 194 15009547 10.1111/j.1440-1754.2004.00335.x
Camargo CA JrWeiss ST Zhang S Willett WC Speizer FE Prospective study of body mass index, weight change, and risk of adult-onset asthma in women Arch Intern Med 1999 159 2582 2588 10573048 10.1001/archinte.159.21.2582
Behren JV Kreutzer R Hernandez A Self-Reported Asthma Prevalence in Adults in California J Asthma 2002 39 429 440 12214897 10.1081/JAS-120004036
Beckett WS Jacobs DR JrYu X Iribarren C Williams OD Asthma is associated with weight gain in females but not males, independent of physical activity Am J Respir Crit Care Med 2001 164 2045 2050 11739133
Chen Y Dales R Tang M Krewski D Obesity may increase the incidence of asthma in women but not in men: longitudinal observations from the Canadian National Population Health Surveys Am J Epidemiol 2002 155 191 197 11821241 10.1093/aje/155.3.191
Kraut A Walld R Mustard C Prevalence of physician-diagnosed asthma by occupational groupings in Manitoba, Canada Am J Ind Med 1997 32 275 282 9219658 10.1002/(SICI)1097-0274(199709)32:3<275::AID-AJIM14>3.0.CO;2-S
Whelan EA Lawson CC Grajewski B Petersen MR Pinkerton LE Ward EM Schnorr TM Prevalence of respiratory symptoms among female flight attendants and teachers Occup Environ Med 2003 60 929 934 14634183 10.1136/oem.60.12.929
Environmental Protection Agency Managing Asthma in the School Environment: Asthma in Schools Accessed Feb 17, 2005
Teacher to Teacher: Issues Affecting the Classroom Teacher by AFT President Sandra Feldman Accessed Feb 17, 2005
Eduard W Douwes J Omenaas E Heederik D Do farming exposures cause or prevent asthma? Results from a study of adult Norwegian farmers Thorax 2004 59 381 386 15115863 10.1136/thx.2004.013326
Kogevinas M Anto JM Sunyer J Tobias A Kromhout H Burney P Occupational asthma in Europe and other industrialized areas: A population-based study. European Community Respiratory Health Survey Study Group Lancet 1999 353 1750 1754 10347988 10.1016/S0140-6736(98)07397-8
Mendonca EM Algranti E de Freitas JB Rosa EA dos Santos Freire JA de Paula Santos Ud U Pinto J Bussacos MA Occupational asthma in the city of Sao Paulo, 1995–2000, with special reference to gender analysis Am J Ind Med 2003 43 611 617 12768611 10.1002/ajim.10210
Huss K Naumann PL Mason PJ Nanda JP Huss RW Smith CM Hamilton RG Asthma severity, atopic status, allergen exposure and quality of life in elderly persons Ann Allergy Asthma Immunol 2001 86 524 530 11379803
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-981618149110.1186/1471-2458-5-98Research ArticleHIV prevention programmes for female sex workers in Andhra Pradesh, India: outputs, cost and efficiency Dandona Lalit [email protected] Pratap [email protected] SG Prem [email protected] YK [email protected] A Anod [email protected] M Chalapathi [email protected] Elliot [email protected] M [email protected] Nell [email protected] James G [email protected] Health Studies Area, Centre for Human Development, Administrative Staff College of India, Hyderabad, India2 Institute for Health Policy Studies and AIDS Research Institute, University of California, San Francisco, USA2005 24 9 2005 5 98 98 13 7 2005 24 9 2005 Copyright © 2005 Dandona et al; licensee BioMed Central Ltd.2005Dandona 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
Female sex workers and their clients play a prominent role in the HIV epidemic in India. Systematic data on the outputs, cost and efficiency for HIV prevention programmes for female sex workers in India are not readily available to understand programme functioning and guide efficient use of resources.
Methods
Detailed output and cost data for the 2002–2003 fiscal year were obtained using standardised methods at 15 HIV prevention programmes for female sex worker in the state of Andhra Pradesh in southern India. The services provided and their relation to the total and unit economic costs were analysed using regression techniques. The trends for the number of sex workers provided services by the programmes since inception up to fiscal year 2004–2005 were examined.
Results
The 15 programmes provided services to 33941 sex workers in fiscal year 2002–2003 (range 803–6379, median 1970). Of the total number of contacts with sex workers, 41.6% were by peer educators and 58.4% by other programme staff. The number of sex worker contacts in a year by peer educators varied 74-fold across programmes as compared with a 2.7-fold variation in sex worker contacts by other programme staff. The annual economic cost of providing services to a sex worker varied 6-fold between programmes from Indian Rupees (INR) 221.8 (US$ 4.58) to INR 1369 (US$ 28.29) with a median of INR 660.9 (US$ 13.66) and mean of INR 517.8 (US$ 10.70). Personnel salaries made up 34.7% of the total cost, and recurrent goods made up 38.4% of which 82.1% was for condoms. The cost per sex worker provided services had a significant inverse relation with the number of sex workers provided services by a programme (p < 0.001, R2 = 0.75; power function). There was no correlation between the full time equivalents of programme staff and the number of sex workers provided services by the programmes, but there was a modest inverse correlation between the number of sex workers served and the average time spent with each sex worker in the year adjusted for the full-time equivalents of programme staff (p = 0.011, R2 = 0.40; exponential function). The average number of sex workers provided services annually by the first batch of 7 programmes started in early 1999 plateaued after the fourth fiscal year to 3500, whereas the 8 second-batch programmes started in late 2000 reached an average of 2000 sex workers in 2004–2005 with an increasing trend up to this fourth fiscal year.
Conclusion
The HIV prevention efforts in this Indian state would benefit from standardisation of the highly variable services provided by peer educators, who form an important part of the sex worker programmes. The cost per sex worker served decreases with increasing number of sex workers served annually, but this has to be weighed against an associated modest trend of decrease in time spent with each sex worker in some programmes.
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Background
The number of people infected with HIV in India is estimated to be one of the highest in the world [1,2]. The state of Andhra Pradesh in southern India has a population of 80 million, and the sentinel surveillance suggests that it has one of the highest estimated burden of HIV among the Indian states [3]. It has been suggested that female sex workers and their clients play a very prominent role in the HIV epidemic in India, and that effective interventions for them could help substantially curb this epidemic [4]. In the sentinel surveillance of female sex workers at seven locations in Andhra Pradesh in 2004, the HIV prevalence ranged from 8% to 41% with a mean of 16% [5].
HIV prevention programmes for female sex workers form a major component of the HIV prevention efforts in Andhra Pradesh. With recent substantial funding from the Gates Foundation for HIV prevention [6], the number of intervention programmes for female sex workers is increasing rapidly at present in Andhra Pradesh. However, there are few data assessing the outputs, cost and efficiency of the various strategies to control HIV in India [7,8]. Such data are needed for informed planning and efficient utilisation of resources available for HIV control. As part of a study to assess the cost-efficiency of various HIV prevention strategies in Andhra Pradesh, we report data on the outputs, cost and efficiency of HIV prevention programmes for female sex workers.
Methods
This study forms part of a multi-country effort to study cost and efficiency of HIV prevention in India, Mexico, Russia, South Africa and Uganda by the Prevent AIDS Network for Cost-Effectiveness Analysis (PANCEA) [9]. The methods for the overall multi-country study are described in detail elsewhere [10]. The methods relevant for this report follow.
Selection of programmes
At the time of starting data collection for this study in mid-2003, 15 HIV prevention programmes for female sex workers were being run by non-governmental organisations in Andhra Pradesh with the support of the Andhra Pradesh State AIDS Control Society. As the HIV epidemic in Andhra Pradesh was initially estimated to be more prevalent in the eastern Coastal region of the state with nine districts, 13 of the 15 programmes were in this region. The other two programmes were in the northern Telangana region of the state that has the state capital and nine other districts, whereas the smaller southern Rayalseema region with four districts had none of these programmes. For this study, we included all the 15 programmes, of which 7 had started functioning in February 1999 and 8 in December 2000.
Data collection procedures
The initial versions of the data collection instruments from the global PANCEA study were reviewed and refined to suit the context of Andhra Pradesh. The data collection team, consisting of six investigators with background in economics or finance, was involved with the refinement of the instruments and received extensive training to ensure a standardised approach to data collection. A pilot study was done to make final refinements in the data collection format and approach.
Detailed data were initially collected for the April 2002 – March 2003 fiscal year at the 15 sex worker programmes, which included a history of the evolution of the programme, and output and cost data by month. Formal consent of the senior-most person responsible for each programme, generally the director of non-governmental organisation, was obtained to collect data. The programme project director, project manager, accounts officer, outreach coordinator, outreach workers and counsellor were interviewed and the available written records reviewed to obtain the programme data. Each visit started with an interview containing structured open-ended questions on the history of the programme, and operational or community factors that may have affected the programme. Data collection at a programme by three investigators lasted one week. Data were recorded in the field on laptop computers in MS Excel and MS Word files, which after review were entered into an MS Access database.
The number of sex workers provided services annually by each programme since inception up to the 2004–2005 fiscal year was documented in another round of data collection that finished in April 2005.
Output data
Detailed data were obtained from the written records at the sex worker programmes regarding the services provided by month. The Andhra Pradesh State AIDS Control Society categorises the services delivered by sex worker intervention programmes into four major components: behaviour change communication, sexually transmitted infections care, condom promotion, and creating an enabling environment [11]. Behaviour change communication comprises several types of sessions by outreach workers and peer educators to teach and encourage the sex worker individually and in groups to follow safe sex practices. Sexually transmitted infection care includes the programme staff taking or referring sex workers and occasionally their partners for treatment of sexually transmitted infections (the cost of this is covered by the programme), HIV counselling and referrals to voluntary counselling and testing centres, help in utilising the services of HIV care and support centres, and organising training programmes and sensitisation meets for sexual health service providers such as registered medical practitioners. Condom promotion includes free condom distribution and sale of condoms under social marketing. Creating an enabling environment for sex workers includes meeting with a variety of external stakeholders by organising sensitisation meets with the general public, advocacy meets with the police, media and policy makers, establishing linkages with relevant governmental and non-government organisations, and also assisting sex workers to organise and form community based organisations and with some of their non-sexual health needs. The average time spent by the programme staff with a sex worker for each of the different types of contact was estimated based on information given by programme staff.
Cost data
The cost of the sex worker programme was divided into five categories: salaries, recurrent goods, recurrent services, office space rentals and capital goods. These cost data were collected for each month, as far as possible. Economic cost was computed, i.e. the true resource cost incurred rather than just the financial cost, as described below.
Salary cost was recorded for all personnel contributing to the work of sex worker programme, which included the project director, project manager, accounts officer, outreach coordinator, outreach workers, counsellor and attender. In addition, the compensation paid by the programme to sex worker peer educators in cash or kind was included in salary costs. Personnel compensation was noted from the official records of the programme. There were no non-salary personnel costs.
The major component of recurrent goods was male condoms. The majority of condoms distributed to the sex workers at these programmes are of a particular brand and are provided free by the Andhra Pradesh State AIDS Control Society. The market price of this brand of condoms is subsidised by 70% by the government. We considered the economic cost of these condoms as what it would have been without the subsidy. Some condoms of other brands are also sold to sex workers under the condom social marketing effort at the cost at which they are procured. Since these condoms are not subsidised, the market price of these condoms was used for economic cost calculations. The other recurrent goods utilised by the sex worker programme included medications for sexually transmitted infections, behaviour change communication materials, stationery and condom outlet boxes. The cost of the other recurrent goods was noted from the official records of the programmes, except for behaviour change communication materials about which information was obtained from the Andhra Pradesh State AIDS Control Society as it supplies these materials to the sex worker programmes.
Recurrent services included local travel, organising various workshops and meets, training for staff and peer educators, computer rentals and maintenance, consultation fees for sexually transmitted infections (investigations were usually not done, diagnosis and treatment were based on symptoms and signs), educational tour for staff, organising special events such as the World AIDS Day and candle light movement, cleaning and building maintenance, telephone, out of station travel, printing, photocopying, electricity, postage and courier, gas and some miscellaneous items. The cost of recurrent services was noted from official records of the programme. The cost for staff training was calculated by including travel fare, per diem, trainer fees, training materials, and training facility cost, information about which was obtained not only from the programme, which incurred some of these costs, but also from the Andhra Pradesh State AIDS Control Society, which incurred many of these costs.
The office of all these programmes run by non-government organisations were located in rented buildings. The details of the monthly rent paid were obtained from the programme records.
Capital goods used for the work of the sex worker programmes included computer and accessories, office furniture, electrical fixtures, telephone, white/black board, scooter, television, bicycle, public address system, video recorder, type writer, stove, refrigerator, audio system, inverter, projector, fax machine, lamination machine, sewing machine, spiral binding machine, water filter, camera, cash box and wall clock. If information about the cost of the capital goods was not available from the sex worker programme or its parent organisation, the market price was determined from retail sellers of these goods. Three quotations for these goods were obtained from the market for the 2002–2003 fiscal year and the average of these taken as the cost. The life of the capital goods was assumed to be five years, and therefore, one-fifth of the cost was allocated to the 2002–2003 fiscal year if the good was used for the full year. If a good was used only for part of the year, the cost was pro-rated for that use. If a capital good, for example a projector or a public address system, was also being used by the parent organisation for work other than that of the sex worker programme, a cost was allocated to the sex worker programme according to the proportion use reported by programme staff.
The average exchange rate of Indian Rupees (INR) 48.40 to a US$ for the 2002–2003 fiscal year was used for INR to US$ conversions [12].
Quality control
Quality control measures included a thorough pilot study before commencing formal data collection, comprehensive training of a qualified data collection team including their conceptual understanding of all data issues, full back-up and justification for any data recorded, supervision of data collection at each programme by the project coordinator, thorough review by the study team of the data obtained at each programme, and contacting programmes again to obtain information about data issues that needed clarification after the review.
Data analysis
Data were analysed using SPSS statistical software. The total number of sex workers provided services by each programme, different types of services, services provided per full time equivalent of programme staff, and services provided per sex worker, were analysed. The average economic cost per sex worker served in that fiscal year was taken as the main measure of cost-efficiency, and its relation assessed with the total number of sex workers served. The cost per contact with sex workers was also assessed. Incremental costs for each of the four major types of services provided by the programmes were assessed using a multiple regression model. Statistical significance was defined as p < 0.05.
The relation between the number of sex workers provided services and the intensity of services in the form of time spent with each sex worker was assessed. Comparison was done between the services provided by programmes that were started in the first phase in February 1999 (first batch) and those started later in December 2000 (second batch).
Results
A total of 33941 female sex workers were provided services by the 15 programmes during the 2002–2003 fiscal year. The managers for these programmes estimated that there were a total of 42900 female sex workers in the areas covered by these programmes in this fiscal year. The number of sex workers provided services annually by each programme ranged from 803 to 6379, with a median of 1970 and mean of 2263 (Table 1). Based on estimates of the types of female sex workers served by each programme, of the total sex workers served by all the programmes considered together 57% were street-based, 27% brothel-based and 16% home-based. There were a total of 245367 contacts with sex workers. The average number of contacts of programme staff with each sex worker in a year ranged 6.5-fold from 2.6 to 16.9 with a median of 8 for the different programmes (Table 1). There were a total of 49142 contacts with external stakeholders. The average number of contacts of programme staff other than peer educators with the external stakeholders in a year ranged 15-fold from 98 to 1463 with a median of 215 (Table 1).
Table 1 Number and intensity of services provided by the sex worker programmes in fiscal year 2002–2003.
Programme number* Number of sex workers provided services Total number of contacts with sex workers Number of contacts with each sex worker Total full time equivalents of staff Number of contacts with sex workers per staff full time equivalent Total number of contacts with all types of external stakeholders† Total full time equivalents of staff other than peer educators Number of contacts with external stakeholders per staff full time equivalent other than peer educators‡
8 6379 57077 8.9 26.00 2196 1002 8.50 118
10 4690 12477 2.7 18.75 665 1989 10.00 199
5 3847 9953 2.6 20.04 497 2880 9.79 294
9 2552 26976 10.6 15.66 1722 14869 10.16 1463
7 2336 18920 8.1 26.91 703 2015 11.66 173
11 2156 18358 8.5 14.35 1279 1111 6.85 162
1 2102 10833 5.2 14.78 733 1320 10.03 132
12 1970 14327 7.3 19.91 719 8244 10.41 792
15 1442 13064 9.1 20.33 643 1793 8.33 215
4 1350 22766 16.9 17.00 1339 2636 8.25 320
13 1274 9043 7.1 28.83 314 3016 10.33 292
2 1229 8867 7.2 11.58 766 816 8.33 98
6 915 10568 11.5 18.33 577 3433 8.33 412
14 896 7207 8.0 26.08 276 3248 8.33 390
3 803 4931 6.1 15.25 323 770 7.75 99
Total 33941 245367 7.2 293.80 835 49142 137.05 359
* Programmes arranged in decreasing order of the number of sex workers to whom they provided services in fiscal year 2002–2003.
† External stakeholders include general public, police, media, policy makers, and governmental and non-governmental agencies.
‡ Peer educators excluded here as contact with external stakeholders is made by other programme staff.
Of the total number of contacts with sex workers by the 15 programmes, 58.4% were by staff other than peer educators and 41.6% by peer educators. The full time equivalents of peer educators with the different programmes had a wider range of 3.3 to 18.5 (median 9.5) as compared with the range of 6.9 to 11.7 (median 8.5) for the programme staff other than peer educators (Table 2). In addition, the number of sex worker contacts in a year per full time equivalent of peer educators ranged 74-fold from 36 to 2626 (median 259) whereas those per full time equivalent of programme staff other than peer educators ranged 2.7-fold from 568 to 1520 (median 958) (Table 2). There was no significant correlation between the number of full time equivalents of peer educators in a programme and the number of sex worker contacts per peer educator (p = 0.66, R2 = 0.01; linear function), or between full time equivalents of programme staff other than peer educators in a programme and the number of sex worker contacts per staff (p = 0.26, R2 = 0.09; linear function), or between sex worker contacts per full time equivalents of programme staff other than peer educators in a programme and sex worker contacts per full time equivalents of peer educators (p = 0.20, R2 = 0.12; linear function).
Table 2 Intensity of services provided by peer educators and other programme staff in fiscal year 2002–2003.
Programme number* Number of sex workers provided services Full time equivalents of staff other than peer educators Total number of contacts with sex workers other than peer educators Contacts with sex workers per staff other than peer educators Full time equivalents of peer educators Total number of contacts with sex workers by peer educators Contacts with sex workers per peer educator
Project director Project manager Accounts officer Outreach coordinator Outreach worker Counsellor Office assistant Total
8 6379 0.33 1.08 1.00 1.00 4.00 1.00 0.08 8.50 11114 1308 17.50 45963 2626
9 2552 0.33 1.00 1.00 0.92 4.00 1.00 1.92 10.16 15332 1509 5.50 11644 2117
4 1350 0.33 1.00 1.00 0.00 4.00 0.92 1.00 8.25 8426 1021 8.75 14340 1639
11 2156 0.27 0.75 0.58 0.00 3.34 1.08 0.83 6.85 7503 1095 7.50 10855 1447
2 1229 0.33 1.00 1.00 0.00 4.00 1.00 1.00 8.33 5507 661 3.25 3360 1034
12 1970 0.33 1.00 1.00 1.00 5.08 1.00 1.00 10.41 9581 920 9.50 4746 500
6 915 0.33 1.00 1.00 0.00 4.00 1.00 1.00 8.33 7918 951 10.00 2650 265
1 2102 1.00 1.00 1.03 1.00 4.00 1.00 1.00 10.03 9605 958 4.75 1228 259
13 1274 0.33 1.00 1.00 0.00 6.00 1.00 1.00 10.33 6830 661 18.50 2213 120
5 3847 0.33 1.04 1.00 1.00 4.25 1.17 1.00 9.79 8901 909 10.25 1052 103
15 1442 0.33 1.00 1.00 0.00 4.00 1.00 1.00 8.33 11960 1436 12.00 1104 92
7 2336 0.33 1.00 1.00 1.00 6.33 1.00 1.00 11.66 17728 1520 15.25 1192 78
3 803 0.33 1.00 1.00 0.00 3.75 0.67 1.00 7.75 4405 568 7.50 526 70
10 4690 1.00 1.00 1.00 1.00 4.00 1.00 1.00 10.00 11996 1200 8.75 481 55
14 896 0.33 1.00 1.00 0.00 4.00 1.00 1.00 8.33 6574 789 17.75 633 36
Total 33941 6.23 14.87 14.61 6.91 64.75 14.84 14.83 137.05 143380 1046 156.75 101987 651
* Programmes arranged in decreasing order of the number of contacts with sex workers per peer educator in fiscal year 2002–2003.
The major portion of the contacts with sex workers by programme staff was related to behaviour change communication (93.7%) (Table 3). The number of such contacts per sex worker in a year ranged from 2 to 16.6 (median 7.4) for the different programmes. The number of sexually transmitted infection care related contacts in a year ranged from 0.15 to 0.84 (median 0.39) per sex worker provided services by the different programmes (Table 3). Of these contacts related to sexually transmitted infections, 77.9% were for referral of sex workers for treatment of sexually transmitted infections, 14.9% for HIV counselling, referral to voluntary counselling and testing centres, and referral to care and support centres, 5.5% for training of sexual health service providers, and 1.7% for referral of partners of sex workers for treatment of sexually transmitted infections. The number of condoms distributed by the different programmes in a year per sex worker provided services ranged from 46 to 432 with a median of 168 (Table 3). Of all the condoms distributed, 95% were given free and 5% sold as part of the condom social marketing effort.
Table 3 Components of services provided by sex worker programmes in fiscal year 2002–2003.
Programme number* Number of sex workers provided services Behaviour change communication contacts Behaviour change communication contacts per sex worker Sexually transmitted infection care related contacts Sexually transmitted infection care related contacts per sex worker Condoms distributed Condoms distributed per sex worker Contacts for creating enabling environment Contacts for creating enabling environment per sex worker
8 6379 56141 8.8 987 0.15 590922 93 951 0.15
10 4690 10688 2.3 1326 0.28 215861 46 2452 0.52
5 3847 7556 2.0 746 0.19 419583 109 4531 1.18
9 2552 25400 10.0 2045 0.80 356658 140 14400 5.64
7 2336 18221 7.8 1449 0.62 393102 168 1265 0.54
11 2156 18173 8.4 347 0.16 140285 65 949 0.44
1 2102 6671 3.2 1222 0.58 554146 264 4260 2.03
12 1970 14155 7.2 509 0.26 448004 227 7907 4.01
15 1442 12074 8.4 1214 0.84 448704 311 1569 1.09
4 1350 22409 16.6 369 0.27 173858 129 2624 1.94
13 1274 8647 6.8 654 0.51 300750 236 2758 2.16
2 1229 8458 6.9 485 0.39 165670 135 740 0.60
6 915 10316 11.3 292 0.32 179390 196 3393 3.71
14 896 6600 7.4 655 0.73 387237 432 3200 3.57
3 803 4498 5.6 443 0.55 138490 172 760 0.95
Total 33941 230007 6.8 12743 0.38 4912660 145 51759 1.52
* Programmes arranged in decreasing order of the number of sex workers to whom they provided services in fiscal year 2002–2003.
The number of contacts by the programmes related to creating an enabling environment for sex workers ranged from 0.15 to 5.64 (median 1.18) per sex worker provided services (Table 3). Of these contacts, 77% were with the community at large in the form of mass or special events, 12.1% were with other external stakeholders in the form of advocacy meets and with agencies to enhance linkages, and 10.9% were with sex workers regarding formation of community based organisations and facilitation of non-sexual health needs. There was no significant correlation between behaviour change communication contacts per sex worker by a programme on the one hand and sexually transmitted infections related contacts per sex worker provided services (p = 0.98, R2 = 0.00; linear function) or number of condoms distributed per sex worker provided services (p = 0.95, R2 = 0.00; linear function) or number of contacts related to creating an enabling environment for sex workers (p = 0.30, R2 = 0.08; linear function) on the other hand.
The total economic cost of services provided by the 15 programmes during the 2002–2003 fiscal year was INR 17,575,858 (US$ 363,138), of which personnel cost made up 34.7%, recurrent goods 38.4% (82.1% of this was for condoms and 12.6% for medicines for sexually transmitted infections), recurrent services 21.1% (the largest component was local travel, which made up 23.2% of this), rentals 4.1%, and capital goods 1.7%. There were modest variations in these proportional costs among some of the programmes (Table 4). The total financial cost was 20.8% less than the economic cost due to the subsidy on condoms.
Table 4 Cost and efficiency of sex worker programmes in fiscal year 2002–2003.
Programme number* Number of sex workers provided services Total number of contacts with sex workers Total economic cost Percent of economic cost Cost per sex worker provided services Cost per sex worker contact
INR US$ Personnel Recurrent goods Recurrent services Rentals Capital goods INR US$ INR US$
10 4690 12477 1040254 21493 46.9 19.6 24.3 5.8 3.5 221.8 4.58 83.4 1.72
8 6379 57077 1969785 40698 34.1 41.4 20.5 2.8 1.2 308.8 6.38 34.5 0.71
11 2156 18358 725710 14994 36.6 27.4 28.1 5.8 2.1 336.6 6.95 39.5 0.82
5 3847 9953 1327582 27429 33.1 37.6 22.0 4.5 2.7 345.1 7.13 133.4 2.76
9 2552 26976 1482412 30628 32.2 39.0 23.6 3.2 2.0 580.9 12.00 55.0 1.14
12 1970 14327 1278085 26407 35.9 45.0 14.3 3.6 1.1 648.8 13.40 89.2 1.84
7 2336 18920 1526201 31533 32.4 37.4 24.0 3.9 2.3 653.3 13.50 80.7 1.67
2 1229 8867 812295 16783 45.4 30.3 18.9 4.9 0.6 660.9 13.66 91.6 1.89
4 1350 22766 930607 19228 36.2 32.3 26.9 3.9 0.8 689.3 14.24 40.9 0.84
15 1442 13064 1065021 22005 27.3 52.3 14.8 4.5 1.1 738.6 15.26 81.5 1.68
1 2102 10833 1605711 33176 29.2 52.0 13.3 4.1 1.4 763.9 15.78 148.2 3.06
3 803 4931 673355 13912 45.4 24.3 24.2 4.9 1.1 838.5 17.33 136.6 2.82
13 1274 9043 1089778 22516 34.5 33.9 26.2 3.9 1.5 855.4 17.67 120.5 2.49
6 915 10568 822442 16993 38.7 36.8 18.2 4.4 1.9 898.8 18.57 77.8 1.61
14 896 7207 1226620 25343 27.6 44.5 22.6 3.8 1.4 1369.0 28.29 170.2 3.52
Total 33941 245367 17575858 363138 34.7 38.4 21.1 4.1 1.7 517.8 10.70 71.6 1.48
* Programmes arranged in increasing order of cost per sex worker provided services in fiscal year 2002–2003.
The economic cost for each sex worker provided services varied 6-fold between the 15 programmes from INR 221.8 (US$ 4.58) to INR 1369 (US$ 28.29), with a median of INR 660.9 (US$ 13.66) (Table 4). The average cost of providing services to each sex worker at all the 15 programmes combined was INR 517.8 (US$ 10.70). The economic cost for each sex worker contact varied 5-fold between the 15 programmes from INR 34.5 (US$ 0.71) to INR 170.2 (US$ 3.52), with a median of INR 83.4 (US$ 1.72) (Table 4). The average cost of each sex worker contact at all the 15 programmes combined was INR 71.6 (US$ 1.48). Both, the cost per sex worker provided services and the cost per sex worker contact for the different programmes, were significantly inversely associated with scale. The best fit for this relation was obtained with the power function (Figures 1 and 2), with which scale explained 75% of the variability in cost per sex worker provided services and 73% of the variability in cost per sex worker contact by programme staff. There was also a statistically significant direct linear relation between the cost of providing services to each sex worker and the cost of each sex worker contact at a programme, and this explained 35% of the variability (p = 0.02, R2 = 0.35).
Figure 1 Relation between the number of sex workers provided services in fiscal year 2002–2003 by the 15 programmes and the cost per sex worker (p < 0.001). INR is Indian Rupee (1 US$ = INR 48.40 in 2002–2003).
Figure 2 Relation between the number of sex worker contacts in fiscal year 2002–2003 by the 15 programmes and the cost per contact (p < 0.001). INR is Indian Rupee (1 US$ = INR 48.40 in 2002–2003).
There was no significant correlation between the full time equivalents of programme staff and the number of sex workers provided services by the programmes (p = 0.42, R2 = 0.05; linear function). Not only was there no inverse relation between the number of sex workers provided services and the contacts per sex worker by the programmes, there was a modest positive linear relation (p = 0.02, R2 = 0.34). However, there was also a borderline inverse correlation between the number of sex workers served and the average time spent with each sex worker by the programme staff during the year (p = 0.08, R2 = 0.22; exponential function) (Figure 3), which became statistically significant after adjusting for the number of full time equivalents of staff in each programme (p = 0.01, R2 = 0.40) (Figure 4).
Figure 3 Relation between the number of sex workers provided services in fiscal year 2002–2003 by the 15 programmes and the average time spent with each sex worker in the year (p = 0.079).
Figure 4 Relation between the number of sex workers provided services in fiscal year 2002–2003 by the 15 programmes and the average time spent with each sex worker in the year adjusted for the full time equivalents of programme staff (p = 0.011).
The 15 programmes had started in two phases, 7 in February 1999 (first batch) and 8 in December 2000 (second batch), resulting in a difference of a 1.75 years in their duration. The first batch programmes were started in places estimated to have the highest numbers of sex workers in the state, followed by the second batch programmes at places also estimated to have sizeable number of sex workers. All 7 first batch programmes (numbers 8, 10, 5, 9, 7, 1, 12 in Table 1) fell among the 8 programmes that served the highest number of sex workers in the 2002–2003 fiscal year. Of the 7 programmes with the least cost per sex worker served in this fiscal year, 6 were first batch programme (numbers 10, 8, 5, 9, 12, 7 in Table 4).
Since scale was a major determinant of efficiency, we studied the trends of the average number of sex workers provided services annually by the first and second batch programmes since inception (Figure 5). The fiscal year 1999–2000 was mostly spent in planning and establishing the first batch of sex worker programmes in Andhra Pradesh. The average number of sex workers served by each of the first batch programmes increased over the first four fiscal years and then settled around 3500 annually. The second batch of programmes, established at places estimated to have the next highest number of sex workers after the places where the first batch was started, has now finished the first four fiscal years over which it has shown an increasing trend so far that has reached an annual average of about 2000 sex workers per programme served in fiscal year 2004–2005.
Figure 5 Changes in the average number of sex workers provided services annually by each of the first and second batch programmes since inception.
Considering the 2002–2003 fiscal year data from each programme as a data point, multiple regression was used to assess the relationship between total economic cost and the four major functions of these programmes, i.e. behaviour change communication contacts with sex workers, condoms distributed, sexually transmitted infections related contacts, and contacts to enhance enabling environment, which revealed the following relation:
= 372887 + 8.10 P + 1.67 Q + 136.78 R + 3.64 S
where Ĉ is the total economic cost in INR, P is the number of behaviour change communication contacts with sex workers, Q is the number of condoms distributed, R is the number of sexually transmitted infections related contacts, and S is the number of contacts to enhance enabling environment. This model explained 88% of the variability in the economic cost (R2 = 0.88, standard error = 125362) and the fit was significant at p < 0.001. In this model, the constant was significant at p = 0.001 (F = 27.38; degrees of freedom: 4 for regression, 10 for residual, 14 total). Two of the predictor variables were statistically significant (number of behaviour change communication contacts with sex workers and number of condoms distributed) whereas the other two predictor variables were not (number of sexually transmitted infections related contacts and number of contacts to enhance enabling environment at) (Table 5). This model suggests that apart from the constant cost of INR 372887 (US$ 7704) for a programme, the additional cost for each behavioural change communication contact was INR 8.10 (US$ 0.17), for each condom distributed INR 1.67 (US$ 0.03), for each sexually transmitted infections related contact INR 137 (US$ 2.83), and for each contact to enhance enabling environment INR 3.64 (US$ 0.08).
Table 5 Coefficients in the multiple regression model and their significance.
Variable Coefficient (βi) Standard error t Significance
Constant 372887.07 83932.49 4.443 0.001
Number of behaviour change communication contacts with sex workers 8.10 2.84 2.852 0.017
Number of condoms distributed 1.67 0.27 6.298 0.000
Number of sexually transmitted infections related contacts 136.78 85.31 1.603 0.140
Number of contacts to enhance enabling environment 3.64 10.91 0.334 0.745
Dependent variable: Total economic cost
Applying these incremental costs to the total services provided by the 15 programmes (Table 3), of the total economic cost in 2002–2003, 68.3% was variable. Of the variable cost, 15.5% was for behavioural change communication, 14.5% for sexually transmitted infections, 68.4% for condoms, and 1.6% for enhancing enabling environment.
Discussion
In this study of 15 HIV prevention programmes for female sex workers in the Indian state of Andhra Pradesh, the most important determinant of cost per sex worker provided services was scale. As the number of sex workers provided services increased, the cost per sex worker decreased. This was observed for the entire range of scale of the 15 programmes – 803 to 6373 sex workers provided services in the 2002–2003 fiscal year. However, this should be interpreted with certain caveats. There was a trend towards a modest decrease in the average time spent with each sex worker as the total number of sex workers served increased. The distribution of this relationship (Figure 4) implies that at least for some low-to-medium scale programmes it should be possible to increase the efficiency without compromising time spent with each sex worker. Interestingly, there was also a modest increase in the number of contacts with each sex worker as the total number of sex workers served increased. However, no standardised estimates are available about how many contacts and how much time with each sex worker would be optimal for providing effective HIV prevention services in India. Our data imply that although the efficiency increases with the scale of the programme, further development of sex worker programmes should also include assessment of the optimal number of contacts and time spent with sex workers for the various types of services provided in order to make these programmes not only efficient but also effective. This would have to include variables such as geographical coverage of the programme and transport availability, relative mix of fresh and old sex workers covered by the programme, and relative mix of street-based, home-based and brothel-based sex workers covered.
The first batch of 7 programmes started in early 1999 had reached an average scale of about 3500 sex workers per year per programme by the fourth fiscal year since inception and this was maintained for three fiscal years so far. The second batch of 8 programmes started in late 2000 showed an increasing trend in scale over the four fiscal years so far since inception, with an average of about 2000 sex workers served per year per programme. Since the second batch of programmes were started in places that had on average somewhat fewer sex workers than the first batch of programmes, it is possible that the second batch may not reach the average scale reached by the first batch. There was an increase of 57% for the average scale from 1258 to 1976 from fiscal year 2002–2003 to 2004–2005 for the second batch programmes. The average cost per sex worker provided services by the second batch programmes in 2002–2003 was INR 730 (US$ 15.08). If the equation in Figure 1 for the relation of scale with efficiency were applied, the cost per sex worker served by the second batch programmes in 2004–2005 would have decreased by 21% to INR 576 (US$ 11.90) at the 2002–2003 constant INR/US$. On the other hand, the average cost per sex worker provided services by the first batch programmes in 2002–2003 was INR 428 (US$ 8.85), which would have likely remained stable up to 2004–2005 as these programmes had a stable average scale of about 3500 per year over this period.
Another study of 17 sex worker programmes, 9 in Andhra Pradesh and 8 in Tamil Nadu – another state in southern India, found a U-shaped relationship between scale and efficiency for the 2001–2002 fiscal year with the cost per sex worker being lowest in the range of 1000–1700 sex workers served per year by a programme and then rising [13]. This study reported a median cost of US$ 19.20 (range 9.86–50.70) per sex worker served for a range of 200 to 2008 sex workers served per year by the 17 programmes. Direct comparison of this study with ours may not be possible because of methodological differences, such as the different scale of programmes in the two studies. Our study included much larger programmes; the range of sex workers served per year in the 2002–2003 fiscal year was 803 to 6373, with 7 of the 15 programmes having served over 2000 sex workers and 3 programmes substantially above this number (3847, 4690 and 6379). We did not find a U-shaped relation between scale and efficiency in our data, as the efficiency continued to improve with scale, though the rate of improvement decreased with increasing scale. The values for the cost per sex worker served in our data (median US$ 13.66, range US$ 4.58–28.29) were also lower than in the above study, again likely due to the higher scale in our study. The relationship between scale and efficiency of sex worker programmes is more likely to be revealed if the range of scales of programmes studied is wide. For our study, we included all the 15 HIV prevention programmes for female sex workers that were being run in the fiscal year 2002–2003 by non-governmental organisations in Andhra Pradesh with the support of the Andhra Pradesh State AIDS Control Society.
Peer educators, sex workers who are trained to deliver HIV prevention services to other sex workers, are an important component of HIV prevention programmes for female sex workers in this Indian state with 41.6% of all contacts with sex workers in the fiscal year 2002–2003 done by peer educators for the 15 programmes studied. As the peer educators are themselves sex workers, they are likely to have good access to and rapport with other sex workers. There was a very high 74-fold variation between the programmes for the number of sex worker contacts in a year per peer educator, whereas the variation for the number of sex worker contacts per programme staff other than peer educators was much smaller (2.7-fold). If attempts were made to standardise the output of peer educators, the overall output and efficiency of sex worker programmes in this Indian state would improve. It should be noted though that our study did not assess effectiveness, which would also have to receive attention in attempts to increase efficiency.
The contact by programme staff with sex workers for behavioural change communication forms a major portion of the work of the programme (Table 3). This activity also overlaps with condom distribution. Though the number of contacts for behavioural change communication for all programmes considered together was 18 times higher than the contacts related to sexually transmitted infection care in 2002–2003, the total variable costs for these two activities were similar as the cost of each behaviour change contact (INR 8.10, US$ 0.17) was 17 times lower than the cost of each sexually transmitted infection care related contact (INR 137, US$ 2.83). Based on the multiple regression model for economic cost, apart from the fixed cost needed to run the programmes (31.7% of the total economic cost), of the variable cost two-third was accounted for by the 4.91 million condoms distributed by the 15 programmes in 2002–2003. This suggests that availability of low-cost condoms of reasonable quality is very important for increasing efficiency of sex worker programmes.
The estimates of outputs, cost and efficiency of HIV prevention services in India are generally only scantily available [7,8]. The outputs, cost and efficiency estimates, relationship of efficiency with scale, and the unit incremental costs for each of the major activities of the sex worker programmes, presented in this paper could be useful for planning sex worker programmes and estimating the resources needed by them in Andhra Pradesh and other states in India. This is particularly relevant as there has been a major recent increase in the number of sex worker programmes in Andhra Pradesh and a few other Indian states with substantial funding from the Gates Foundation [6]. The data presented in this paper also highlight that the role of peer educators needs to be standardised and that the relationship between efficiency and effectiveness needs to be understood further.
This study is part of the multi-country PANCEA study [9,10]. The outputs and cost-efficiency estimates of HIV prevention programmes for sex workers from India can be compared with similar estimates from other countries in the PANCEA study using similar methodology. Policy-relevant comparisons of these estimates would also be possible with other HIV prevention strategies in Andhra Pradesh that we have undertaken with similar methodology. For example, we have recently reported cost-efficiency estimates for HIV voluntary counselling and testing in Andhra Pradesh [14]. Standardised comparisons of cost-efficiency estimates for HIV prevention services are needed across countries and across the different types of prevention in the background of the considerable attention recently to estimating the resources needed for HIV control, which would enable more firm local and global estimates for planning.
Conclusion
The annual economic cost of providing services to a sex worker varied 6-fold between the 15 HIV prevention programmes for female sex workers that were being run in the fiscal year 2002–2003 by non-governmental organisations in the southern Indian state of Andhra Pradesh. The cost per sex worker served decreased with increasing number of sex workers served annually by a programme, and this was associated with a modest trend of decrease in time spent with each sex worker in some programmes. However, it should be possible for at least some low-to-medium scale programmes to increase scale and efficiency without compromising time spent with each sex worker. The services provided by peer educators, who form an important part of the sex worker programmes, were highly variable, standardisation of which would be useful. The data of this study imply that although the efficiency increases with the scale of the programme, further development of sex worker programmes should also include assessment of the optimal number of contacts and time spent with sex workers for the various types of services provided in order to make these programmes not only efficient but also effective.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LD led the PANCEA study in India, guided the design, data collection and analysis, and wrote the initial draft of this paper. PS contributed to the design, data collection and analysis. SGPK, YKR, AAK, MCR and SM contributed to data collection and analysis. NM participated in the design of data collection instruments and the review of collected data. EM and JGK oversaw the PANCEA design and contributed to the analytical design and presentation. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We thank the Andhra Pradesh State AIDS Control Society and its Technical Resource Unit for facilitating this study, and the sex worker programmes for participating in this study. This study was supported by the U.S. National Institutes of Health through Task Order #7 contract 282-98-0026 and grant R01 DA15612. The views expressed in this paper are those of the authors and do not necessarily reflect the views of the funding agency, organisations that facilitated this study, or the institutions employing the authors.
==== Refs
Joint United Nations Programme on HIV/AIDS (UNAIDS) 2004 Report on the Global AIDS Epidemic Geneva 2004
National AIDS Control Organisation (NACO) HIV Estimates (2003) New Delhi: NACO, Ministry of Health & Family Welfare, Government of India.
National AIDS Control Organisation (NACO) Observed HIV prevalence levels state wise: 1998–2004 New Delhi: NACO, Ministry of Health & Family Welfare, Government of India.
Nagelkerke NJ Jha P de Vlas SJ Korenromp EL Moses S Blanchard JF Plummer FA Modelling HIV/AIDS epidemics in Botswana and India: impact of interventions to prevent transmission Bull World Health Organ 2002 80 89 96 11953786
Andhra Pradesh State AIDS Control Society (APSACS) VIIIth round of national annual sentinel surveillance for HIV, Andhra Pradesh, 2004 Hyderabad: APSACS
Bill & Melinda Gates Foundation Avahan: India AIDS Initiative
Walker D Cost and cost-effectiveness of HIV/AIDS prevention strategies in developing countries: is there an evidence base? Health Policy Plan 2003 18 4 17 12582104 10.1093/heapol/18.1.4
Dandona L Enhancing the evidence base for HIV/AIDS control in India Natl Med J India 2004 17 160 166 15253404
Prevent AIDS Network for Cost-Effectiveness Analysis Home page
Marseille E Dandona L Saba J McConnel C Rollins B Gaist P Lundberg M Over M Bertozzi S Kahn JG Assessing the efficiency of HIV prevention around the world: methods of the PANCEA project Health Services Research 2004 39 1993 2012 15544641 10.1111/j.1475-6773.2004.00329.x
Andhra Pradesh State AIDS Control Society Strategies
Reserve Bank of India Exchange rate of the Indian Rupee: Table 145
Guinness L Kumaranayake L Bhuvaneswari R Sankaranarayanan G Vannela G Raghupathi P George A Does scale matter? The costs of HIV prevention interventions for commercial sex workers in India Bull World Health Organ
Dandona L Sisodia P Ramesh YK Kumar SGP Kumar AA Rao MC Someshwar M Hansl B Marshall N Marseille E Kahn JG Cost and efficiency of HIV voluntary counselling and testing centres in Andhra Pradesh, India Natl Med J India 2005 18 26 31 15835489
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BMC PhysiolBMC Physiology1472-6793BioMed Central London 1472-6793-5-151618149010.1186/1472-6793-5-15Research ArticleThe relationship between ciliary neurotrophic factor (CNTF) genotype and motor unit physiology: preliminary studies Conwit Robin A [email protected] Shari [email protected] Stephen [email protected] Daniel [email protected] Ben [email protected] Robert [email protected] E Jeffrey [email protected] National Institute on Neurological Disorders and Stroke, Rockville, MD, USA 208922 Clinical Research Branch, National Institute on Aging, National Institute on Aging Intramural Research Program, Harbor Hospital, 5th Floor, Baltimore, MD, USA 212253 Department of Kinesiology, University of Maryland, College Park, MD, USA4 University of Waterloo, Department of Systems Design Engineering, Waterloo, Ontario, Canada5 Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA2005 23 9 2005 5 15 15 31 1 2005 23 9 2005 Copyright © 2005 Conwit 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
Ciliary neurotrophic factor (CNTF) is important for neuronal and muscle development, and genetic variation in the CNTF gene has been associated with muscle strength. The effect of CNTF on nerve development suggests that CNTF genotype may be associated with force production via its influence on motor unit size and firing patterns. The purpose of this study is to examine whether CNTF genotype differentially affects motor unit activation in the vastus medialis with increasing isometric force during knee extension.
Results
Sixty-nine healthy subjects were genotyped for the presence of the G and A (null) alleles in the CNTF gene (n = 57 G/G, 12 G/A). They were tested using a dynamometer during submaximal isometric knee extension contractions that were from 10–50% of their maximal strength. During the contractions, the vastus medialis was studied using surface and intramuscular electromyography with spiked triggered averaging to assess surface-detected motor unit potential (SMUP) area and mean firing rates (mFR) from identified motor units. CNTF genotyping was performed using standard PCR techniques from DNA obtained from leucocytes of whole blood samples. The CNTF G/A genotype was associated with smaller SMUP area motor units and lower mFR at higher force levels, and fewer but larger units at lower force levels than G/G homozygotes. The two groups used motor units with different size and activation characteristics with increasing force generation. While G/G subjects tended to utilize larger motor units with increasing force, G/A subjects showed relatively less increase in size by using relatively larger units at lower force levels. At higher force levels, G/A subjects were able to generate more force per motor unit size suggesting more efficient motor unit function with increasing muscle force.
Conclusion
Differential motor unit responses were observed between CNTF genotypes at force levels utilized in daily activities.
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Background
Sarcopenia, the progressive loss of strength and muscle mass with increasing age, is an important contributor to frailty, poor mobility function, and mortality [1,2]. Neuromuscular alterations, and specifically changes in the peripheral nerve, motor unit, and muscle composition, are likely contributors to sarcopenia [3]. Genetic contributions are being explored for both frailty and sarcopenia [4-6].
We recently determined that heterozygotes for the ciliary neurotrophic factor (CNTF) "null" A allele exhibited significantly higher knee extensor concentric (shortening) isokinetic (3.14 rad/s) peak torque, as well as significantly higher knee extensor concentric (shortening) muscle quality (strength per unit of muscle), than G/G homozygotes [4]. The null A allele results in a truncated and presumably inactive mutant CNTF protein [5]. CNTF is a neurotrophic factor important for neuronal and muscle development and growth [7]. The observation of strength differences by CNTF genotype and the known role of CNTF on nerve development suggest that genetic differences are likely to impact on neuromuscular organization by altering muscle, nerve or both and may impact on the development of sarcopenia [8]. These effects may occur by altering the size and firing patterns of motor units, the fundamental organizational unit for muscle activation. We are unaware of any studies that have examined whether CNTF genotype differences impact on motor unit activation in humans.
Muscle force generation is dependent on the number of active motor units and the rate at which they discharge, with recruitment similar to muscle fiber recruitment based on Henneman's size principle [9], i.e. recruited in an orderly sequence according to size, with smaller units activated before larger units. As more motor units are recruited or as motor unit firing rates increase the force of contraction increases. The force produced by a motor unit is dependent on its cross-sectional area, its innervation ratio and the specific tension of its fibers [10]. Different muscles use different strategies to increase force production. Smaller muscles tend to recruit a motor unit, increase its firing rate and subsequently recruit additional units. Larger muscles tend to progressively recruit motor units and only at high force levels increase firing rates.
Clinical techniques have been developed to study motor unit activity during muscle activation [11,12]. The focus of these methods has been on estimating the number of motor units within a muscle. In previous work, we have modified a method developed by Stashuk [13] that decomposed an intramuscular needle EMG signal to identify individual motor units and used spike-triggered averaging of the surface electromyogram (SEMG) to estimate the surface projection of the unit as the surface-detected motor unit potential (SMUP) to examine the distribution of motor unit size and firing rates in the vastus medialis during submaximal isometric contractions of the knee extensors. In previous work examining the vastus medialis [14,15], we found that sampling 15 motor units at force levels corresponding to 10% and 20% of isometric knee extensor maximal voluntary contraction gave a test-retest coefficient of variation of approximately 10%, with test-retest correlations between trials above 0.65 for most comparisons. As muscle force increased during isometric knee extension, the average size of measured motor units increased consistent with an orderly recruitment. Mean firing rate showed only a modest increase up to 30% of maximal voluntary contraction (mean firing rate increasing from 10.1 to 10.8 Hz). A nonlinear relationship was found between muscle force generation and motor unit size with an explained variance of 0.67. We found that the force generated during knee extension was directly related primarily to the size of active motor units and secondarily to firing rate [15].
In the present study, we analyzed data from two studies that included participants in whom CNTF genotype was determined, and motor unit function was assessed. Given the apparent influence of CNTF genotype on muscle strength [4], we hypothesized that subjects with G/A genotype would demonstrate a different motor unit activation pattern with increasing muscle force generation in the vastus medialis than homozygotes with G/G genotype.
The primary focus of this study was to determine the relationship between motor unit size and firing rate in the vastus medialis during knee extensor muscle isometric force generation by sampling the active motor unit pool using the spike triggered averaging and decomposition of needle EMG and SEMG. The primary measures resulting from this approach are an estimate of individual motor unit size from the SMUP and individual motor unit mean firing rates (mFR). Using these direct measures several calculated measures were created to explore the relationship between motor unit size, mean firing rate and muscle isometric force generation (Table 1). In previous work, we have observed that the direct motor unit size measurements, SMUP amplitude and SMUP area can be used to estimate motor unit size, and are highly correlated (r= 0.97) [14], SMUP area provides some advantages when considering the ratios described below. The firing rate is represented by the mFR for the motor unit during the measured part of the contraction. In addition, the product of SMUP area and mFR was calculated and termed the motor unit mean voltage (MUmV). The MUmV represents the average contribution of a motor unit to the acquired surface EMG signal as well as to the resulting generated force, when ignoring contributions by twitch potentiation, nonlinear summation, and other related factors [14]. Jabre and Salzsieder [16,17] defined a similar property that they designated as electrotwitch that was calculated from the instantaneous motor unit firing rate times the macro motor unit potential area and was related directly to the instantaneous force production by a muscle. The MUmV reflects the property that a motor unit increases force production as the firing rate increases to a point where further increases in firing rate have no effect. The average force produced by a muscle is directly related to the product of the mean SMUP area and mean mFR of a representative sample of motor units or in other words by the mean MUmV[15].
To examine the extent of muscle activation, the surface SEMG was rectified, and an average value was obtained over the 30-second contraction. It represents the average electrical activity of the muscle measurable by the electrode during the specific contraction. Using the SEMG, motor unit properties can be expressed relative to the average measured activity of the muscle which allows for the estimation of several properties of the contracting muscle (See Table 1 for a list of terms). During a fatigue study [18], we previously developed and published an index of the number of motor units active during the contraction that can be calculated by dividing the SEMG by the mean MUmV hereafter referred to as the motor unit relative index (MURI). MURI is an estimate of the relative number of motor units being measured by the active recording electrode. SEMG reflects the activity of the whole muscle, while the mean MUmV represents the average motor unit activity during the contraction. MURI is an index and not a direct estimate of the active units, since the ratio does not account for synchronization, nonlinear summation and other factors that could affect the relationship between size and activity. The ratio has some validity, as Suzuki et al [19] found that an increase in mean MUmV was directly related to the relative increase in the SEMG with force generation at increasing percentage of maximal voluntary contraction (MVC).
A second measure is the ratio of motor unit size (mean SMUP area) to force generation, which estimates the average motor unit size per Newton force (N) produced by the knee extensors. The force produced by a single motor unit is dependent on the cross-sectional area of its muscle fibers and the specific tension of its muscle fibers [10]. Presumably, as more motor units are recruited with higher levels of force generation, the type of motor unit will gradually change from the less fatigable type I units to the larger type IIa and IIb units that are capable of generating more force. As this occurs, the force generated per unit of motor unit size should increase to the extent that type II motor units are capable of generating greater force than type I motor units.
A third measure is a ratio of MURI to force which estimates the average number of motor units per N within the field of the surface electrode. This provides an estimate of the force capabilities of the motor units per unit size.
In this preliminary study, we demonstrate that subjects who are heterozygous for the null A allele (i.e. G/A genotype) as compared to subjects homozygous for the common G allele (i.e. G/G genotype) activate motor units with different size and activation characteristics with increasing force generation in the range of 10% to 50% of their maximal knee extensor isometric strength. While G/G subjects tended to utilize larger motor units with increasing force, G/A subjects showed relatively less increase in size by using relatively larger units at lower force levels. At higher force levels, G/A subjects were able to generate more force per motor unit size suggesting more efficient motor unit function with increasing muscle force.
Results
The subjects included 57 (83%) with the CNTF G/G and 12 (17%) with the G/A genotypes, with no A/A homozygotes represented. These genotype frequencies are similar to those reported previously [4] and the genotype frequencies were in expected Hardy Weinberg equilibrium. Subjects' characteristics are shown in Table 2. No difference or interaction with gender was found between G/A and G/G subjects for MVC, age, height, or weight.
The relationships between motor unit properties and CNTF genotype were examined using mixed effects models adjusted for percent of MVC, force generated, age and gender. The models included an interaction between CNTF genotype and force to test whether the motor unit size utilized by subjects with the G/G and G/A genotypes differed based on force level. The statistical significance levels for direct and calculated motor unit measures are presented in Table 3.
Significant differences by CNTF genotype and the interaction between CNTF genotype and muscle force generation were present in essentially all models while controlling for age and gender. Only firing rate was not associated with CNTF genotype. The accompanying figures (Figures 1, 2, 3, 4, 5, 6) show the relationships between the motor unit parameter and force generation without adjustments. The figures do not directly reflect the statistical models as the regression lines are based on loess nonparametric smoothing regression which help determine the shape of the regression relationship[20]. The figures show the clear interaction between the EMG measures and muscle force generation between GA and GG CNTF genotypes with an intersection at approximately 200 N.
To address whether the motor units differed in size at lower force levels (< 200 N) and at higher force levels (>= 200 N), separate analyses were examined for both force levels. CNTF genotype and an interaction between CNTF genotype and force were significant when examining only force levels lower than 200 N and greater than 200 N (Table 3). mFR did not differ by CNTF genotype for either force level.
The CNTF G/A and G/G group difference persisted with adjustments for multiple comparisons using false discovery rate control when considering all force levels, levels < 200 N, and levels >= 200 N. P values for false discovery rate control are given in the parentheses in Table 3. For all force levels, the first number represents an adjustment for 9 tests from our initial analyses which only included all force levels. The second p level is for all 21 tests considered for Table 3. For < 200 N and >= 200 N, only an adjustment for 21 tests is given, as this represented a post hoc analysis. Several interactions for all force levels tests lost their significant difference or showed a variable significant difference depending on the number of comparison tests.
To better demonstrate the differences in motor unit sizes between G/A and G/G at forces less than and greater than 200 N, density distributions were plotted for SMUP and MUmV (Figure 7). The density distributions for G/A and G/G differed for SMUP (p = 0) and MUmV (p = 0) for units less than 200 N, with a clear difference apparent with G/A being more dispersed with its mode at a higher SMUP and MUmV than G/G. For forces greater than 200 N, G/G is more dispersed than G/A for both SMUP (p = 0) and MUmV (p = 0).
SMUP area
The G/A genotype was associated with activation of larger SMUP area at lower force levels and much smaller SMUP at levels above 200 N with a slower increase in SMUP area with increasing force output than the G/G genotype (Figure 1). A significant interaction was found between CNTF genotype by force when adjusting for age and gender (Table 3).
mFR
mFR data revealed a significant interaction between force and CNTF genotype. Individuals with the G/A genotype were slower to increase mFR with increasing force than those from G/G subjects (Figure 2). No statistical difference or interaction was found between CNTF genotype and force (Table 3).
MUmV
For G/A subjects, the MUmV was larger at low force levels, and smaller at high force levels than for G/G subjects (Figure 3). MUmV revealed a significant interaction between CNTF genotype by force similar to what was observed for SMUP area (Table 3).
MURI
MURI, an index of the number of active units, was smaller at lower force levels and larger at higher force levels in G/A subjects than G/G subjects (Figure 4). A significant CNTF by force interaction was found (Table 3).
SMUP area per unit force
The motor unit size (SMUP area) per unit force in G/A subjects declines with increasing force levels as would be expected with the progressive recruitment of larger motor units that are capable of generating greater force levels overall, as well as per MU fiber (Figure 5). In contrast, G/G subjects show a different pattern with little difference in motor unit size (SMUP area) per unit force at low and high force levels. SMUP area per unit force differed by force and CNTF genotype with a significant interaction (Table 3).
MURI per unit force
At low force levels G/A subjects appear to use fewer motor units per unit force than G/G subjects (Figure 6). This is consistent with their use of larger units per unit force (Figure 1). At higher force levels, G/G and G/A subjects show the use of similar numbers of motor units per unit force. A significant interaction was found between CNTF genotype and force (Table 3).
Discussion
Based on this preliminary study, CNTF genotype appears to influence the characteristics of the motor units of the vastus medialis active during submaximal knee extensor muscle contractions. G/A and G/G subjects appear to have different compositions of motor units that are reflected in the different sizes and firing patterns of motor units active during different levels of force generation. The strategy of motor unit activation appears to use Henneman's size principle with motor units becoming active based on their size. We speculate that G/A subjects show greater motor unit efficiency with increasing levels of force output than G/G individuals. G/A individuals appear to require less motor unit input per relative force level at higher force generation suggesting the utilization of smaller, potentially less fatigable units, which may be considered to be more efficient. At low force levels with the use of smaller, likely less fatigable motor units, G/A use fewer but larger units than G/G. Together the observations at low and high force levels suggest greater relative force generation per motor unit in participants of G/A genotype, which may lead to some loss of finer motor control in a muscle where the finest motor control may not be of prime importance. The force levels examined in this study are comparable to those used during daily activities and the differences in motor unit organization may have an impact on these activities.
In our previous report regarding CNTF and muscle strength, Roth et al [4] had found that G/A individuals were significantly stronger during isokinetic concentric testing at 3.14 rads/sec, but not at 0.52 rads/sec. In the current study, subjects were found not to differ in strength during isometric strength testing (i.e. 0 rads/sec). Roth et al [4] noted that the difference between 3.14 rads/sec and 0.52/sec suggested that the difference between G/G and G/A subjects may reflect differences in muscular power generation. The differences were not explained by differences in muscle mass, so Roth et al [4] suggested that the expressed null CNTF protein competed with the active CNTF protein for the CNTF receptor within muscle. How this would lead to increased strength was not clear. Muscular power is dependent on maximizing the use of strength and speed of movement, which is dependent on both the capability of muscle to generate force, and on the nervous system to maximize the coordination between force and speed. The observations in this report, while not directly addressing the power issue, suggest that neural organization in the peripheral motor system shows differences between G/A and G/G individuals that could result in differential maximal power generation.
CNTF is a gp130 cytokine member of the IL-6 superfamily that is present in both muscle and nerve. It appears to exert anabolic and catabolic effects that are important for muscle adaptation responses to injury [21]. In addition, the application of CNTF to female rats increases levator ani muscle volume and the number of muscle fibers, while not affecting muscle fiber size in younger rats [22]. CNTF administration in older rats resulted in a 17% increase in muscle fiber area [7]. CNTF also has known trophic, survival and regenerative effects on motor units [23], protects nerve conduction in diabetic rats [24], and is involved with motor neuronal sprouting [25]. In nerves, CNTF is predominantly localized to the Schwann cells of larger myelinated fibers [26,27], and enhances myelin formation [28]. These observations suggest that neural and myotrophic roles could explain the differences between G/A and G/G genotypes we observed in motor unit physiology. The differences between G/A and G/G subjects noted by Roth et al [4], and here, which could reflect differences in muscle power generation, may have potential importance in understanding the development of sarcopenia and frailty. Muscle power is related to functional disability in the elderly[29], and has been found to be related to longevity independent of isometric muscle strength[30].
The observations in this study are based on the motor unit properties as assessed using spiked triggered averaging and the decomposition program developed by Stashuk [13]. The methodology is based on approaches used to estimate the number of motor units in a muscle. Such methods give reasonable and reliable estimates of the number of motor units in a muscle [11]. We modified one of these approaches to examine the motor units during fixed levels of force generation during knee extension. The major advantage of the approach is the ability to study large numbers of subjects and to study them over time. In previous reports [14,15], we have noted limitations of the method. The main limitation stems from sampling bias. At any force level, the decomposition program is more likely to see larger than smaller active motor units. At low force levels this does not appear to be a problem. At higher levels, 30% and 50%, of MVC the program does not identify most small units (see Figure 1). However, the units being observed appear to account for most of the force generation, and are strongly related to the SEMG [19]. This issue should equally affect both G/G and G/A genotypes. However, the G/G subjects had smaller units at lower force levels, which if not observed would increase the regression estimate at higher force levels. The G/G subjects, in general had larger units at the higher force levels than did the G/A (Figure 1b). This can be seen by noting that in the 200 N to 450 N range, the 95th percentile for the G/A group was equivalent to the 81st percentile for G/G group, while the G/A group actually had the smallest observed units in this force range. In this higher force range, the G/G group had both the smallest and largest recorded units. Any bias in unit selection does not appear to impact on G/G or G/A subjects in a way that would markedly alter the observations.
While CNTF genotype appears related to the pattern of motor unit activation in the vastus medialis, only one allele was studied, the A ("null") allele. Many genes are involved in the development, maintenance, and activation of the neural and muscular tissues that make up the motor unit. The observations in this study represent only a single component of this overall organization. A more thorough understanding of the process will require examining the interaction of a number of genes that are involved in motor unit activation.
Conclusion
In summary, in this preliminary investigation, the presence of an inactive null allele in the CNTF gene resulted in a different pattern of motor unit activation during force generation between 10% and 50% of MVC compared to the most common CNTF genotype, force levels at which activities of daily living are performed. Subjects with the G/A genotype activated fewer but larger units at low force levels, and a greater number of relatively smaller units at higher force levels than G/G subjects. Whether the observation results in functional performance differences could not be determined from this study, however, could have important clinical implications and warrant further study at force levels used during daily activities.
Methods
Subjects
Two groups of subjects were used for the present analysis: 1) 36 women and men (aged 30 to 94 years) from the Baltimore Longitudinal Study of Aging [31], and 2) a cohort of 33 young and older men and women (23–73 years) who were evaluated prior to an exercise intervention study [32]. All subjects were healthy. Informed consent was obtained prior to each visit. Both studies were approved by the Johns Hopkins University Bayview Institutional Review Board.
Strength
Maximal isometric force of the knee extensors was measured while participants were securely seated on the Kin-Com 125E isokinetic dynamometer (Chattecx, Chattanooga, TN) using methodology described previously [14,32]. Maximal voluntary isometric force in Newtons (N) was measured at a knee angle of 2.09 rad. Three maximal isometric contractions were performed with a ten second rest period between efforts. The average of the two best trials was used to represent MVC.
Motor unit measurements
The methods for obtaining the motor unit measurements in the vastus medialis have been described in detail by Conwit et al. [14]. Subjects were tested while on the Kin-Com device as described above. The active surface detection electrode was placed over the motor point of the vastus medialis. To maximize the rise time of motor unit action potentials generated during low-level contractions, the concentric-needle electrode was inserted into the muscle body near the active surface electrode. Subjects then extended their knee against resistance with enough isometric force to achieve a specified percentage of their MVC. Visual and verbal cues were provided to the participant to help them achieve and maintain that level of force for 20–30 seconds. Simultaneously detected intramuscular and surface EMG signals were acquired using bandpass filtering from 10 Hz to 10 kHz and 5 Hz to 1000 Hz and sampling rates of 25 kHz and 2.5 kHz, respectively. The intramuscular EMG signal was decomposed using algorithms developed by Stashuk [13] to obtain accurate estimates of mean MU firing rates, and were combined with the surface EMG signal to provide estimates of surface-detected motor unit action potentials (SMUPs) using spike-triggered-averaging. After movement and readjustment of the position of the intramuscular electrode in order to minimize MU potential rise time, subjects were asked to generate similar force level contractions until SMUPs from 15–20 motor units were sampled at each force level studied. Previous reports using this method have shown that analysis of 15–20 motor units results in a coefficient of variation of approximately 10% [14]. Motor unit measurements were obtained during contraction levels of 10, 20, 30, and 50% of MVC for the Baltimore Longitudinal Study of Aging cohort, while measures were available only for 10 and 30% MVC for the exercise intervention cohort.
Genotype
Genomic DNA was extracted from whole blood samples and CNTF genotype was determined as described previously [4]. Subjects were categorized as exhibiting the G/G, G/A, though no A/A homozygotes were observed in the present study.
Data analysis
Mixed effects models were used to examine CNTF genotype differences in the relationship between motor unit variables with force generation while controlling for age, and gender. Mixed effects models are flexible models that can be used to deal with multiple data collected from groups or individuals as seen with longitudinal and repeated measures [33]. In the current study, each subject was tested at multiple force levels with approximately 15 motor units collected at each force level. The force levels were at 10, 20, 30 and 50% of MVC. Each subject may differ to some degree from the group as a whole, and this is dealt with by introducing random effects which were included for the intercept (i.e. how much the intercept from a subject's individual regression line deviated from the overall intercept), and for the percent of MVC) to allow for differences in how an individual responded to increasing force generation. All models had the following form
Yij = (β0+bj0)+ β1*CNTFj+(β2+ bj2)*percentj+ β3*forcej+ β4*CNTFj *forcej+ β5*agej+ β6*genderj+ eij
with Yij being the motor unit property for the ith motor unit and jth subject and bj0 and bj2 being the random effect for subject j and percent j. All dependent variables were examined for deviation from normality. Terms that included SMUP were markedly skewed, and were log transformed for the analysis. We assumed that SMUP variance would increase with increasing force, as larger units are sequentially activated with increasing variance, and modeled variance as a power function in relationship to the percent of effort [33]. This assumption was found not to influence the reported findings.
Significant interactions were observed between CNTF and force, which suggested that at both higher and lower forces levels motor unit properties differed by genotype, with G/A using larger units at lower force levels. To directly test this hypothesis, the mixed effects model was applied only to data with force levels less than or greater/equal to 200 N. This force level was approximately where the curves for GA and GG intersected. In addition, density plots were graphed for SMUP and MUmV for GA and GG for force levels less than and greater/equal to 200 N. Differences between the GA and GG densities were tested using the Kolmogorov-Smirnov goodness-of-fit test.
At the initial submission, Table 3 contained 9 specific statistical tests which raise issues regarding the extent of need for consideration of multiple comparisons to control for falsely rejecting the null hypothesis. A traditional approach would be to adjust the significance level using the Bonferonni adjustment by dividing the test p by the number of tests to control type 1 or familywise error. However, this is an extremely conservative approach that increases the risk of accepting a false null hypothesis (type 2 error). An alternative approach is to control the false discovery rate, i.e. the expected proportion of type I errors among all significant tests. Attempting to hold this constant is different from the familywise error rate, where the goal is to avoid any type 1 errors [34]. We have added the adjusted p values to the models using the entire dataset and 2 adjustments, first for the 9 tests performed for Table 3 in the initial submission (3 of which were excluded in subsequent revisions), and for the 21 tests when including tests for force levels below and above 200 N.
Analyses were completed in SPLUS 6.2 (In Sightful, Seattle, WA). Chi-square and t-tests were used to compare subject characteristics. Data are means ± SE. Statistical significance was accepted at P < 0.05. Graphs show scatter plots with loess nonparametric regression lines to show the nature of the relationships between the motor unit variables and force generation [20].
List of abbreviations used
CNTF: ciliary neurotrophic factor
mFR: mean firing rate
MUmV: motor unit mean voltage
MURI: motor unit relative index
MVC: maximal voluntary muscle contraction
SEMG: surface electromyography
SMUP: surface motor unit potential
Authors' contributions
RAC and EJM developed the motor unit protocol that was used in the study. They were responsible for the collection of the motor unit data that was used in the analysis. They were the principal writers of the manuscript, and EJM did the analyses. SL was involved with the planning of the analyses and in the thinking that resulted in Table 1. She contributed to the manuscript preparation. SR and RF did the DNA work and with BH were responsible for initiating our interest in the impact of CNTF on muscle and motor unit function. All three contributed their expertises to the preparation of the manuscript. DS developed the spiked trigger averaging methodology, worked with RAC and EJM in developing and implementing the motor unit protocol, and contributed to the manuscript preparation.
Acknowledgements
The work was completed as part of the Intramural Research Program of the National Institute on Aging, National Institutes of Health contract 1AG42148, and grant AG022791.
Figures and Tables
Figure 1 Relationship between knee extension force and motor unit size (SMUP area) for CNTF GG and GA genotypes. Data represent individual measurements with a loess regression line for each genotype. Statistical analyses are given in Table 3.
Figure 2 Relationship between knee extension force and mean motor unit firing rate (mFR) for CNTF GG and GA genotypes. Data represent individual measurements with a loess regression line for each genotype. Statistical analyses are given in Table 3.
Figure 3 Relationship between knee extension force and average motor unit contribution considering motor unit size and firing rate (MUmV) for CNTF GG and GA genotypes. Data represent individual measurements with a loess regression line for each genotype. Statistical analyses are given in Table 3.
Figure 4 Relationship between knee extension force and the relative number of motor units active (MURI) for CNTF GG and GA genotypes. Data represent individual measurements with a loess regression line for each genotype. Statistical analyses are given in Table 3.
Figure 5 Relationship between knee extension force and motor unit size per unit force (SMUP area/force) for CNTF GG and GA genotypes. Data represent individual measurements with a loess regression line for each genotype. Statistical analyses are given in Table 3.
Figure 6 Relationship between knee extension force and relative number of motor units active per unit force (MURI/force) for CNTF GG and GA genotypes. Data represent individual measurements with a loess regression line for each genotype. Statistical analyses are given in Table 3.
Figure 7 Density distribution of motor unit size for CNTF GG and GA genotypes for force levels less than and greater than 200 N for SMUP and MUmV. The graphs show the frequency distribution of motor units based on either SMUP or MUmV levels for genotypes GG and GA. The graphs show the interaction between CNTF genotype and motor unit properties.
Table 1 Explanation of the assessed motor unit parameters
Measure Calculation Conversion-Units Assessment
Individual Motor Unit Measures
SMUP area Surface motor unit potential area uV*msec Motor unit size
mFR Mean firing rate /sec Motor unit mean firing rate
Estimates of motor unit relationship to muscle activation
MUmV (SMUP area)*FR 0.001* uV Motor unit mean contribution to contraction
MURI SEMG/1000*MUmV Relative index of number of active units in field of measurement during contraction
Estimate of overall muscle characteristics per unit force
(SMUP area)/N Surface motor unit potential area per Newton force generated uV*msec/N Mean motor unit size per Newton force generated during a contraction
mFR/N Mean firing rate per Newton force 1/sec*N Mean motor unit firing rate per Newton force generated during a contraction
MUmV/N (SMUP area)*mFR/N uV/N Mean motor unit contribution per Newton force
MURI/N MURI/N 1000/N Relative index of number of active units per unit force
Table 2 Subject Characteristics
G/A G/G P
Sample size 12 58
Age 52.0 (19.3) 51.2 (19.8) 0.89
Percent Female 42 33 0.35
Height 173.1 (9.5) 171.1 (7.8) 0.48
Weight 78.7 (14.2) 76.7 (18.1) 0.68
Isometric MVC 522.5 (160.9) 542.2 (173.6) 0.71
Table 3 Significance levels for mixed effects models examining force to motor unit relationships
Dependent Variables All Force Levels
CNTF Percent+ Force+ CNTF*Force
SMUP area* 0.01 (0.02, 0.02) 0.00 0.00 0.02 (0.06, 0.04)
mFR 0.32 (0.32,0.36) 0.01 .00 0.13 (0.14, 0.15)
MUmV* 0.00 (0.01, 0.02) 0.02 0.00 0.00 (0.06, 0.03)
MURI* 0.01 (0.02, 0.02) 0.07 0.013 0.01 (0.06, 0.03)4
(SMUP area)/force* 0.01 (0.02, 0.02) 0.00 .00 0.03 (0.07, 0.03)
MURI/force* 0.0 (0.00, 0.00) 0.00 0.00 0.05 (0.07, 0.07)
Force < 200 N
SMUP area* 0.00 (0.02) 0.07 0.00 0.01 (0.03)
mFR 0.37 (0.39) 0.10 0.00 0.23 (0.26)
MUmV* 0.00 (0.01) 0.19 0.00 0.00 (0.02)
MURI* 0.00 (0.02) 0.69 0.65 0.01 (0.03)
(SMUP area)/force* 0.01 (0.02) 0.09 0.59 0.03 (0.05)
MURI/force* 0.01 (0.02) 0.01 0.00 0.01 (0.03)
Force > -200 N
SMUP area* 0.01 (0.02) 0.22 0.00 0.00 (0.00)
mFR 0.57 (0.57) 0.45 0.00 0.96 (0.96)
MUmV* 0.03 (0.04) 0.42 0.00 0.00 (0.00)
MURI* 0.28 (0.32) 0.12 0.00 0.12 (0.15)
(SMUP area)/force* 0.01 (0.02) 0.15 0.00 0.00 (0.00)
MURI/force* 0.19 (0.24) 0.09 0.00 0.08 (0.12)
Each model was adjusted for age and gender. Numbers within parentheses are p values adjusted for multiple comparisons based on 9 and 21 tests for all force levels, and 12 tests for force< 200 N and force >= 200 N using the false discovery rate adjustment. *Variable was studied with natural log transform. + Percent refers to the force generated as a percent of MVC during motor unit collection. Force refers to the force generated during data collection.
==== Refs
Marcell TJ Sarcopenia: causes, consequences, and preventions J Gerontol A Biol Sci Med Sci 2003 58 M911 6 14570858
Metter EJ Talbot LA Schrager M Conwit R Skeletal muscle strength as a predictor of all-cause mortality in healthy men J Gerontol A Biol Sci Med Sci 2002 57 B359 65 12242311
Conwit R Metter EJ Brown WF, Bolton CF and Aminoff MJ Age related changes in peripheral and central conduction Neuromuscular function and disease, basic, clinical and electrodiagnostic aspects 2002 New York, Saunders 602 617
Roth SM Schrager MA Ferrell RE Riechman SE Metter EJ Lynch NA Lindle RS Hurley BF CNTF genotype is associated with muscular strength and quality in humans across the adult age span J Appl Physiol 2001 90 1205 1210 11247915
Takahashi R Yokoji H Misawa H Hayashi M Hu J Deguchi T A null mutation in the human CNTF gene is not causally related to neurological diseases Nat Genet 1994 7 79 84 8075647 10.1038/ng0594-79
Seibert MJ Xue QL Fried LP Walston JD Polymorphic variation in the human myostatin (GDF-8) gene and association with strength measures in the Women's Health and Aging Study II cohort J Am Geriatr Soc 2001 49 1093 1096 11555072 10.1046/j.1532-5415.2001.49214.x
Forger NG Roberts SL Wong V Breedlove SM Ciliary neurotrophic factor maintains motoneurons and their target muscles in developing rats J Neurosci 1993 13 4720 4726 8229194
Guillet C Auguste P Mayo W Kreher P Gascan H Ciliary neurotrophic factor is a regulator of muscular strength in aging J Neurosci 1999 19 1257 1262 9952403
Henneman E Somjen G Carpenter DO Functional Significance of Cell Size in Spinal Motoneurons J Neurophysiol 1965 28 560 580 14328454
Burke RE Levine DN Salcman M Tsairis P Motor units in cat soleus muscle: physiological, histochemical and morphological characteristics J Physiol 1974 238 503 514 4277582
Doherty T Simmons Z O'Connell B Felice KJ Conwit R Chan KM Komori T Brown T Stashuk DW Brown WF Methods for estimating the numbers of motor units in human muscles J Clin Neurophysiol 1995 12 565 584 8600172
McComas AJ Motor unit estimation: anxieties and achievements Muscle Nerve 1995 18 369 379 7715621 10.1002/mus.880180402
Stashuk DW Decomposition and quantitative analysis of clinical electromyographic signals Med Eng Phys 1999 21 389 404 10624736 10.1016/S1350-4533(99)00064-8
Conwit RA Tracy B Jamison C McHugh M Stashuk D Brown WF Metter EJ Decomposition-enhanced spike-triggered averaging: contraction level effects Muscle Nerve 1997 20 976 982 9236788 10.1002/(SICI)1097-4598(199708)20:8<976::AID-MUS7>3.0.CO;2-3
Conwit RA Stashuk D Tracy B McHugh M Brown WF Metter EJ The relationship of motor unit size, firing rate and force Clin Neurophysiol 1999 110 1270 1275 10423192 10.1016/S1388-2457(99)00054-1
Jabre JF Salzsieder BT The volitional unit: a functional concept in cortico-motoneuronal connections in humans Electroencephalogr Clin Neurophysiol 1997 105 365 369 9363001 10.1016/S0924-980X(97)00043-X
Jabre JF Salzsieder BT An EMG study of functional cortico-motoneuronal connections in humans J Physiol Paris 1999 93 147 154 10084718 10.1016/S0928-4257(99)80145-4
Conwit RA Stashuk D Suzuki H Lynch N Schrager M Metter EJ Fatigue effects on motor unit activity during submaximal contractions Arch Phys Med Rehabil 2000 81 1211 1216 10987164 10.1053/apmr.2000.6975
Suzuki H Conwit RA Stashuk D Santarsiero L Metter EJ Relationships between surface-detected EMG signals and motor unit activation Med Sci Sports Exerc 2002 34 1509 1517 12218747 10.1097/00005768-200209000-00018
Harrell FEJ Regression modeling strategies with applications to linear models, logistic regression, and survival analysis 2001 New York, Springer 24 25
Zoico E Roubenoff R The role of cytokines in regulating protein metabolism and muscle function Nutr Rev 2002 60 39 51 11852969 10.1301/00296640260085949
Peroulakis ME Forger NG Ciliary neurotrophic factor increases muscle fiber number in the developing levator ani muscle of female rats Neurosci Lett 2000 296 73 76 11108984 10.1016/S0304-3940(00)01649-9
Sendtner M Stockli KA Thoenen H Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration J Cell Biol 1992 118 139 148 1618901 10.1083/jcb.118.1.139
Mizisin AP Vu Y Shuff M Calcutt NA Ciliary neurotrophic factor improves nerve conduction and ameliorates regeneration deficits in diabetic rats Diabetes 2004 53 1807 1812 15220205
Siegel SG Patton B English AW Ciliary neurotrophic factor is required for motoneuron sprouting Exp Neurol 2000 166 205 212 11085886 10.1006/exnr.2000.7528
Rende M Muir D Ruoslahti E Hagg T Varon S Manthorpe M Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve Glia 1992 5 25 32 1531807 10.1002/glia.440050105
Friedman B Scherer SS Rudge JS Helgren M Morrisey D McClain J Wang DY Wiegand SJ Furth ME Lindsay RM Ip NY Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo Neuron 1992 9 295 305 1497895 10.1016/0896-6273(92)90168-D
Stankoff B Aigrot MS Noel F Wattilliaux A Zalc B Lubetzki C Ciliary neurotrophic factor (CNTF) enhances myelin formation: a novel role for CNTF and CNTF-related molecules J Neurosci 2002 22 9221 9227 12417647
Rantanen T Avela J Leg extension power and walking speed in very old people living independently J Gerontol A Biol Sci Med Sci 1997 52 M225 31 9224434
Metter EJ Talbot LA Schrager M Conwit RA Arm-cranking muscle power and arm isometric muscle strength are independent predictors of all-cause mortality in men J Appl Physiol 2004 96 814 821 14555682 10.1152/japplphysiol.00370.2003
Shock NW Gruelich RC Andres RA Arenberg D Costa PTJ Lakatta EG Tobin JD Normal Human Aging. The Baltimore Longitudinal Study of Aging 1984 Washington, DC:, US Government Printing Office
Ivey FM Tracy BL Lemmer JT NessAiver M Metter EJ Fozard JL Hurley BF Effects of strength training and detraining on muscle quality: age and gender comparisons J Gerontol A Biol Sci Med Sci 2000 55 B152 7; discussion B158-9. 10795719
Pinheiro JC Bates DM Mixed-effects models in S and S-PLUS 2000 New York, Springer 206 225
Verhoeven KJF Simonsen KL McIntyre LM Implementing false discovery rate control: increasing your power OIKOS 2005 108 643 647 10.1111/j.0030-1299.2005.13727.x
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BMC Struct BiolBMC Structural Biology1472-6807BioMed Central London 1472-6807-5-171616475910.1186/1472-6807-5-17Research ArticleProtein secondary structure assignment revisited: a detailed analysis of different assignment methods Martin Juliette [email protected] Guillaume [email protected] Antoine [email protected] Jean-François [email protected] Brevern Alexandre G [email protected] Jean-François [email protected] INRA, Unité Mathématiques Informatique et Génome, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France2 INSERM U726, Equipe de Bioinformatique Génomique et Moléculaire, Université Paris 7, case 7113, 2 place Jussieu, 75251 Paris cedex 05, France2005 15 9 2005 5 17 17 26 5 2005 15 9 2005 Copyright © 2005 Martin et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
A number of methods are now available to perform automatic assignment of periodic secondary structures from atomic coordinates, based on different characteristics of the secondary structures. In general these methods exhibit a broad consensus as to the location of most helix and strand core segments in protein structures. However the termini of the segments are often ill-defined and it is difficult to decide unambiguously which residues at the edge of the segments have to be included. In addition, there is a "twilight zone" where secondary structure segments depart significantly from the idealized models of Pauling and Corey. For these segments, one has to decide whether the observed structural variations are merely distorsions or whether they constitute a break in the secondary structure.
Methods
To address these problems, we have developed a method for secondary structure assignment, called KAKSI. Assignments made by KAKSI are compared with assignments given by DSSP, STRIDE, XTLSSTR, PSEA and SECSTR, as well as secondary structures found in PDB files, on 4 datasets (X-ray structures with different resolution range, NMR structures).
Results
A detailed comparison of KAKSI assignments with those of STRIDE and PSEA reveals that KAKSI assigns slightly longer helices and strands than STRIDE in case of one-to-one correspondence between the segments. However, KAKSI tends also to favor the assignment of several short helices when STRIDE and PSEA assign longer, kinked, helices. Helices assigned by KAKSI have geometrical characteristics close to those described in the PDB. They are more linear than helices assigned by other methods. The same tendency to split long segments is observed for strands, although less systematically. We present a number of cases of secondary structure assignments that illustrate this behavior.
Conclusion
Our method provides valuable assignments which favor the regularity of secondary structure segments.
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Background
In 1951, Pauling and Corey predicted the existence of two periodic motifs in protein structures: the α-helix [1] and the β-sheet [2] which turned out to be major features of protein architecture. Secondary structures, because they allow a simple and intuitive description of 3D structures, are widely employed in a number of structural biology applications. For instance, they are used for structure comparison [3] and structure classification [4,5]. They also provide a natural frame for structure visualization [6,7].
In recent years, secondary structures have come to play a major role in a number of methods aiming at predicting protein 3D-structures. Indeed, being able to predict accurately secondary structure elements along the sequence provides a good starting point toward elucidating the 3D-structure [8,9]. Current algorithms for predicting the secondary structure provides accuracy rates of about 80% for a 3 state prediction: α-helix, β-strand and coils [10-12], using neural networks and evolutionary information. The maximum achievable prediction has been estimated to lie in the range 85% [13] to 88% [14].
The divergence between observed and predicted secondary structure has been noticed early [15]. It took more time, though, for the structuralist community, to realize that obtaining an accurate and objective secondary structure assignment was not a trivial task, due to the variations observed in secondary structures when compared to ideal ones. As noted by Robson and Garnier [16]: "In looking at a model of a protein, it is often easy to recognize helix and to a lesser extent sheet strands, but it is not easy to say whether the residues at the ends of these features be included in them or not. In addition there are many distorsions within such structures, so that it is difficult to assess whether this represents merely a distortion, or a break in the structure. In fact the problem is essentially that helices and sheets in globular proteins lack the regularity and clear definition found in the Pauling and Corey models." For instance, as found by Barlow and Thornton [17] and Kumar and Bansal [18,19], a majority of α-helices in globular proteins are smoothly curved. Therefore, a group of experts (NMR spectroscopists and crystallographers), asked to assign the secondary structure of a particular protein, is likely to come up with different assignments.
To cope with this problem, as well as the increase in the number of experimentally solved 3D structures, the need for automatic secondary structure assignment programs was felt in the mid seventies. Such programs are intended to embody expert's knowledge and to provide consistent and reproducible secondary structure assignments. Periodic secondary structures generate regularities that can be used as criteria to define them, e.g., Cα distances, dihedral angles, like α angles or pairs of (Φ/Ψ) angles, and specific patterns of hydrogen bonds. Along the years, various methods using these criteria have been proposed. The first implementation of such methods, allowing automatic secondary structure assignment from 3D coordinates, was done by Levitt and Greer [20]. The algorithm was mainly based on inter-Cα; torsion angles.
A few years later, Kabsch and Sander developed a method called DSSP [21] that still remains one of the most widely-used program for secondary structure assignment. The DSSP algorithm is based on the detection of hydrogen-bonds defined by an electrostatic criterion. Secondary structure elements are then assigned according to characteristic hydrogen-bond patterns. This methodology has been widely accepted as the gold standard for secondary structure assignment. A number of software packages make use of DSSP when they need to assign secondary structures. For instance rasmol [6], the most widely distributed visualization software, assigns the repetitive structures with a fast DSSP-like algorithm. Similarly GROMACS analysis tools use the DSSP software [22].
STRIDE [23] is a software related to DSSP. It makes a very similar use of hydrogen-bond patterns to what is done in DSSP, although the definition of hydrogen-bonds is slightly different. In addition STRIDE takes into account (Φ/Ψ) angles to assign secondary structures. STRIDE is used by the visualization tool VMD [7] to assign secondary structures.
SECSTR [24] belongs to the same family of methods. It has been developed specifically to improve the detection of π-helices. Indeed, SECSTR's authors found dssp and STRIDE unable to detect several π-helices they were able to characterize with their method.
Other methods have been developed that use different criteria to assign secondary structures. DEFINE [25] relies on Cα coordinates only and compares Cα distances with distances in idealized secondary structure segments. It also provides a description of super-secondary structures. P-CURVE approach [26] is based on the definition of helicoidal parameters for peptide units and generates a global peptide axis. PSEA [27] only considers Cα atoms. It is based on distance and angle criteria. XTLSSTR [28] has been developed to assign secondary structures " in the same way a person assigns structure visually", from distances and angles calculated from the backbone geometry. It is concerned with amide-amide interactions. The most recent method, to the best of our knowledge, is VoTAP [29] which employs the concept of Voronoi tessellation, yielding new contact matrices.
Let us notice that structure files provided by the Protein Data Bank (PDB) [30] contain secondary structure descriptions in the HELIX, SHEET and TURN fields (see the PDB Format Description Version 2.2 [31]). These secondary structure descriptions are either provided by the depositor (optional) or generated by DSSP. Approximately 90% of the PDB files do have secondary structure fields. However, even though these fields are used, it may happen that only a few secondary structure elements, of interest for the depositor, are described, the others being ignored.
The variety of available methods illustrates the fact that there are several legitimate ways to define secondary structures. It is hardly surprising that these different methods provide different assignments, especially at the edges of secondary structure segments. For example, Colloc'h and co-workers [32] showed that the percentage of agreement is only 63% between DSSP, P-CURVE and DEFINE and that DEFINE tends to assign too many repetitive secondary structure segments. XTLSSTR authors noted that DSSP assigns more β-strands than XTLSSTR does [28]. SECSTR is logically more sensitive for π-helix detection than DSSP or stride [24].
In this paper we want to focus on how well some of the above methods handle the secondary structure irregularities mentioned by Robson and Gamier [16]. We are particularly interested in the way these different methods process the edges of secondary structure elements and deal with the various structure distorsions occurring in proteins. For structures solved by X-ray diffraction, it is well known that the resolution has a direct effect upon the quality of the resulting model. One expects the secondary structure assignment to be less accurate for low resolution structures [23]. It is thus interesting to assess the effect of the resolution upon the secondary structure assignment proposed by the different methods. It is also worth comparing secondary structure assignments for structures solved by X-ray crystallography and by NMR techniques. Structures solved by NMR correspond to proteins in solution and provide a more "dynamic" representation of the protein conformation than X-ray structures do. NMR structures are therefore more prone to local distorsions and constitute difficult, and interesting, cases for secondary structure assignment methods.
In the following we present a new method for secondary structure assignment, called KAKSI (KAKSI means "two" in Finnish) based on Cα distances and (Φ/Ψ) angles. These characteristics are intuitively used when examining visually a 3D structure. Our main purpose in developing this method was to deal, in a satisfactory way, with the structure irregularities. For instance we consider that regions of the polypeptide chain that show an abrupt change in their curvatures (such as kinks in a helices) should be considered as breaks in periodic secondary structures. The objective of an assignment method is to provide accurate and reliable assignment. Demonstrating that our methodology is an improvement over existing methods would be difficult since there is no standard of truth to benchmark methods with. We then carry out comparisons of the assignments of this new method with a number of other methods that use different criteria to define secondary structures: DSSP, STRIDE, SECSTR, XTLSSTR and PSEA, as well as with the descriptions found in PDB files. These comparisons are performed on 4 different datasets: 3 X-ray datasets with, respectively, high, medium and low resolution and an NMR dataset. This allows us to evaluate the effect of the resolution and experimental method upon the different secondary structure assignment methods.
We address the problem of inclusion of residues at the edges of helices and strands by examining the length of segments assigned by different methods. We also study the problem of correctly defining segments in case of distortions. More specifically, for helices, we appraise the geometry of helical segments using HELANAL [33], a software dedicated to this task.
Finally, we illustrate how KAKSI deals with distorted secondary structures by comparing its assignments with STRIDE assignments for a number of difficult cases.
Results and discussion
KAKSI parameters
In KAKSI secondary structure detection depends on a number of parameters (see Method section).
To test the robustness of the method to the choice of these parameters, we examined the effect of changing εH, εb and σb upon the secondary structure contents of the comparison sets. We let εH and εb vary in the range 1.29 to 3.30, and σb in the range 3 to 6. Each parameter is tested separately, while keeping other parameters to the selected values given in Methods section.
The effects are similar on all sets of structures. The decrease of εH below 1.96 results in a moderate diminution of the percentage of α-helix, whereas this percentage slightly increases when εH is greater than 1.96. Fewer β-sheets are assigned when εb, is lower than 2.58. On the contrary, the percentage of β-sheets increases when εb, is greater than 2.58. Slightly more β-sheets are assigned when σb is lower than 5, and there is a diminution of β-sheets assignment when σb is greater than 5.
Two different behaviors are observed: KAKSI assignments are not very sensitive to variations of α-helix detection thresholds, but quite sensitive to variations of β-sheets detection thresholds. This is easily explained by the detection heuristic: the detection of α-helix is achieved by the distance or the angle criteria, moderate changes of εH are balanced by other criteria. On the contrary, the β-sheet detection is achieved by the satisfaction of both, distance and angle, criteria.
The two criteria implemented in KAKSI for kink detection in α-helices, K1 based on (Φ/Ψ) angles and K2 based on axes, are also tested. To evaluate the efficiency of each criterion, we analyze the geometry of kinked helices with the HELANAL software. We monitor the fraction of helices classified as kinked by HELANAL. This fraction is reduced when each criterion is used separately showing that both criteria are able to detect kinks (data not shown). Results obtained with K1 agree better with HELANAL results than those obtained with K2. However the best agreement with HELANAL is obtained when criterion K1 and K2 are used sequentially. Hereafter, KAKSI assignments are obtained with the parameter values given in Material and Methods and both criteria K1 and K2 applied for kink detection.
Secondary structure content
The secondary structure content is used to assess the sensitivity of different assignment methods to the structure resolution. Table 2 shows the secondary structure content in all our comparison sets, according to five available assignment softwares, KAKSI and the PDB description.
There is no absolute consensus, even for the HRes set, about secondary structure content according to different methods. STRIDE and DSSP figures are very close, as expected due to the similarity of these methods [21,23]. PSEA systematically assigns less helices and more strands than other methods. PDB assignments are always richer in α-helix than any automatic procedure. KAKSI assigns a fraction of periodic secondary structures comparable to STRIDE and DSSP on the HRes set.
Secondary structure contents in the HRes and the MRes sets are similar according to different methods. Assignments on the LRes and the NMR sets result in smaller contents in regular secondary structures. This is true for every assignment methods, but more or less marked, depending on the method. β-assignment is lower on the LRes set for a majority of methods. Only PSEA assignments show a proportion of β comparable for all datasets. It must be noted that this method consistently assigns more β-strands than all other methods, whatever the dataset considered. Overall, though, the influence of the resolution upon the assignments of the methods is moderate. The type of technique use to solve the structure (X-ray vs NMR) appear to have a more pronounced effect.
The decrease in β-sheets assignment on the LRes and NMR sets indicates that less stringent parameter values are required when dealing with structures belonging to these sets. For example, KAKSI assignment on the LRes set with σb = 3 result in a proportion of 22.3% residues in β-sheet and 20.7% with σb = 3.30 (data not shown). In the same way, the percentage of β-sheet residues in the NMR sets is about 17.7% with σb = 3 or εb = 3.30. Consequently, we suggest to adapt the β-sheet detection parameters when dealing with low resolution and NMR structures.
Measures of global agreement between methods
C3 scores
Table 3 shows the C3 scores obtained for the HRes set (the overall agreement between the different assignment methods show the same tendencies for the different comparison sets, [see Additional file 1]). A group of methods shows a strong agreement: C3 scores within the group DSSP, STRIDE, SECSTR and PDB are all in the range 87.4% (SECSTR versus PDB) to 95.4% (STRIDE versus DSSP). The strong similarity between DSSP and STRIDE assignments, which both used a hydrogen-bond criterion, has been noted in previous studies [27,29,34]. The SECSTR method is strongly related to the DSSP algorithm and logically belongs to this group. As was expected, PDB descriptions are very close to DSSP assignments due to the way secondary structure assignments are performed.
Assignments given by XTLSSTR are the most different from others: C3 scores with DSSP, STRIDE, SECSTR and PDB are all below 81%. KAKSI and PSEA show an intermediate behavior of the other methods [see Additional file 2]. The C3 scores are all in the same range, between 81.5% (KAKSI/PSEA) and 83.5% (KAKSI/STRIDE), excluding XTLSSTR (78.3%).
SOV criterion
The SOV criterion is usually employed for secondary structure prediction evaluation, whereas here, comparisons are made between alternative structure assignments. SOV values depend on which structure ischosen as reference. To allow comparison, KAKSI is taken as reference. Table 4 shows SOV values computed from the HRes set for helices and strands, between KAKSI and other methods. SOV values for other datasets are available, [see Additional file 3].
For helical segments, the highest SOV with KAKSI assignment is obtained with DSSP (91.7%). It lies in the same range for STRIDE. It is slightly lower for other methods but remains above 87%. For the strands, a good agreement is seen with DSSP, STRIDE and PDB (SOV scores about 90%). Lower SOV (about 83%) are found with PSEA and SECSTRC. Moderate agreement is seen with XTLSSTR (75.8% only). C3 score between XTLSSTR and KAKSI is only 78.3%(see table 3). SOV values are high for helices and slightly lower for strands, showing that differences between both methods mainly concern β-sheets assignments. Hereafter we will restrict our comparisons to KAKSI, STRIDE, and PSEA assignments on the HRes set. STRIDE is a widely-used method whose results are very similar to DSSP and PDB, as shown by the C3 scores. STRIDE is chosen because it exhibits the largest C3 score with KAKSI. PSEA is chosen because its algorithm fairly differs from other methods, but SOV values remain consistent when compared to KAKSI'S.
Segment length distribution
The length distributions of helices and strands assigned by KAKSI, PSEA and STRIDE on the HRes set are shown on Figure 3.
In helix distributions, three zones can be distinguished. (i) For helices shorter than 8 residues, the distributions are very different: STRIDE assigns many 3 residue long helices, whereas PSEA and KAKSI do not assign helices shorter than 5 residues. PSEA assignments results in slightly larger number of short helices than STRIDE. KAKSI distribution shows a very high peak at 7 residues. (ii) In the range 8 to 15 residues, small differences are observed: KAKSI distribution shows a peak about 12 residues, unlike PSEA and STRIDE distributions. (iii) For helices longer than 15 residues, distributions are similar.
Similarly, 3 distinct zones appear in the strand distributions. (i) Up to 6 residues, PSEA and KAKSI curves show larger peaks than STRIDE distribution, at 3 to 5 residues for KAKSI, and 4 and 5 residues for PSEA. PSEA and KAKSI do not assign strands shorter than three residues, whereas STRIDE assignment result in a large number of 1-residue long strands. These segments are isolated β-bridges (state b in stride assignments). (ii) Between 6 and 9 residues, psea and KAKSI segments are more numerous than STRIDE segments. (iii) After 9 residues, the distributions are identical.
Global measures, such as C3 and SOV scores, show that KAKSI assignments are globally consistent with those given by other existing methods. The length distributions of helices and strands indicates that segment distribution is also roughly similar across methods. This broad consensus was expected. In the following sections we now turn toward the study of details of the assignments, in particular, as mentioned in the introduction, we compare the way different methods deal with the edges of secondary structures and cope with local distorsions.
Detailed comparison
Pair length
The SOV criterion is a measure of the global overlapping of secondary structure segments. It gives no information about the effect of length of segments or about the respective length of facing segments. Figure 4 shows the plot of lengths for pair of corresponding repetitive structure segments between STRIDE and KAKSI, and PSEA and KAKSI assignments. The pairs are those used for the SOV computation: a pair is considered when there is at least one residue in the same state for the two assignments. Unpaired segments are ignored.
Taking KAKSI assignment as our reference, three different cases occur: (i) One segment according to KAKSI corresponds to a single segment in another method assignment: these are one-to-one events. (ii) One segment assigned by KAKSI corresponds to two or more segments in another method assignment. We call this a fusion event. (iii) The symmetric case, several segments in KAKSI assignment corresponding to a single segments in another method assignment, is called a division event. The three cases are available plotted on separate graphs [see Additional file 4].
Helix length
The strong accumulation of points along the diagonal, on both plots (KAKSI versus STRIDE and KAKSI versus PSEA) and for every segment lengths shows that KAKSI often agrees with other methods about the length of helices. There are more points below the diagonal than above, indicating that KAKSI tends to assign slightly longer segments than STRIDE and PSEA (one or two residue longer). This occurs for all segment lengths, but it is more striking on the PSEA/KAKSI comparison.
The points appearing far from the diagonal correspond to division and fusion events, as shown by the squared correlation coefficients r2. Correlations are calculated on the pairs (PSEA or STRIDE length/KAKSI length) and are used as indicators for the dispersion about the diagonal. On the KAKSI/STRIDE comparison, r2 = 0.28 for all the 5146 pairs, but reaches 0.88 when only the 3755 one-to-one events are considered. The remaining pairs correspond to 142 cases of fusion and 1249 cases of division events. Division events are responsible for the numerous observations of pairs of short helices in KAKSI assignment (5 to 9 residues) with longer helices in PSEA and STRIDE assignments (10 to 20 residues).
Similarly, for the KAKSI/PSEA comparison there are 4762 pairs (r2 = 0.23), distributed in 3443 one-to-one events (r2 = 0.85), 150 fusion and 1169 division events. Numerous cases of divisions appear on the plot as pairs of 5 to 9 residue helices for KAKSI and 10 to 20 residue helices for PSEA.
For both comparisons (KAKSI/STRIDE and PSEA/KAKSI), the number of division events is greater than the number of fusion events, showing that KAKSI tends to split long segments into shorter ones. This is a direct consequence of the kink detection mechanism used in KAKSI. It also explains why short helices are more abundant in KAKSI assignments than in STRIDE and PSEA. Some examples of this phenomenon are illustrated in Fig 5.
Strand length
The situation is less clear than for helices. The points are more dispersed and there is no clear accumulation of points accounting for division events. In the KAKSI/STRIDE comparison, the 5974 pairs yield a r2 equal to 0.35. This value increases to 0.69 when only the 5403 one-to-one events are considered. Amongst the remaining pairs 214 correspond to fusion events, and 357 to division events. The splitting of long segments is thus less systematic than for helices. This makes senses since there is no mechanism similar to the kink detection in helices for β-strands. 52% of the one-to-one events fall above the diagonal (longer segments in KAKSI assignment) and 22 % fall below the diagonal (shorter segments in KAKSI assignment). The remaining 26% are on the diagonal. It shows that KAKSI tend to assign longer strands than STRIDE.
In the KAKSI/PSEA comparison, r2 equals 0.23 on the 5041 pairs and 0.44 on the 4694 one-to-one events. There are 214 fusion events and 133 division events. The numbers of division and fusion events are close, indicating that there only a slight splitting effect. 27% of the one-to-one events are on the diagonal, 50% are above (greater length in PSEA assignment) and 23% are below (greater lenght in kaksi assignment). In a majority of case, KAKSI assigns shorter strand segments concerning one-to-one events.
For both kind of segments and both comparisons, we also checked for the existence of systematic shifts of the segments toward the N-ter or C-ter termini of the secondary structure elements. No such systematic bias was found (data not shown).
Helix geometry analysis with HELANAL
In KAKSI we pay a special attention to the detection of kinks in α-helices by applying angle and axis criteria. This motivates the study of the geometry of helices with an external tool, according to alternative definitions of helix locations. We check the geometry of helices assigned by the different assignment methods with the HELANAL software. We are interested in the distribution of helices into the three classes: linear (L), curved (C) or kinked (K). Unclassified helices represent less than 1% in our datasets.
When analyzed by HELANAL, helices assigned by all methods show a high proportions of kinks. On the HRes set, for example, about 20% (DSSP, STRIDE, KAKSI) up to 30% (SECSTR, XTLSSTR) helices appear classified as kinked. This ratio is 16% only for the PDB assignments, and less than 10% for PSEA. When the resolution gets worse, this proportion increases [see Additional file 5]. On the NMR set, we observe as much as 40% kinked helices for PSEA assignment and more 50% kinked helices for STRIDE, SECSTR and PDB.
This high ratio of irregular helices (curved or kinked) is in agreement with previously published results [17]. However, the high ratio of kinked helices found here is larger than previously reported by Kumar and Bansal [19]. There is a difference between Kumar and Bansal's work and our study: they modified helix assignment given by DSSP before submission to HELANAL. Using distance and axis criteria, they corrected helix boundaries to avoid distortions at the termini. Consequently, the high ratio of kinked helices is likely due to these terminal residues. Rather than applying the correction used by Kumar and Bansal, we apply a systematic correction before submitting helices to HELANAL, i.e., one residue is removed at each helix terminus. The reason for applying a systematic correction rather than a correction based on geometrical criteria is that we want to make a statistical comparison of helices assigned by various softwares. The goal is not to correct potentially wrong helices boundaries. We want to evaluate the assignments as they are produced by the softwares and used in later applications.
Table 5 shows the results obtained on the HRes set, before and after correction, for helices defined by the seven methods. Results for other datasets are available [see Additional file 5].
As HELANAL can handle only helices longer than nine residues, we restrict our analysis to helices longer than eleven residues. When removing the first and last residues of helices, the ratio of kinked helices decreases, showing that part of the kinks are due to distortion at the termini. After correction, the geometry of helices assigned by KAKSI (14.5% of kinked helices) is the closest to the geometry of helices described in the PDB (12% kinked helices). The KAKSI method also assigns the highest ratio of linear helices (12.3%). PSEA has only 7.8% kinked helices but it should be noted that the number of helices submitted to analysis is slightly lower.
It is interesting to investigate the geometry of helices when KAKSI assigns several helices in a region where STRIDE assign a single long helix, i.e., the division events. If we consider the division events involving pair of helices longer than nine residues, we find 128 pairs where a kinked helix assigned by stride corresponds to curved or linear helices assigned by KAKSI. The symmetric case, kinked helices in KAKSI assignment paired with a curved or linear helices in STRIDE assignment concerns only 7 cases. This indicates that splitting long helices into several short ones helps to define helices devoid of kink.
All these observations suggest that the kink detection implemented in KAKSI is efficient and leads to more reliable helix locations. The major feature of KAKSI assignments is then the geometry of α-helices: while assigning slightly longer helices than stride, the global geometry of helices remains satisfactory, with more linear helices than other assignments and a limited ratio of kinked helices, very close to PDB assignments. This is accomplished by dividing long distorted helices when appropriate. Some examples are shown in the following section.
Some examples of assignment disagreements
Figure 5 shows some interesting examples of disagreement between STRIDE and KAKSI assignments. The first three examples in Figure 5 concern disagreement about helix assignments. In example (a), the long helix assigned by STRIDE shows a sharp kink. In KAKSI assignment it is replaced by two helices from residues 4 to 19 and 21 to 34. The first helix is classified as curved by HELANAL. The second one is classified as kinked, but it becomes linear after removal of terminal residues. The angle between two global axes fitted in these two helices is 83°. The second example (b), is even more striking: a 33-residue long helix defined by STRIDE from residues 308 to 340 exhibits a reverse turn near its N-terminal edge. The definition given by KAKSI is two helices from 308 to 315 and 320 to 341. The first helix is too short to be analyzed by HELANAL and the second one is classified as linear. The third example is the case of a division of a long helix assigned by STRIDE into three segments in KAKSI assignment. Although less marked than for the first two examples, the kinks are well apparent. The three helices defined by KAKSI are all classified as curved by HELANAL, with their global axes making angles equal to 135 and 120° between the first and the second, and the second and the third helix respectively.
The last example 5(d) is an example of disagreement on a β-strands assignment. β-strands assigned by STRIDE are fairly curved, allowing a change of direction of the backbone. No specific routine is implemented in KAKSI to split distorted strands, as it is done for helices. Nonetheless, the criteria of β-sheet assignment being fairly strict, some cases of division in long β-strands can also occur. These examples illustrate the fact that a small disagreement on a per-residue basis can result in a radical change in the structure description. In the examples shown on Fig. 5 we believe that KAKSI assignments provide a more pertinent description of the protein structure.
Conclusion
We have developed a new automatic procedure to assign secondary structures from 3D coordinates. Our method, KAKSI, uses Cα distances and (Φ/Ψ) angles and pay a special attention to kink detection in helices. Like other methods (except PSEA), it is sensitive to the resolution, and the type of experimental technique used to solve the structure. Consequently, we propose to choose detection parameters according to the structure resolution or technique and the nature of the secondary structure, since β-sheets are more difficult to detect. The careful comparison of KAKSI assignments with assignments produced by five available methods and the description provided by the PDB highlights the similarities and differences between the different methods. Good general agreement are observed between methods, especially on α-helices. The length of α-helices and β-strands, in case of agreement on the number of segments, are very similar when compared to STRIDE and PSEA. When different lengths are assigned, we observe slightly longer α-helices and β-strands than the STRIDE definition. When two methods disagree on the number of segments, we observe more division events than fusions, i.e., several short helices assigned by KAKSI in front of a unique long helix assigned by STRIDE or PSEA. Division events are also slightly predominant in the comparison of β-strand length with STRIDE and PSEA. The study of α-helix geometry with an external tool reveals that KAKSI helices are less kinked that helices assigned by other methods, except PSEA. KAKSI is also the method that assigns helices with geometrical characteristics in best agreement with helices described in the PDB, and, maybe more important, the highest proportion of linear helices. As stated by Andersen and co-workers [35], each method reflects its own definition of secondary structures. Our definition favors a certain regularity of secondary structure elements, as illustrated by the examples on Fig. 5.
Methods
Datasets
The KAKSI method uses geometrical characteristics of α-helices and β-sheets extracted from available protein structures. A reference set (Ref set), consisting of 2880 structural domains taken from ASTRAL 1.63 [36] is used to estimate these geometrical characteristics. The list of domains with less than 40% identity provided by the ASTRAL server [37] is filtered to keep only X-ray structures with a resolution better than 2.25 Å and longer than 50 residues.
KAKSI assignments are compared with secondary structure assignments done by other methods. For the reasons mentioned above four different sets of structures are used. Hereafter we refer to these datasets as the Comparison sets.
The number of structures reported below refer to the files that are successfully processed by all assignment programs and contain a secondary structure description provided by the PDB.
• A High Resolution set (HRes set): X-ray structures with resolution better than 1.7 Å, R-factor < 0.19, identity percentage between sequences less than 30%, obtained from the WHATHIF website [38,39]. There are 689 structures in this set, corresponding to 151922 residues with a defined secondary structure, i.e., excluding missing coordinates.
• A Medium Resolution set (MRes set): X-ray structures with resolution between 1.7 Å and 3 Å, R-factor < 0.3, identity percentage between sequences less than 30%, minimum length of 40 residues, provided by the PISCES website [40,41]. There are 624 structures in this set, corresponding to 160 276 residues with a defined secondary structure.
• A Low Resolution set (LRes set): X-ray structures with resolution worse than 3 Å, R-factor > 0.3, identity percentage between sequences less than 30%, minimum length of 40 residues, provided by the PAPIA website [42]. There are 332 structures in this set, corresponding to 97852 residues with a defined secondary structure.
• A NMR set: structures with less than 30% sequence identity, extracted from all NMR entries obtained on the PDB website [43]. The redundancy of the set is reduced to 30% sequence identity with PISCES. There are 296 structures in this set, corresponding to 27533 residues with a defined secondary structure.
These lists are available on the web [see Additional file 6].
KAKSI method
The assignment of repetitive secondary structures by KAKSI is based on a set of characteristic values of Cα distances and (Φ/Ψ) dihedral angles. The parameters of KAKSI have been chosen to best fit the secondary structure assignments obtained from the PDB files (HELIX and SHEET fields). These fields, when present, are automatically generated with the DSSP method or are provided by the depositor who might have used some secondary structure assignment program and/or might have inspected visually the 3D structure and assigned himself the secondary structures. We use these PDB assignments as our gold-standard for the sake of parameter calculations, keeping in mind that the data are partly similar to DSSP assignments. Assignment is done by sliding windows along the sequence. α-helices are assigned first, followed by β-sheets. Two windows are slid for the β-sheet detection because we only want to assign β-strands involved in β-sheets. Residues once assigned in α-helix cannot be re-assigned in β-sheets.
Secondary structure characteristics used by the KAKSI heuristic
As mentioned earlier, α-helices and β-strands being periodic structures, their backbone geometry exhibits a number of regularities. This periodicity leads to characteristic distances between Cα atoms as well as characteristic values of (Φ/Ψ) dihedral angles.
More precisely, we have estimated from the Ref set:
• distances between Cα in α-helices and β-sheets. Different statistical distributions are computed for terminal residues and cores of secondary structure segments because greater variations are observed at segment termini. For α-helices, 4 distances are considered between residues i and j along the sequence, with j ∊ [i + 2, i + 5]. Table 1 shows the means and standard deviations obtained on the Ref set. For β-sheets, three different types of distances are considered. Figure 1 illustrates these distances and reports the values obtained on the Ref set.
• (Φ/Ψ) values for residues involved in α-helices and β-strands. Densities of (Φ/Ψ) angles are computed using Ramachandran maps. These maps are divided into 10 by 10 degree squares. This yields two population maps: one specific of α-helices and the other specific of β-strands [see Additional file 7]. For the α-helix map, we only consider angles lying in the area (Φ < 0° and -90° < Ψ < 60°) and we set to zero square frequencies that are too low (frequency <δH). In this study, the threshold δH is fixed, empirically, to 20 × nmean, nmean being the mean frequency for a square in the Ramachandran map.
As mentioned above we are particularly interested in the detection of kinks in α-helices. Kinks are frequent and not easy to detect with usual distance and angle criteria. In a regular helix, (Φ/Ψ) angles should remain located in a narrow region of the Ramachandran map. One way to detect kinks (criterion K1 below), is to compute distances between (Φ/Ψ) pairs of successive residues j and j + 1 in the Ramachandran map. We use the 95-percentile of the distance distribution in α-helices. The kink detection is only performed in helix cores, terminal residues of segments being disregarded in the computation.
KAKSI heuristic for helix and strand assignment
Figure 2 illustrates the heuristic implemented in KAKSI. We have tested several criteria and combinations of criteria. The final heuristic presented here shows a good agreement with PDB assignments. The principle of the assignment is to test the Cα distances along the protein to check if they are close to the typical distances in regular secondary structure. The (Φ/Ψ) angles are tested in the same manner. α-helix assignment is achieved according to a distance or an angle criterion. The β-sheet detection requires the satisfaction of both angle and distance criteria. α-helix assignments are corrected whenever kinks are detected. Criteria applied at each step shown on Figure 2 are explained below, in the order they appear in the assignment process. Characteristic values extracted from the Ref set are shown in capital. The parameters of the method are : εH and εb are used to define thresholds for Cα distances and ηH and σb are used to define thresholds for the constraints on (Φ/Ψ) angles.
• Distance criterion for α-helices (C1). All Cα distances in a sliding window of length w1 (fixed to 6 in this study) must lie within the interval [Mα - ε H × SDα; Mα+ εH × SDα]. Mα and SDα represent the mean and standard deviation of Cα distance distributions in α-helices.
• Angle criterion for α-helices (C2). All (Φ/Ψ) pairs in a sliding window of length w2 (fixed to 4 in this study) must satisfy the condition (Φ < 0° and -90° < Ψ < 60°) and one pair at least must fall in the highly populated zone of the population matrix, i.e with density> δH.
• Kinks in α-helices are detected using two criteria.
- Kink criterion K1 is based on the values of (Φ/Ψ) dihedral angles. A helix is interrupted at residue j + 1 if the sum dΦ/Ψ (j, j + 1) + dΦ/Ψ (j + 1, j + 2) is greater than . dΦ/Ψ (j, j + 1) is analogous to the root mean square deviation on angular value described by Shuchhardt and coll [44]. It measures the distance between dihedral angle pairs of residues j and j + 1 in the Ramachandran map. is the 95-percentile of the distribution of such distances.
- Kink criterion K2 relies on axes. An axis is fitted along the helix, by minimizing the function with n the number of residues in the helix, di the distance from the ith Cα to the axis, and dm the mean of the dis. For a perfect (linear) helix the value of Daxis is zero and the corresponding vector is the axis of the cylinder circumscribed by backbone atoms. A helix is interrupted if it appears better to fit it with two axes. These two axes must make an angle greater than θk (θk fixed to 25° in this study).
• Distance criterion for β-sheets (C3). All the Cα distances in two sliding windows of length w3 (here w3 = 3) must be in the interval [Mβ - εb × SDβ; Mβ + εb × SDβ]. Mβ and SDβ represent the mean and standard deviation of Cα distance distributions in β-sheets.
• Angle criterion for β-sheets (C4). For each (Φ/Ψ) angle pair falling in the populated zone of the Ramachandran map (density > 0), we increment a counter score(sheet) by 1. If a (Φ/Ψ) angle pair of the central residue of a sliding window verifies -120° < Ψ < 50°, then score(sheet) is reset to zero. The final score(sheet) must be greater or equal to σ b.
• Contiguous segments correction, criterion (C5). If a helix and a strand are adjacent, a coil is introduced in between, shortening the helix by one residue.
Empirically, the optimal parameter values are: εH = 1.96, ηH = 2.25, εb = 2.58 and σb = 5.
Comparative methods for secondary structure assignment and reduction to three states
KAKSI assignments are compared to the assignments given by five available methods on the Comparison sets: DSSP [21], STRIDE [23], PSEA [27], XTLSSTR [28] and SECSTR [24]. HELIX and SHEET records in PDB files are also considered as an independent assignment method.
When needed, secondary structure assignments are reduced to three classes (H for α-helix, b for β-strand, c for coil) as follows: DSSP, STRIDE and SECSTR: (H,G,I) = H, (E,b) = b, others (S,T,blank) = c; XTLSSTR: (G,g,H,h) = H, (E,e) = b, others (T,N,P,p,-) = c. PSEA assigns only three states. XTLSSTR possibly provides several alternative assignments for one residue. In that case, only the first assignment is considered. When dealing with NMR structures, only the first model is analyzed.
Comparison measures
Secondary structure content
The secondary structure content of a dataset is measured by the percentage of residues involved in the three structural classes: α-helix, β-strand and coil.
Overall agreement
The C3 score is the percentage of residues assigned in the same state when comparing two different assignments: C3 = Nid/Ntot with Nid the number of residues for which both assignments are identical, and Ntot the total number of residues with defined secondary structure. It is analogous to the Q3 score used to evaluate secondary structure prediction.
Segment based-agreement
• The mean agreement based on secondary structure segments is measured by the percentage of Segment OVerlap (SOV). We use the SOV definition described by Zemla and coworkers [45]. For state i (α-helix, β-strand or coil) the segment overlap measure is defined as:
with the normalization value N(i) defined as:
The sums on S(i) are taken over all the segment pairs in state i which overlap by at least one residue. The sum on S'(i) is taken over the remaining segments in state i found in the reference assignment 1, len(s1) is the number of residues in segment s1, minov(s1, s2) is the length of overlap of s1 and s2, maxov(s1, s2) is the total extend for which either of the segments S1 and s2 has a residue in state i, and delta(s1, s2) is defined as:
min {maxov(s1, s2) - minov(s1, s2); minov(s1, s2); int(len(s1)/2); int(len(s2)/2)},
where min {x1; x2; x3;...; xn} is the minimum of n integers. This formula is usually employed to compare a secondary structure prediction (S2) with a secondary structure description (S1) taken as reference. The roles of S1 and S2 are thus not symmetrical.
• Length of pair of segments used for the SOV computation are collected. A pair is defined each time there is at least one residue in common between assignment X and Y. Unpaired secondary structure elements are ignored in this analysis. These length pairs can be viewed on a bi-plot (length(X) versus length(Y)).
Helix geometry analysis with an external software
The HELANAL software developed by Kumar and Bansal [33] is dedicated to helix geometry analysis. HELANAL takes as input a PDB file and a description of helix boundaries. It calculates local axes every four residues. The geometry of a helix is determined by the angles between axes and the goodness of fit of the helix trace with a circle or a line. Helices are then classified as kinked (K), linear (L) or curved (C). HELANAL can leave a helix unclassified if its geometry is ambivalent. The minimum length for a helix to be analyzed is nine residues.
In this study, HELANAL is used as an external control of helix geometry. All α-helices in the comparison sets are submitted to HELANAL analysis. Different assignment methods are used to provide alternate definition of helices boundaries.
Availability and requirements
• Project name: KAKSI
• Project home page: http://migale.jouy.inra.fr/mig/mig_fr/servlog/kaksi/
• Operating system: Linux
• Programming langage: C
• Other requirements: libxml2 >= 2.6, see ftp://xmlsoft.org/
• License: GNU GPL
• Any restrictions to use by non-academics: no
• Implementation: the software is composed of 2 programs: KAKSI takes a PDB file as input and prints the assigned secondary structure (and other data of intereset) in an XML output K2R reads a KAKSI XML output file and outputs the data in various FASTA format files by default. K2R allows users to easily implement any new output format they whish. a lot of different informations in raw formats (mainly FASTA format).
The source code is available on the project home page.
List of abbreviations used
3D: three-dimensional, Cα: backbone α-carbon, NMR: Nuclear Magnetic Resonance, PDB: Protein Data Bank.
Authors' contributions
JM and AM developped the program. GL carried out the comparison between different assignments. JM GL and JFT carried out the analysis. JM, AdB and JFG conceived the study and participated in its design and coordination
Supplementary Material
Additional File 1
C3 scores for all datasets.
Click here for file
Additional File 2
Graphical views of C3 scores for the HRes set.
Click here for file
Additional File 3
SOV scores for all datasets.
Click here for file
Additional File 4
Length of pairs of helices and strands on separate plots.
Click here for file
Additional File 5
Helix geometry analysis on all datasets.
Click here for file
Additional File 6
Urls to retrieve the list of structures used in this study.
Click here for file
Additional File 7
Φ/Ψ repartition in helices and strands defined by the PDB.
Click here for file
Acknowledgements
This research was funded in part by the 'ACI Masse de données'. We are grateful to INRA for awarding a doctoral Fellowship to JM and to the Ministère de l'Education Nationale, de l'Enseignement supérieur et de la Recherche for awarding a doctoral Fellowship to JFT.
Figures and Tables
Figure 1 Typical Cα distance in β-sheets. Typical Cα distances computed from the Ref set in parallel (left part) and anti-parallel β-sheets. Mean distances are indicated in Å with their standard deviations within parentheses. Separate statistics were computed for distances involving only residues in strand cores (italic) and distances involving residues at strand edges (bold). For the intra-strand distance (type i to i + 2), no distinction is made on the sheet orientation.
Figure 2 Flow-chart of the kaksi heuristic for secondary structure assignment. Minimum length for helices is set to LH = 5. The criteria C1, C2, C3, C4, C5, K1 and K2 are detailed in the text.
Figure 3 Length distribution of helices and strands assigned by stride, psea and kaksi. Length distribution of helical (top) and extended (bottom) segments assigned by STRIDE (plain line and crosses), PSEA (dashed line and open circles), and KAKSI (dotted line and filled circles), on the HRes set. The STRIDE assignment generates a large number of 3 residue-long helices (1238 segments) and 1 residue-long strands (corresponding to 1800 β-bridges).
Figure 4 Length for pair of segments assigned by stride vs kaksi and psea vs kaksi. Length for pair of helices (upper part) and strands (lower part) when comparing STRIDE and KAKSI assignments, and PSEA and KAKSI assignments. We report a pair when we found at least one residue in the same state in both assignments. Data are shown as a "sunflower plot": a point stands for a single observation, then the number of "leaves" is proportional to the number of additional observations. The diagonal x = y (same length for two assignments) is shown.
Figure 5 Examples of disagreement between kaksi and stride. The divergent assignments are drawn in cartoon representation and highlighted in purple (helix and strand) and cyan (coil assigned by KAKSI). Images are generated with Molscript [46]. Average bending angles (AverBA) between local axes computed by HELANAL in long helices are reported, (a): hemoglobin I from the clam Lucina pectinata, PDB code:1b0b, resolution 1.43 Å. STRIDE assignment: α-helix from residues 4 to 35, AverBA = 15.4°. KAKSI assignment: two helices from 4 to 19, AverBA = 3.84° and 21 to 34, AverBA = 9.0°. (b): chain A of L(+)-mandelate dehydrogenase from Pseudomonas putida, PDB code: 1p4c, resolution 1.35 Å. STRIDE assignment: helix from 308 to 340, AverBA = 24.7°. KAKSI assignment: two helices from 308 to 315 and 320 to 341, AverBA = 4.3°. (c): chain B of C-phycocyanin from the thermophylic cyanobacterium Synechococcus elongatus, PDB code: 1jbo, resolution: 1.45 Å. STRIDE assignment: helix from residues 21 to 62, AverBA = 13.1°. KAKSI assignment: 3 helices from 21 to 33, AverBA = 4.5°, 35 to 46, AverBA = 3.0°, and 48 to 61, AverBA = 6.6°. (d): chain A from endo-xylanase from Clostridium stercorarium, PDB code: 1od3, resolution: 1 Å. STRIDE assignment: two β-strands from 61 to 82 and 116 to 135. KAKSI assignment: four β-strands from 61 to 69, 75 to 83, 115 to 122, and 128 to 136.
Table 1 Distances in α-helices. Core: distances not involving residues at helix edge. Termini: distances involving at least one residue at helix edge. Mean distances, computed on the HRes set, are indicated in Å with their standard deviations within parentheses.
Type Core Termini
i to i + 2 5.49(0.20) 5.54(0.25)
i to i + 3 5.30(0.64) 5.36(0.39)
i to i + 4 6.33(0.71)
i to i + 5 8.72(0.63)
Table 2 Secondary structure content according to different assignment methods. %H: percentage of residues assigned in α-helix. %b: percentage of residues assigned in β-strand. See the text for β-strand assignment with kaksi using different parameter values on the LRes and the NMR sets.
Dataset HRes set MRes set LRes set NMR set
Method %H %b %H %b %H %b %H %b
KAKSI 36.8 22.0 38.0 22.5 35.1 19.0 33.5 15.2
PDB 40.5 20.3 41.7 20.9 39.3 18.2 35.5 17.3
DSSP 35.9 22.5 37.3 22.9 35.4 20.4 32.2 17.3
STRIDE 36.4 22.6 38.6 23.3 36.3 21.2 33.7 18.8
PSEA 32.1 23.7 34.2 25.0 33.0 24.4 30.6 22.8
SECSTR 37.2 20.1 38.5 20.4 37.0 18.6 33.3 16.3
XTLSSTR 40.4 19.7 40.9 19.6 35.9 14.4 34.3 14.8
Table 3 C3 scores between different methods on the HRes set
DSSP STRIDE PSEA SECSTR XTLSSTR PDB
KAKSI 82.1% 83.5% 81.5% 81.7% 78.3% 83.4%
DSSP 95.4% 80.1% 93.4% 80.4% 90.8%
STRIDE 81.1% 91.9% 80.8% 89.9%
PSEA 79.8% 75.8% 78.1%
SECSTR 79.6% 87.4%
XTLSSTR 80.7%
Table 4 SOV measures between kaksi and other methods on the HRes set. SOVH: SOV for α-helix. SOVb: SOV for β-strand. KAKSI is taken as reference.
Method SOV
H
SOV
b
DSSP 91.7% 92.1%
STRIDE 91.2% 91.9 %
SECSTR 89.0% 83.9%
PSEA 87.5% 82.7%
XTLSSTR 89.3% 73.4%
PDB 88.4% 89.4%
Table 5 Helix geometry analyzed by HELANAL on the HRes set. Correction: assignments are corrected by shortening each helix by one residue at each terminus. %L: percentage of helices that are linear according to HELANAL. %C: percentage of helices that are curved according to HELANAL. %K: percentage of helices that are kinked according to HELANAL. N: number of helices submitted to HELANAL.
Method No correction With Correction
Minimum length 11 9 after correction
%L %C %K N %L %C %K N
DSSP 8.3 70.0 21.2 2215 10.9 70.8 17.8 2215
STRIDE 10.1 65.9 23.6 2431 10.8 68.5 20.2 2431
PSEA 10.9 78.5 10.0 2260 11.5 80.0 7.8 2260
SECSTR 8.0 55.7 36.0 2349 10.0 59.7 29.9 2349
XTLSSTR 8.7 58.9 32.1 2618 9.5 61.4 28.9 2618
KAKSI 10.2 66.5 22.8 2442 12.3 72.6 14.5 2442
PDB 11.4 71.1 17.0 2565 11.3 71.5 12.0 2565
==== Refs
Pauling L Corey RB Branson HR The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain Proc Natl Acad Sci USA 1951 37 205 211 14816373
Pauling L Corey RB The pleated sheet, a new layer configuration of polypeptide chains Proc Natl Acad SciU S A 1951 37 251 256
Gibrat JF Madej T Bryant SH Surprising similarities in structure comparison Curr Opin Struct Biol 1996 6 377 385 8804824 10.1016/S0959-440X(96)80058-3
Murzin AG Brenner SE Hubbard T Chothia C SCOP: a structural classification of proteins database for the investigation of sequences and structures J Mol Biol 1995 247 536 40 7723011 10.1006/jmbi.1995.0159
Orengo CA Michie AD Jones S Jones DT Swindells MB Thornton JM CATH-a hierarchic classification of protein domain structures Structure 1997 5 1093 1108 9309224 10.1016/S0969-2126(97)00260-8
Sayle RA Milner-White EJ RASMOL: biomolecular graphics for all Trends Biochem Sci 1995 20 374 7482707 10.1016/S0968-0004(00)89080-5
Humphrey W Dalke A Schulten K VMD: visual molecular dynamics J Mol Graph 1996 14 33 38 27-28. 8744570 10.1016/0263-7855(96)00018-5
Sali A Blundell TL Comparative protein modelling by satisfaction of spatial restraints J Mol Biol 1993 234 779 815 8254673 10.1006/jmbi.1993.1626
Bradley P Chivian D Meiler J Misura KM Rohl CA Schief W R Wedemeyer W Schueler-Furman O Murphy P Schonbrun J Strauss C Baker D Rosetta predictions in CASP5: successes, failures, and prospects for complete automation Proteins 2003 53 457 468 14579334 10.1002/prot.10552
Pollastri G Przybylski D Rost B Baldi P Improving the prediction of protein secondary structure in three and eight classes using recurrent neural networks and profiles Proteins 2002 47 228 235 11933069 10.1002/prot.10082
Petersen TN Lundegaard C Nielsen M Bohr H Bohr J Brunak S Gippert GP Lund O Prediction of protein secondary structure at 80% accuracy Proteins 2000 41 17 20 10944389 10.1002/1097-0134(20001001)41:1<17::AID-PROT40>3.0.CO;2-F
Jones DT Protein secondary structure prediction based on position-specific scoring matrices J Mol Biol 1999 292 195 202 10493868 10.1006/jmbi.1999.3091
Frishman D Argos P The future of protein secondary structure prediction accuracy Fold Des 1997 2 159 62 9218953 10.1016/S1359-0278(97)00022-9
Rost B Review: protein secondary structure prediction continues to rise J Struct Biol 2001 134 204 218 11551180 10.1006/jsbi.2001.4336
Schulz GE Barry CD Friedman J Chou PY Fasman GD Finkelstein AV Lim VI Pititsyn OB Kabat EA Wu TT Levitt M Robson B Nagano K Comparison of predicted and experimentally determined secondary structure of adenyl kinase Nature 1974 250 140 2 4367211 10.1038/250140a0
Robson B Garnier J Introduction to Proteins and Protein Engineering 1986 Amsterdam: Elsevier Press
Barlow DJ Thornton JM Helix geometry in proteins J Mol Biol 1988 201 601 619 3418712 10.1016/0022-2836(88)90641-9
Kumar S Bansal M Structural and sequence characteristics of long alpha helices in globular proteins Biophys J 1996 71 1574 1586 8874031
Kumar S Bansal M Geometrical and sequence characteristics of alpha-helices in globular proteins Biophys J 1998 75 1935 1944 9746534
Levitt M Greer J Automatic identification of secondary structure in globular proteins J Mol Biol 1977 114 181 239 909086 10.1016/0022-2836(77)90207-8
Kabsch W Sander C Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features Biopolymers 1983 22 2577 637 6667333 10.1002/bip.360221211
Berendsen HJC van der Spoel D van Drunen R GROMACS: A message-passing parallel molecular dynamics implementation Comp Phys Comm 1995 91 43 56 10.1016/0010-4655(95)00042-E
Frishman D Argos P Knowledge-based protein secondary structure assignment Proteins 1995 23 566 579 8749853 10.1002/prot.340230412
Fodje MN Al-Karadaghi S Occurrence, conformational features and amino acid propensities for the pi-helix Protein Eng 2002 15 353 358 12034854 10.1093/protein/15.5.353
Richards FM Kundrot CE Identification of structural motifs from protein coordinate data: secondary structure and first-level supersecondary structure Proteins 1988 3 71 84 3399495 10.1002/prot.340030202
Sklenar H Etchebest C Lavery R Describing protein structure: a general algorithm yielding complete helicoidal parameters and a unique overall axis Proteins 1989 6 46 60 2608659 10.1002/prot.340060105
Labesse G Colloc'h N Pothier J Mornon JP P-SEA: a new efficient assignment of secondary structure from C alpha trace of proteins Comput Appl Biosci 1997 13 291 5 9183534
King SM Johnson WC Assigning secondary structure from protein coordinate data Proteins 1999 3 313 320 10.1002/(SICI)1097-0134(19990515)35:3<313::AID-PROT5>3.0.CO;2-1
Dupuis F Sadoc JF Mornon JP Protein secondary structure assignment through Voronoi tessellation Proteins 2004 55 519 528 15103616 10.1002/prot.10566
Berman HM Westbrook J Feng Z Gilliland G Bhat TN Weissig H Shindyalov IN Bourne PE The Protein Data Bank Nucleic Acids Res 2000 28 235 242 10592235 10.1093/nar/28.1.235
PDB Format Description Version 2.2
Colloc'h N Etchebest C Thoreau E Henrissat B Mornon JP Comparison of three algorithms for the assignment of secondary structure in proteins: the advantages of a consensus assignment Protein Eng 1993 6 377 382 8332595
Bansal M Kumar S Velavan R HELANAL: a program to characterize helix geometry in proteins J Biomol Struct Dyn 2000 17 811 819 10798526
Fourrier L Benros C de Brevern AG Use of a structural alphabet for analysis of short loops connecting repetitive structures BMC Bioinformatics 2004 5 58 15140270 10.1186/1471-2105-5-58
Andersen C Rost B Bourne PE, Weissig H Automated Secondary Structure Assignment Structural Bioinformatics 2003 Hoboken: Wiley-Liss 341 363
Brenner SE Koehl P Levitt M The ASTRAL compendium for protein structure and sequence analysis Nucleic Acids Res 2000 28 254 256 10592239 10.1093/nar/28.1.254
ASTRAL website
Hobohm U Scharf M Schneider R Sander C Selection of a representative set of structures from the Brookhaven Protein Data Bank Protein Science 1992 1 409 417 1304348
WHATHIF website
PISCES website
Wang G Dunbrack RLJ PISCES: a protein sequence culling server Bioinformatics 2003 19 1589 1591 12912846 10.1093/bioinformatics/btg224
PAPIA website
PDB website
Schuchhardt J Schneider G Reichelt J Schomburg D Wrede P Local structural motifs of protein backbones are classified by self-organizing neural networks Protein Eng 1996 9 833 842 8931122
Zemla A Venclovas C Fidelis K Rost B A modified definition of Sov, a segment-based measure for protein secondary structure prediction assessment Proteins 1999 34 220 223 10022357 10.1002/(SICI)1097-0134(19990201)34:2<220::AID-PROT7>3.0.CO;2-K
Kraulis PJ MOLSCRIPT: A Program to Produce Both Detailed and Schematic Plots of Protein Structures J Applied Crystallogr 1991 24 946 950 10.1107/S0021889891004399
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==== Front
Genet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central London 1479-0556-3-71611531910.1186/1479-0556-3-7ResearchA combined nucleocapsid vaccine induces vigorous SARS-CD8+ T-cell immune responses Azizi Ali [email protected] Susan [email protected] Helina [email protected] Rita [email protected] Masoud [email protected] Catalina [email protected] Turaya [email protected] Francisco [email protected] Infectious Disease and Vaccine Research Centre, Children's Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Ottawa, ON, K1H 8L1, Canada2 Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, K1H 8M2, Canada2005 22 8 2005 3 7 7 9 5 2005 22 8 2005 Copyright © 2005 Azizi et al; licensee BioMed Central Ltd.2005Azizi 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.
Several studies have shown that cell-mediated immune responses play a crucial role in controlling viral replication. As such, a candidate SARS vaccine should elicit broad CD8+ T-cell immune responses. Several groups of mice were immunized alone or in combination with SARS-nucleocapsid immunogen. A high level of specific SARS-CD8+ T-cell response was demonstrated in mice that received DNA encoding the SARS-nucleocapsid, protein and XIAP as an adjuvant. We also observed that co-administration of a plasmid expressing nucleocapsid, recombinant protein and montanide/CpG induces high antibody titers in immunized mice. Moreover, this vaccine approach merits further investigation as a potential candidate vaccine against SARS.
VaccineSARSNucleocapsidXIAP
==== Body
Introduction
The SARS epidemic had a high mortality rate as well as a huge economic impact worldwide. Treatment with antiviral drugs or an effective vaccine is not available for protection against this disease [1,2]. The SARS-CoV is a single-stranded RNA virus that has been identified as a new type of coronavirus. The genome is approximately 30 kb long and contains four structural proteins: spike, envelope, matrix and nucleocapsid in the same order as other coronaviruses [3,4]. However, the sequence analysis of SARS-CoV with other members of the coronavirus family did not show more than 20% nucleotide homology [5].
The SARS-NC gene encodes a 46 kDa protein that participates in the replication and transcription of the virus and interferes with the cell cycle of host cells [6]. Previous studies in other coronavirus members suggest that this protein is highly immunogenic and could be a good target for the design of an effective vaccine [7-10]. The expression of NC in CHO cells led to the observation that this protein folds spontaneously into viral-like particles (VLPs). These particles are effectively incorporated at several stages of the virus life cycle, including assembly, budding from cells, and receptor-binding leading to membrane fusion. The viral particles also present antigens to the immune system in a structure that mimics the infectious virion [11-13].
DNA vaccines are able to induce both humoral and cellular immune responses and have demonstrated their efficacy in several experimental models [14,15]. There are several eukaryotic vectors that express recombinant proteins efficiently. However, the uptake of antigens and its presentation are critical elements in DNA vaccination strategies. One strategy to increase the potency of DNA vaccines is to prolong the survival of antigen presenting cells (APCs), especially dendritic cells. Previous studies show that survival of dendritic cells is increased in the presence of anti-apoptotic factors such as XIAP. This approach has results in increased amounts of antigen-specific CD8+ T cells [16,17].
Specific CD8+T-cells play an important role in the control of viral infection [18-20]. Activation of specific CD8+ cells results in the secretion of inflammatory cytokines (IFN-γ and TNF-α) [21] and the synthesis of effector molecules, such as perforin and granzymes which kills infected cells, decreasing virus replication and virus load [22,23].
The present study characterizes cellular and humoral immune responses to SARS-CoV in mice receiving a DNA-NC construct alone or in combination with protein and different adjuvants. The combination of DNA-NC, protein and XIAP elicited a significant anti-SARS CD8+ T-cell response independent of CD4+ T-cell immune responses.
Materials and methods
Cell culture
Chinese Hamster Ovary (CHO) cells were grown at 37°C, 5% CO2 in Iscove's Modified Dulbecco's Medium (IMDM: Sigma, St. Louis, MO) and supplemented with 10% Fetal Calf Serum (FCS: Life Technologies, Grand Island, NY), 100 U/ml penicillin and 100 μg/ml gentamyin.
Construction of DNA plasmids
Total RNA was purified using an RNeasy extraction kit (Qiagen, Mississauga, Ont) from the lung tissue of an autopsied patient who died from SARS. The full-length NC (1.2 kb) gene was amplified using specific primers (forward primer: 5'-ggatccatgtctgataatggaccc-3'; reverse primer: 5'-gaattcttatgcctgagttgaatc-3'). The amplicon was purified using the QIAquick gel extraction kit (Qiagen) and cloned into the PCR 2.1 TOPO-TA vector (Invitrogen, Burlington, Ont) according to the manufacturer's instructions. After plasmid digestion, the 1.2 kb band corresponding to the NC gene was sub-cloned into BamHI-and EcoRI sites of pVAX-1 which contains a CMV promoter for high level expression in vivo. The fragment was also sub-cloned into the pEF6-Myc/His (Invitrogen) and pQE (Qiagen) vectors. The pEF6 vector was designed to over produce recombinant proteins in mammalian cell lines and was used to establish a stable cell line by using a resistant blasticidine gene. The pQE Tri system vector was used for production of proteins in bacteria. These vectors ultimately allow for the purification of the protein by immobilized metal affinity chromatography. All expression constructs were confirmed and characterized by restriction enzymes and nucleotide sequence analysis.
Expression of recombinant nucleocapsid protein
CHO cells and JM109 bacteria were transfected with pEF6 and pQE vectors containing NC or vector alone. In order to increase and sustain expression of the NC protein, a stable NC expressing CHO cell line was established using blasticidine-supplemented medium. Cells were harvested, sonicated and lysed in lysis buffer (25 mM Tris base, 2.5 mM Mercaptoethanol, 1% Triton-X100 and a cocktail of protease inhibitors). Cell pellets were centrifuged and the supernatant was incubated with the TALON metal resin (Clontech, Palo Alto, CA) for one hour. After incubation, the mixture of protein-resin was added to the columns and washed three times with 20 bed volumes of Tris-Cl, NaCl (PH 8). The recombinant protein was eluted with 150 mM imidazole.
To confirm the proteins, samples were mixed with Laemmli loading buffer, boiled for 5 minutes and loaded on a 10% polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane by electrophoretic transfer. The membranes were blocked with 5% dried milk in PBS-Tween 20 (PBS-T) and incubated with 1/3000 dilution of a sera from a SARS patient for three hours at room temperature. After washing with PBS-Tween, the blots were incubated with anti-human IgG-HRP conjugate (BioRad, Hercules, CA) for one hour at room temperature. After incubation, the blots were washed and incubated with ECL reagent (Santa Cruz Biotechnology, Santa Cruz, CA) for one minute and exposed to X-ray film (Kodak).
Electron Microscopy
CHO cells transfected with either the DNA vector expressing NC or DNA vector alone were harvested and washed with PBS. Pellets were then fixed with 2% glutaraldehyde. Cells were rinsed twice in 0.1 M sodium cacodylate buffer at 4°C. The cells were then fixed with 2% Osmium Tetroxide for 2 hr at 4°C. After washing with distilled water, the cells were dehydrated with increasing concentrations of ethanol and embedded in spur resin. Thin sections were stained with uranyl acetate and lead citrate. The sections were screened by using a JEOL 1010 Transmission Electron Microscope (TEM).
Adjuvants
CpG oligodeoxynucleotide (5'-TCCATGACGTTCCTGACGTT-3') was provided by Coley (Ottawa, ON). Montanide ISA-51 mineral oil adjuvant was purchased from Seppic Inc. (Paris, France). The pcDNA3 construct expressing 1.5 kb XIAP gene encoding an anti-apoptotic gene product was a kind gift from Dr. R.G. Korneluk [24].
Animal Immunization
Six to eight week-old female B6/C3/F1 mice (Charles River, St. Constant, PQ) were immunized subcutaneously at the base of the tail with 50 μg of DNA construct expressing the nucleocapsid gene, 5μg of nucleocapsid protein and 50 μl of montanide ISA-51 (Seppic)/30 μg CpG (Coley), or 50 μg pcDNA3-XIAP at each vaccination. Each mouse was boosted three times, at one month intervals. Fourteen days after the last boost, the mice were sacrificed and their spleens and blood was collected for further testing or for long-term storage in cryopreservation medium.
Antibody measurement by ELISA
96 well ELISA plates were coated overnight at 4°C with NC protein, and the wells were washed with PBS containing 0.05% Tween 20 and then blocked with 1% BSA in PBS. Serially diluted sera was added and incubated for 2 h at 37°C. The plates were washed and incubated for 2 h with a 1/2000 dilution of a peroxidase-conjugated affinity-purified rabbit anti-mouse secondary antibody (Bio-Rad, Richmond, CA). The plates were washed three times and developed with O-phenylendiamine dihydrochloride (OPD) substrate (Sigma, St. Louis, MO). The color reaction was stopped with 1N HCl and absorbance was read at 490 nm with an ELISA plate reader (Bio-Rad).
Proliferation assay
Splenocytes from immunized mice were resuspended at 2 × 106 cells/ml in RPMI 1640 containing 10% FCS, 50 μM β-mercaptoethanol and 100 U/ml penicillin/streptomycin. A 100 μl aliquot containing 2 × 105 cells was added to each well of a 96 well plate. The NC protein (100 μl at 20μg/ml) was added to each well in triplicate. As a positive control, cells were also stimulated with phorbol 12-myristate 13-acetate and ionomycin (PMA/ION). After 72 h of culture, 1 μCi [3H] thymidine (Amersham, Arlington Heights, IL) was added to each well. Following 16 h of incubation, cells were harvested onto glass fibre filtermats and thymidine incorporation was measured with a Microbeta beta counter (Wallac, Turku, Finland).
Intracellular cytokine staining
Fresh blood and splenocytes from immunized mice were cultured in IMDM media in the presence of 10 μg/ml brefeldin A (Sigma) and stimulated in vitro with the NC protein (10 μg/ml) expressed in bacteria. In every experiment, a negative control (without stimulation), positive control (PMA/ION) and an irrelevant protein (HIV-1 gp120 protein) was included to control for spontaneous production of IFN-γ. Sixteen hours after incubation, the cells were washed once (1600 rpm for 5 min) with 3 ml PBS / 2% FCS / 0.01% Azide and surface-stained for 15 min with PE-labeled Ab to mouse CD3, TC-labeled Ab to mouse CD8α or CD4 (Caltag Laboratories, Hornby, ON). The cells were washed as above, fixed and permeabilized using 100 μl each of A and B fixation-permeabilization solution (Caltag Laboratories). The cells were stained intracellularly with anti-mouse IFN-γ FITC-labeled Ab and incubated for 30 min (in the dark) at 4°C. Following washing, cells were analyzed by FACScan (Becton Dickinson, Mississauga, ON). An increase of 0.1% of IFN-gamma producing cells over the unstimulated control was considered as positive response to vaccination.
ELISPOT assay
Multiscreen-HTS plates (Millipore, Bedford, MA) were coated with 10 μg/ml of anti-mouse IFN-γ antibody (mAb AN18, Mabtech, Mariemont, OH) in PBS over night at 4°C. The plates were then washed with PBS and blocked with IMDM containing 10% FCS and 100 U/ml penicillin/streptomycin for 1 h at room temperature. The medium were removed and 4 × 105 cell suspension (100 μl/well) including NC SARS protein expressed in bacteria (10 μg/ml) or irrelevant antigens at the same concentration were added and incubated for 30 h at 37°C. After incubation, cells were removed; plates were washed with PBS+0.05% Tween 20 and incubated with 1 μg/ml of biotinylated anti-mouse IFN-γ antibody (mAb R4-6A2-Biotin, Mabtech) for 2 hr at room temperature. After further washings, 100 μl/well of 1/2000 Streptavidin-ALP-PQ (Mabtech) in PBS+ 0.5% FCS was added and incubated for 1 hr at room temperature. The plates were washed as above and developed with 100 μl per well BCIP/NBT alkaline phosphatase (Moss Inc) for 20 minutes at room temperature. The reaction was stopped with rinsing the plates with tap water. The numbers of spots were analyzed with an ELISPOT reader.
Statistical analysis
Results were expressed as mean ± S.D. In each experiment four animals were used per group. The t-test was applied for the statistical analysis of the data. The p value equal to or less than 0.05 was considered significant.
Results
Construction of the DNA vectors and expression of SARS-nucleocapsid protein in mammalian and bacteria cells
To increase the potency of the specific immune response, the full-length NC was amplified by RT-PCR and ligated into plasmid pVAX-1 under the control of the human cytomegalovirus promoter. For the expression and purification of the recombinant NC protein in CHO and bacteria cells, the amplified NC gene was also sub-cloned into pEF6-Myc/His and pQE-Tri system vectors. To express NC protein, CHO cells and E.coli (JM109) were transfected with pEF6 and pQE vectors encoding the NC gene, respectively. To increase the yield of the recombinant protein, a stable CHO cell line was created using a selective resistant blasticidine gene, allowing for efficient purification of the recombinant protein. Cells were harvested, lysed and the recombinant proteins were purified according to standard methods. The expression of the NC protein in transfected cells was verified by western blotting (Fig 1) and immunofluorescence staining of CHO cells infected with the vector-NC or the vector alone. Antibody raised in rabbits to the NC protein expressed in bacteria reacted strongly in the perinuclear region of the SARS-NC-CHO cell line (data not shown).
Figure 1 Western blot analysis of recombinant SARS-CoV-NC protein. Lane 1: purified protein from JM 109 cells transfected with pQE vector encoding nucleocapsid gene. Lane 2: represents cells transfected with the vector alone. The blot was probed with sera from a SARS patient.
The assembly of NC protein into virus like particles (VLPs)
The CHO cells transfected with pEF6-NC or vector alone were examined by transmission electron microscopy. We observed bundles of VLP of the same morphology as wild type particles both inside and outside cells infected with pEF6-NC. However, neither the mock-transfected cells nor the cells transfected with the vector alone showed viral-like particles (Fig 2). These observations demonstrate that our construct expressing the NC protein synthesised sufficient protein within infected cells to facilitate the formation of VLPs.
Figure 2 Production of viral-like particles shown by electron microscopy. The CHO cells were transfected with DNA-NC or vector alone. Arrows indicate VLPs in the transfected cell lines.
Detection of antibody titer in mice immunized with the candidate vaccine combinations
In order to analyze the antibody titer against NC, five groups of mice were primed and boosted with SARS-nucleocapsid immunogen alone or in combination. Two weeks after the last boost, sera were collected and antibody titer was measured by ELISA. The group received protein and montanide/CpG showed a higher mean IgG antibody titer compared to the group receiving vector DNA+XIAP and DNA-NC alone. This group (NC protein + montanide/CpG) also showed a slightly higher antibody titer compared to the group received DNA-NC + NC protein and XIAP. However, the highest SARS-CoV-specific antibody response was detected in mice immunized with a combination of DNA-NC, protein and montanide/CpG (Fig 3).
Figure 3 Antibody titers were determined in mice (n = 4) two weeks after the last immunization. The 96-well plates were coated with SARS-NC protein and mouse sera were serially diluted in wells for the endpoint titration of anti-NC antibody. Results are shown as mean concentration ± S.D. The symbol * indicates a significant difference (P = 0.01–000.1) compared with all other groups. The symbol † indicates a significant difference (P ≤ 0.001) when compared to animals immunized with DNA+XIAP and DNA-NC alone.
Combination of DNA, recombinant protein and XIAP induce higher level of CD8+T-cell immune responses
To assess whether vaccination with nucleocapsid increases cell-mediated immune responses, splenocytes and fresh blood from immunized mice was retrieved, stimulated, and stained for surface CD4 and CD8+T cells as well as intracellular interferon gamma. The level of IFN-γ producing CD4+ T cell in fresh blood (Fig 4) and splenocytes (data not shown) from immunized mice did not demonstrate a significant CD4+T cell response against the SARS-NC protein. However, the group receiving DNA-NC, protein and montanide/CpG demonstrated higher levels of IFN-γ producing CD4+ T cells.
Figure 4 SARS-CoV-NC specific CD4+ T cell responses in mice immunized with the candidate vaccines. Fresh peripheral blood cells were cultured, stimulated with NC protein and stained for CD4, CD3 and IFN-γ. Flow cytometry was used to analyse the NC-specific CD4+T cell response. A negative control (without stimulation) and a positive control (phorbol myristate acetate + ionomycin) were included to control for the spontaneous production of IFN-γ(data not shown). Results are shown as mean ± S.D. The symbol † indicates a significant difference (P < 0.05) compared with the control group (DNA+XIAP).
Splenocytes were also stimulated with NC protein, and CD4 lymphocyte proliferation was performed with tritiated thymidine. However, a high T-cell proliferation was not detected with this assay (data not shown).
Cell-mediated immune responses were evaluated by intracellular cytokine staining. The group receiving DNA-NC + NC protein and Montanide/CpG elicited higher levels of CD8+T-cells to nucleocapsid in comparison to groups receiveing DNA-NC or NC protein plus adjuvant. However, the highest NC-specific CD8+T-cell response was detected in both splenocytes (data not shown) and fresh blood (Fig 5) in mice that received the DNA construct, recombinant NC protein and adjuvant XIAP.
Figure 5 SARS-CoV-NC specific CD8+ T cell responses in mice immunized with the candidate SARS vaccines. Fresh peripheral blood cells from immunized mice were stimulated with various antigens and stained for CD8, CD3 and IFN-γ with labeled monoclonal antibodies. After staining, flow cytometry was used to analyze the NC-specific CD8+ T cells. A negative control (without stimulation) and a positive control (phorbol myristate acetate + ionomycin) were included to control for the spontaneous production of IFN-γ. Cells were also stimulated with an irrelevant protein, HIV-1 gp120 (data not shown). A: Dot plots show results from individual representative animals from each group of mice. B: Results are shown as mean ± S.D. The symbol * indicates a significant difference (P = 0.01–000.1) compared to all other immunized groups. The symbol † indicates a significant difference (P = 0.026) compared to the control group.
To confirm the results obtained by intracellular cytokine staining, we performed an IFN-γ ELISPOT assay to measure NC-specific T-cell responses of splenocytes from immunized mice. The groups DNA+XIAP, DNA-NC and NC protein + montanide/CpG did not show a high number of spot forming cells (SFC). Potent IFN-γ responses were observed in mice immunized with combination of DNA-NC+NC protein and adjuvants (Fig 6). However, following substitution of adjuvant montanide/CpG with XIAP, SFCs were more than two fold higher (p = 0.01). Although, IFN-γ may be produced by both antigen-stimulated CD4+ and CD8+ T cells, most likely the observed IFN-γ response was generated by effector CD8+ T-cells, since flow cytometry demonstrated CD8+ T cells as the main producers of IFN-γ in this study.
Figure 6 The number of IFN-γ producing cells was measured by an ELISPOT assay. The plates were coated with an anti-mouse IFN-γ antibody. The cells were cultured in the presence of recombinant NC protein or an irrelevant antigen (gp120 protein). NC-specific IFN-γ were detected as described in Materials and Methods. The mean ± S.D. is shown for each group. The symbol * indicates a significant difference (P = 0.01–0.001) between the indicated group and all other immunized groups. The symbol † indicates a significant difference (P < 0.05) between the indicated group and the control group (DNA+XIAP).
Discussion
The SARS epidemic is currently under control. However, the absence of an effective therapeutic agent against this lethal virus, compounded by the threat of its re-emergence, has triggered research efforts to develop an effective vaccine. Previous studies indicate that the spike protein is responsible for the binding of the virus to angiotensin-converting enzyme 2 (ACE2) [25-27]. The spike protein contains epitopes that might elicit neutralizing antibodies in the host species thus making it a good target for vaccine development against SARS [28-31]. However, mutation of this protein could affect the virulence by allowing the virus to escape from specific immune response[32,33]. Other research groups have made efforts to develop vaccines based on viral nucleocapsids since these viral proteins have conserved regions. Milich and McLachlan showed that the viral nucleocapsid contains T-cell dependent and independent epitopes. Nude (athymic) mice immunized with HBV-nucleocapsid alone develop high titers of IgM, IgG2a and IgG2b antibodies which are the predominant antibodies in Th1 responses [34]. There is evidence that the specific structure folding of viral nucleocapsids is responsible for its high immunogenicity [35].
The success of immunization depends on several factors, such as type of antigen, route of administration and usage of adjuvants. Mittal et al. showed [36] that mice immunized intramuscularly, intraperitoneally or subcutaneously have higher antibody titers than mice immunized orally or intranasally. In this study, mice were immunized subcutaneously as this route of administration has been used successfully in the past [37-39].
We promoted the immune responses with adjuvants montanide ISA-51/CpG or XIAP. Montanide is a mineral oil based adjuvant that increases the immune response non-specifically[40,41]. It has been tested in clinical trials and it has a good reactogenicity profile, making it an ideal adjuvant for human use. [42-44]. In an HIV vaccine candidate study, we showed that montanide can induce strong antibody titers against HIV-1 structural genes (gp120, gag and pol). CpG is also among the most frequently used experimental adjuvants; this adjuvant stimulates dendritic cells through Toll-like receptor 9 (TLR9), inducing cell maturation and enhancing antigen presentation and Th1 responses. [45-47]. The combination of montanide and CpG was investigated in light of a recent study demonstrating that this combination is more effective than the use of any of the adjuvants alone [48]. A group of mice received XIAP as adjuvant based on the finding by Kim et al. that mice immunized with DNA encoding XIAP exhibit a strong cell mediated immune response against melanoma. Kim et al. hypothesize that this strong response may be due to increased survival of dendritic cells or T cells in vivo [16,17].
Nucleocapsid has a fundamental role in the viral life-cycle and could be a potential target for enhancing the immune responses. It is also of interest as a particulate carrier for conserved CD8+T-cell epitopes that might be suitable for the development of an effective vaccine for SARS-CoV.
In order to characterize specific immune responses in our candidate SARS vaccines, we used a recombinant protein expressed in bacteria for in vitro assays to detect CD4+ and CD8+T-cell responses, while the vaccine candidates contained a recombinant protein expressed in CHO cells. Ideally, peptides are used to stimulate CD8+effector responses, however, this is not yet feasible since NC CTL epitopes are not yet characterized in this strain of mice. It is likely that VLPs are processed by antigen presenting cells and the epitopes presented in an MHC I context, as suggested by the increased CD8+ T-cell responses observed post-vaccination.
Several studies have assessed the SARS-CoV-NC protein as a candidate vaccine. For instance, Wang et al. [49] showed a low proliferative response to NC in BALB/c mice that receive a DNA vector expressing NC protein. A weak CD4+T cell response was also observed in our study. Two more studies analyzed humoral and cell-mediated immune responses in mice immunized with DNA vaccines expressing NC [50,51]. Kim at al. showed that linkage of NC protein to calreticulin increased humoral and cellular immune responses in vaccinated mice compared to mice receiving DNA-NC alone. We did not detect a high level of CD8+ T cell immune response in mice immunized with DNA-NC or NC protein alone. However, the immunogenicity of our candidate DNA vaccine encoding NC was improved with the co-administration of the recombinant nucleocapsid protein and adjuvants.
Zhu et al. show a high level of antibody titer in mice after three injections of DNA-NC. Surprisingly, we did not detect a high level of antibody titer in mice immunized with DNA-NC alone.
In summary, our results indicate that immunization with different adjuvants could influence the type of immune response. Mice that received DNA, protein and montanide/CpG showed a high level of specific antibody titer against NC. However, vaccination with combinations of DNA-NC, recombinant NC protein and XIAP may add breadth to cell-mediated immune responses. These results suggest a novel approach to produce an effective vaccine against SARS infection.
Abbreviations
XIAP: X-linked inhibitor of apoptosis.
NC: Nucleocapsid
Acknowledgements
We thank the personnel in the animal facility at University of Ottawa for their assistance. We are grateful to Drs. Katrina Gee and Neera Malik for critically reading the manuscript.
==== Refs
Buchholz UJ Bukreyev A Yang L Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity Proc Natl Acad Sci USA 2004 101 9804 9 15210961 10.1073/pnas.0403492101
Spiga O Bernini A Ciutti A Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein Biochem Biophys Res Commun 2003 310 78 83 14511651 10.1016/j.bbrc.2003.08.122
Lu H Zhao Y Zhang J Date of origin of the SARS coronavirus strains BMC Infect Dis 2004 4 3 15028113 10.1186/1471-2334-4-3
Egloff MP Ferron F Campanacci V The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world Proc Natl Acad Sci USA 2004 101 3792 6 15007178 10.1073/pnas.0307877101
He R Leeson A Ballantine M Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus Virus Res 2004 105 121 5 15351485 10.1016/j.virusres.2004.05.002
Surjit M Liu B Kumar P Chow VT Lal SK The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain Biochem Biophys Res Commun 2004 317 1030 6 15094372 10.1016/j.bbrc.2004.03.154
Wege H Schliephake A Korner H Flory E Wege H An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis J Gen Virol 1993 74 1287 94 8393072
Wege H Schliephake A Korner H Flory E Wege H An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis J Gen Virol 1993 74 1287 94 8393072
Wege H Schliephake A Korner H Flory E Wege H Coronavirus induced encephalomyelitis: an immunodominant CD4(+)-T cell site on the nucleocapsid protein contributes to protection Adv Exp Med Biol 1993 342 413 8 7911644
Boots AM Benaissa-Trouw BJ Hesselink W Rijke E Schrier C Hensen EJ Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein Vaccine 1992 10 119 24 1311490 10.1016/0264-410X(92)90028-I
Young KR Smith JM Ross TM Characterization of a DNA vaccine expressing a human immunodeficiency virus-like particle Virology 2004 327 262 72 15351214 10.1016/j.virol.2004.07.009
Doan LX Li M Chen C Yao Q Virus-like particles as HIV-1 vaccines Rev Med Virol 2004
Takamura S Niikura M Li TC DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration Gene Ther 2004 11 628 35 14973544 10.1038/sj.gt.3302193
Pachuk CJ McCallus DE Weiner DB Satishchandran C DNA vaccines–challenges in delivery Curr Opin Mol Ther 2000 2 188 98 11249641
Davis HL McCluskie MJ DNA vaccines for viral diseases Microbes Infect 1999 1 7 21 10594972 10.1016/S1286-4579(99)80009-4
Kim TW Hung CF Ling M Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins J Clin Invest 2003 112 109 17 12840065 10.1172/JCI200317293
Kim TW Hung CF Zheng M A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response J Biomed Sci 2004 11 493 9 15153784 10.1159/000077899
Benito JM Lopez M Soriano V The role of CD8+ T-cell response in HIV infection AIDS Rev 2004 6 79 88 15332430
Gulzar N Copeland KF CD8+ T-cells: function and response to HIV infection Curr HIV Res 2004 2 23 37 15053338 10.2174/1570162043485077
Zhu F Eckels DD Functionally distinct helper T-cell epitopes of HCV and their role in modulation of NS3-specific, CD8+/tetramer positive CTL Hum Immunol 2002 63 710 8 12175725 10.1016/S0198-8859(02)00430-5
Noble A Leggat JA Inderberg EM CD8+ immunoregulatory cells in the graft-versus-host reaction: CD8 T cells activate dendritic cells to secrete interleukin-12/interleukin-18 and induce T helper 1 autoantibody Immunology 2003 109 476 86 12871213 10.1046/j.1365-2567.2003.01687.x
Renner C Held G Ohnesorge S Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells Cancer Immunol Immunother 1997 44 70 6 9177467 10.1007/s002620050357
Pham CT Ley TJ The role of granzyme B cluster proteases in cell-mediated cytotoxicity Semin Immunol 1997 9 127 33 9194223 10.1006/smim.1997.0060
Vitte-Mony I Korneluk RG Diaz-Mitoma F Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines Apoptosis 1997 2 501 9 14646521 10.1023/A:1026434514183
Prabakaran P Xiao X Dimitrov DS A model of the ACE2 structure and function as a SARS-CoV receptor Biochem Biophys Res Commun 2004 314 235 41 14715271 10.1016/j.bbrc.2003.12.081
Xiao X Chakraborti S Dimitrov AS Gramatikoff K Dimitrov DS The SARS-CoV S glycoprotein: expression and functional characterization Biochem Biophys Res Commun 2003 312 1159 64 14651994 10.1016/j.bbrc.2003.11.054
Li W Moore MJ Vasilieva N Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Nature 2003 426 450 4 14647384 10.1038/nature02145
He Y Zhou Y Liu S Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine Biochem Biophys Res Commun 2004 324 773 81 15474494 10.1016/j.bbrc.2004.09.106
Han DP Kim HG Kim YB Poon LL Cho MW Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein Virology 2004 326 140 9 15262502 10.1016/j.virol.2004.05.017
Zhang H Wang G Li J Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies J Virol 2004 78 6938 45 15194770 10.1128/JVI.78.13.6938-6945.2004
Yang ZY Kong WP Huang Y A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice Nature 2004 428 561 4 15024391 10.1038/nature02463
Yoo D Deregt D A single amino acid change within antigenic domain II of the spike protein of bovine coronavirus confers resistance to virus neutralization Clin Diagn Lab Immunol 2001 8 297 302 11238212 10.1128/CDLI.8.2.297-302.2001
Wang L Xu Y Collisson EW Experimental confirmation of recombination upstream of the S1 hypervariable region of infectious bronchitis virus Virus Res 1997 49 139 45 9213388 10.1016/S0168-1702(97)01466-4
Milich DR McLachlan A Moriarty A Thornton GB Immune response to hepatitis B virus core antigen (HBcAg): localization of T cell recognition sites within HBcAg/HBeAg J Immunol 1987 139 1223 31 2440947
Noad R Roy P Virus-like particles as immunogens Trends Microbiol 2003 11 438 44 13678860 10.1016/S0966-842X(03)00208-7
Mittal SK Aggarwal N Sailaja G Immunization with DNA, adenovirus or both in biodegradable alginate microspheres: effect of route of inoculation on immune response Vaccine 2000 19 253 63 10930680
Tobiasch E Kehm R Bahr U Large envelope glycoprotein and nucleocapsid protein of equine arteritis virus (EAV) induce an immune response in Balb/c mice by DNA vaccination; strategy for developing a DNA-vaccine against EAV-infection Virus Genes 2001 22 187 99 11324756 10.1023/A:1008175525254
Du DW Jia ZS Li GY Zhou YY HBV DNA vaccine with adjuvant cytokines induced specific immune responses against HBV infection World J Gastroenterol 2003 9 108 11 12508362
Cui Z Mumper RJ The effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses Eur J Pharm Biopharm 2003 55 11 8 12551699 10.1016/S0939-6411(02)00129-7
Aucouturier J Dupuis L Deville S Ascarateil S Ganne V Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines Expert Rev Vaccines 2002 1 111 8 12908518 10.1586/14760584.1.1.111
Sanderson K Scotland R Lee P Autoimmunity in a phase I trial of a fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma J Clin Oncol 2005 23 741 50 15613700 10.1200/JCO.2005.01.128
Peter K Men Y Pantaleo G Gander B Corradin G Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants Vaccine 2001 19 4121 9 11457536 10.1016/S0264-410X(01)00179-7
Lawrence GW Saul A Giddy AJ Kemp R Pye D Phase I trial in humans of an oil-based adjuvant SEPPIC MONTANIDE ISA 720 Vaccine 1997 15 176 8 9066035 10.1016/S0264-410X(96)00150-8
Barnett PV Pullen L Williams L Doel TR International bank for foot-and-mouth disease vaccine: assessment of Montanide ISA 25 and ISA 206, two commercially available oil adjuvants Vaccine 1996 14 1187 98 8961504 10.1016/S0264-410X(96)00055-2
Jiao X Wang RY Qiu Q Alter HJ Shih JW Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes J Gen Virol 2004 85 1545 53 15166438 10.1099/vir.0.79896-0
Lin L Gerth AJ Peng SL CpG DNA redirects class-switching towards "Th1-like" Ig isotype production via TLR9 and MyD88 Eur J Immunol 2004 34 1483 7 15114682 10.1002/eji.200324736
Zhang Y Palmer GH Abbott JR Howard CJ Hope JC Brown WC CpG ODN 2006 and IL-12 are comparable for priming Th1 lymphocyte and IgG responses in cattle immunized with a rickettsial outer membrane protein in alum Vaccine 2003 21 3307 18 12804862 10.1016/S0264-410X(03)00176-2
Kumar S Jones TR Oakley MS CpG oligodeoxynucleotide and Montanide ISA 51 adjuvant combination enhanced the protective efficacy of a subunit malaria vaccine Infect Immun 2004 72 949 57 14742540 10.1128/IAI.72.2.949-957.2004
Wang Z Yuan Z Matsumoto M Hengge UR Chang YF Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice Biochem Biophys Res Commun 2005 327 130 5 15629440 10.1016/j.bbrc.2004.11.147
Kim TW Lee JH Hung CF Generation and characterization of DNA vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus J Virol 2004 78 4638 45 15078946 10.1128/JVI.78.9.4638-4645.2004
Zhu MS Pan Y Chen HQ Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine Immunol Lett 2004 92 237 43 15081618 10.1016/j.imlet.2004.01.001
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Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-4-401615329810.1186/1475-2875-4-40MethodologyRapid urban malaria appraisal (RUMA) in sub-Saharan Africa Wang Shr-Jie [email protected] Christian [email protected] Thomas A [email protected] Penelope [email protected]é Guéladio [email protected] Diadie A [email protected] Martin [email protected] Deo [email protected] Awash [email protected] Marcel [email protected] Swiss Tropical Institute (STI), P.O. Box, CH-4002 Basel, Switzerland2 Centre Suisse de Recherches Scientifiques (CSRS), 01 B.P. 1303 Abidjan, 01 Côte d'Ivoire3 Centre National de Recherche et de Formation sur le Paludisme, (CNRFP) 01 B.P. 2208, Ouagadougou 01, Burkina Faso4 Centre de Recherche Entomologique de Cotonou (CREC), Ministère de la Santé Publique, B. P. 06-2604, Cotonou, Benin5 Regional/City Medical Office of Health, P.O. Box 9084, Dar es Salaam, Tanzania6 The Earth Institute at Columbia University, 215 West 125th St Suite 301, New York NY, 10027, USA2005 9 9 2005 4 40 40 10 6 2005 9 9 2005 Copyright © 2005 Wang et al; licensee BioMed Central Ltd.2005Wang 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 rapid urban malaria appraisal (RUMA) methodology aims to provide a cost-effective tool to conduct rapid assessments of the malaria situation in urban sub-Saharan Africa and to improve the understanding of urban malaria epidemiology.
Methods
This work was done in Yopougon municipality (Abidjan), Cotonou, Dar es Salaam and Ouagadougou. The study design consists of six components: 1) a literature review, 2) the collection of available health statistics, 3) a risk mapping, 4) school parasitaemia surveys, 5) health facility-based surveys and 6) a brief description of the health care system. These formed the basis of a multi-country evaluation of RUMA's feasibility, consistency and usefulness.
Results
A substantial amount of literature (including unpublished theses and statistics) was found at each site, providing a good overview of the malaria situation. School and health facility-based surveys provided an overview of local endemicity and the overall malaria burden in different city areas. This helped to identify important problems for in-depth assessment, especially the extent to which malaria is over-diagnosed in health facilities. Mapping health facilities and breeding sites allowed the visualization of the complex interplay between population characteristics, health services and malaria risk. However, the latter task was very time-consuming and required special expertise. RUMA is inexpensive, costing around 8,500–13,000 USD for a six to ten-week period.
Conclusion
RUMA was successfully implemented in four urban areas with different endemicity and proved to be a cost-effective first approach to study the features of urban malaria and provide an evidence basis for planning control measures.
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Background
Urbanization has a significant impact on the economy, lifestyles, ecosystems and disease patterns, including malaria [1,2]. An estimated 39% of the population in sub-Saharan Africa (SSA) lived in urban areas in 2003 [3], 198 million Africans lived in urban malaria-endemic areas and 24–103 million clinical attacks occur annually in those areas [4]. An important message addressed in the Pretoria Statement on urban malaria was that the malaria control strategies used in rural areas cannot be directly transferred to the urban context [5]. The epidemiology of urban malaria poses a number of specific challenges: i) the first malaria infection occurs often late in childhood and the acquisition of semi-immunity is delayed [6]; ii) the intensity of the malaria risk is often heterogeneous over small distances, being subjected to the degree of urbanization of particular subdivisions [7,8] and their proximity to possible vector breeding sites [9,10]; iii) rural-urban migration is likely to increase the endemicity of malaria [11]; iv) agricultural and animal husbandry are important economic activities which create a favourable environment for Anopheles breeding [12,13]; v) marginalized populations usually lack access to health care, which hampers the effectiveness of case management and the promotion of intermittent antimalarials during pregnancy [5,14-16]. There is now substantial private sector activity in health care provision in many cities. The private services providers are often untrained or unlicensed, but are seen as a source of inexpensive care by patients. There is not much information about the impact of the private sector on case management.
Around 235 papers related to malaria epidemiology in SSA urban settings were published from 1945 to 2004. Entomological profiles and clinical patterns are known to vary between urban, suburban and rural environments [17]. A review of other studies in SSA urban centres showed that transmission patterns vary greatly by city, season and age group. The overall prevalence of parasitaemia was 4.0% in schoolchildren in Brazzaville [18], 2.4–10.3% in Lusaka [19], 2.0% in a Gambian urban area [20] and 3.6–7.5% in Dakar [21]. It was also reported that malaria prevalence in school children varied from 3.0% to 26.4% in different areas of Ouagadougou [22] and varied from 14% in a central urban area to 65% in peri-urban areas in Kinshasa [23].
Evidence showed that the rate of clinical malaria attacks detected in urban health facilities was high and season-dependent. For example, Hendrickse et al. found that 36.8% of outpatients were parasitaemic in a hospital in Ibadan [24]. In Niamey, the parasite prevalence was 61.9% during the rainy season but only 5.4% in the dry season in 1989 [25]. In Kinshasa, malaria admissions comprised 29.5% of consultations in 1983, then 38.2% in 1985–86 [26]. In Dakar, malaria fever represented 19.7% of consultations and 34.3% of fever cases were caused by malaria in 1988 [27]; the same authors found that 5.3% (dry season) and 58.8% (rainy season) of febrile outpatients were parasitaemic in 1994 [28]. In Ouagadougou, malaria prevalence accounted for 33% of all outpatients [29], while Dabire reported 22% malaria parasitaemia among children aged 0–14 years in the paediatric ward [30].
Transmission and severity of malaria are influenced by the geographic characteristics of a town and by the socio-economic environment. The heterogeneity and seasonal variation of the entomological inoculation rate, depending on both vector densities and sporozoite rates, have been documented [31,32]. Lindsay et al. (1990) showed a difference in the composition of vector species and the vector's adaptation in different subdivisions Banjul [20]. To improve interventions, the determinants of the diversity of transmission levels within subdivisions of a city should be understood. Concerns were raised about the association between urban agricultural activities or local irrigation systems and the creation of breeding sites for Anopheles sp. [12,33,34]. Peri-urban areas often lack infrastructure, including poor water supply and sanitation, which provides an ideal environment for vector breeding [35]. For example, urban Dakar has >5,000 market-garden wells which provide permanent sites for mosquito larvae [13]. An identification of vector species, regular larval inspection and larviciding activities should be implemented in the framework of urban malaria control programmes [36].
This article presents the experience of developing a rapid urban malaria appraisal (RUMA) in SSA, carried out with the support of the Roll Back Malaria Partnership. The aims were i) to develop a rapid assessment package that is explicitly evidence-based and can be carried out within a six to ten weeks timeframe; and ii) to assess how rapid malaria appraisal efforts could be best integrated into the municipal health department supervision and to inform control programmes.
Methods
Study sites
The fieldwork took place in Yopougon municipality/Abidjan (Côte d'Ivoire), Ouagadougou (Burkina Faso), Cotonou (Benin) and Dar es Salaam (United Republic of Tanzania) (Figure 1).
Figure 1 Map of major urban areas in sub-Sahara Africa and the four selected project sites. Major cities (=M) and population density (red >=200, green > 100 and blue = 40 population per square kilometres. Copyright: MARA/ARMA.
Abidjan is the economic capital of Côte d'Ivoire. It is located between latitude 3.7° N–4.0° N and longitude 5.7° E–6.0° E, with a surface area of 454 sq. km. The study was carried out in the large commune of Yopougon (population: 775,000 in 1998) located in the west of Abidjan [37]. The fieldwork in Yopougon municipality (Abidjan) took place from August to September 2002.
Ouagadougou, the capital of Burkina Faso, is situated on the Sahelian border between latitude 12.0° N–13.0° N and longitude 1.15° E–1.40° E. The total surface area was estimated to be around 570–655 sq. km in the year 2000 [38]. The population of Ouagadougou was around 1,100,000 inhabitants in 2002. The fieldwork in Ouagadougou took place from November to December 2002.
Cotonou is the economic capital of Benin. It is located on a strip of land between Lake Nokou and the Gulf of Guinea (between latitude 6.2° N–6.3° N and longitude 2.2° E–2.3° E). The total population was estimated at 780,000 inhabitants on a territory of 73.8 sq. km in 2002 [39]. The fieldwork in Cotonou took place from February to March, 2003.
Dar es Salaam is situated between latitude 6.0° S–7.5° S and longitude 39.0° E–39.6° E on the East African coast. There are 2,500,000 inhabitants on a total surface area of 1,393 sq. km [40]. The fieldwork in Dar es Salaam took place from June to August, 2003.
Study design
In July 2002, a generic RUMA protocol was developed based on existing urban malaria research protocols [41,42]. The relevant institutions in each setting were contacted and city-specific proposals were then produced. Parts of health facilities mapping, school and health facility-based survey activities were integrated into the routine surveillance and health system evaluation at the municipal level. All the fieldwork was completed in August 2003. Final reports were completed in June, 2004.
The six key components of the RUMA were the following (see also Table 1):
Table 1 Study design and methodology of RUMA.
Key measures Epidemiological measures Spatial relationships Individual variations Institutional factors
Methodology Age-specific morbidity and mortality rates Fraction of malaria-attributable fevers Overall endemicity Gradient of malaria risk Environmental risks Travelling history Socio-economic factors Bednet usage Treatment strategy Public/private partnership Coverage of treatment providers Degree of drug resistance
1. Literature review x x x x x x x x
2. Collection of health statistics x x x x x
3. Risk mapping x x x
4. School parasitaemia survey x x x x x x x
5. Health facility-based fever survey x x x x x x x x
6. Brief description of the health care system x x x
1. Literature review. A search of the PUBMED bibliographic database was conducted for the time period from 1960 to April 2004, using the terms "malaria", "urban" and "sub-Saharan Africa". The search was limited to the articles published in English, Chinese, French and Spanish. The reference list of all identified papers was screened. Thesis abstracts filed in the medical libraries of universities and national hospitals were collected at each site and local researchers were also contacted.
2. Collection of routine health statistics. Local experts in ministries of health (MOH) (disease surveillance systems, municipal health departments and national malaria control programmes) and national census and statistics bureaus were contacted to collect demographic data, health system information and statistics, including routine malaria morbidity and mortality reports.
3. Mapping of health care facilities and major Anopheles breeding sites. Three or four trained workers carried out the health facility mapping under the guidance of local health personnel. In order to identify Anopheles breeding sites, simple larvae sampling was performed with the assistance of entomological technicians in Dar es Salaam and Ouagadougou. The duration of these tasks varied by site: 12 weeks during the rainy season in Dar es Salaam and around three weeks during the dry season in Ouagadougou. Due to security issues and technical problems, the mapping of breeding sites and health facilities could not be performed in Yopougon municipality (Abidjan) and Cotonou.
4. School parasitaemia surveys. School surveys were aimed at determining the local endemicity and risk gradient of malaria. In each city, three to four schools with different malaria endemicity (centre/low, intermediate/medium and periphery/high) were investigated. It is a rapid assessment with limited budget; therefore, in each area only one health facility and school were selected for the surveys. The schools were selected near the selected clinics. 200 school children aged 6–10 years were recruited in each school. Additional information on children was collected using a questionnaire with the assistance of teachers (see Additional file 1).
5. Health facility-based surveys (See Additional file 2). The facility-based fever surveys focused on the age-specific fraction of malaria-attributable fevers [43]. Each city was categorised into three to four areas (centre, intermediate, periphery and rural areas) and one clinic from each area was chosen. Health facilities with a high enough volume of outpatients per day were considered for the survey. In urban areas, an estimated 5% to 50% of fever cases among children under 15 years old were due to malaria. A sample size of 200 in each facility gave an estimate of the proportion of cases with parasites with the following approximate lower 95% confidence limits (at 5%, lower 95% CI: 2; at 50%, lower 95% CI: 6). In each clinic, 200 fever cases and 200 non-fever controls were recruited, with half of them being aged <5 years. Outpatients with a history of fever (past 36 hours) or a measured temperature ≥ 37.5°C were defined as cases. Controls were recruited from another department of the same clinic without current or recent past fever, matched by age and residency.
Electronic thermometers were used to measure the armpit temperature. A "normal" body temperature is referred to as an oral temperature of 37°C. An armpit temperature reading is usually 0.3°C to 0.6°C lower than an oral temperature reading. Therefore 0.5°C was added to the temperature displayed on the digital readout. Thick and thin blood films were taken to identify malaria infections. Using 100× magnification to read the thick smears, all malaria trophozoites and gametocytes were counted separately. Parasite density was calculated according to parasites per 200 white blood cells in a thick film (assuming 8000 white blood cells per ml of blood). If 200 white blood cells were counted and less than 9 malarial parasites found, the counting continued until 500 white blood cells were identified.
6. Brief description of the health care system. It focused on i) the municipal malaria control and prevention efforts, ii) the levels and coverage of service delivery, iii) disease surveillance systems, iv) malaria case management and v) trends of parasite resistance to antimalarials.
Quality assurance for blood slides
The diagnostic performance and the quality of blood sample readings were checked twice: first in the field and then at the reference laboratory of the Swiss Tropical Institute (STI) in Basel, Switzerland. The results in Yopougon municipality (Abidjan), Dar es Salaam and Ouagadougou were: sensitivity 87.9%, 83.5% and 98.7%; specificity 89.2%, 99.0% and 98.2%; accuracy rate of slide readings 88.8%, 98.5% and 98.6%. The quality control process was not implemented in Cotonou due to operational problems.
Costing
The financial cost of the resources required for a RUMA were calculated for each site based on local market prices and salary standards, except for the laboratory material that was purchased in Switzerland. All expenses fell into seven categories: salaries, transportation, communications, stationery, laboratory materials, other cost and administrative fees (Table 2). A project team was assembled within the existing structure of partner institutions and then the accountants in each site used a setting-specific cost model to identify the cost factors and determine their local value. The preparation and training cost, programme and administrative costs with the partner institution were estimated and an allowance was added for unforeseen circumstances in the finalized budget. The cost for resources like microscopes and drugs for treatment, vehicles and computers were calculated according to the cost structure of the host institution.
Table 2 Budget categories.
Type of cost Categories Valuation Information source
Human resources Project staff
Health sector staff Gross salary
Per diem Salary slips or personnel records from the project office
Transportation Project vehicles, petrol and maintenance
Taxi, motorbike and bus Shipping and packaging Petrol and maintenance of vehicles based on vehicle logbook
Actual expenditure Freight cost Bills and receipts
Tickets and receipts Invoices
Communication Postage and telephone bills Bills or contract documents
Stationery Office maintenance cost
Survey materials
Photocopies Lap top and printer use Actual expenditure for items Agreement with site
Agreement and receipts
Standard local cost
Agreement with site
Laboratory materials & drugs for treatment International trade good price Invoices
Other items Bills and receipts
Administration Rent of project office, computer and vehicles Agreement with site
Results
One of the principal aims of the present work was to review the feasibility, perceived usefulness and consistency of the collected information. Because RUMA was a cross-sectional assessment the external validity of the findings could not be assessed. However, the internal consistency of the results was assessed.
Below, the strengths and weaknesses of each methodology are presented, bearing in mind the constraints imposed by a rapid assessment. Detailed results for each site will be provided in a series of forthcoming publications.
Literature review (Tables 1 and 3)
Table 3 RUMA methodology strengths and weaknesses.
RUMA Methodology Strengths Weaknesses
Literature review • Time-saving, can be done before and afterwards
• Can identify qualified local expertise
• Comparison of the malaria patterns and trends • Incomplete information in time and space
Collection of health statistics • Good description of malaria burden over a longer time period • Completeness and quality of data
Cross-sectional mapping of healthcare facilities & major Anopheles breeding sites • Visualization of information for policy makers
• Helps to plan urban health programmes and upgrade community infrastructure • Time consuming and only limited scale possible
• Breeding sites may be transient /seasonal
School parasitaemia surveys • Good estimates of local endemicity and local risk factors
• Good description of fever prevalence in school
• Malaria risk gradient • Limited representativeness if only small number of schools were sampled
Health facility-based fever surveys • Estimates malaria-attributable fevers and prevalence of clinical malaria
• Description of fever management • Limited representativeness due to attendance bias
Brief description of the health care system • Understanding of the structure of city health department and of current malaria control activities
• Limited cost
• Review of the efficacy of case management • Only focuses on the available information
• Depends on the efficiency of information dissemination within municipal departments
The systematic review of all literature in each city allowed the collection of background information in a time-efficient manner. A substantial body of information was found in each setting, although it was often incomplete in place (for example covering only a part of the city), in time (few time points, only one season) and in content (not all subject areas covered). For the period 1945 to 2004, a total of 109 papers was found (18, 23, 29 and 39 for Abidjan, coastal Benin, Dar es Salaam and Ouagadougou, respectively), relating to malaria epidemiology, socio-economic risk factors of malaria, entomology and drug resistance [44].
Collection of health statistics (Tables 1, 3 and 4)
Table 4 Reported simple malaria cases among total consultations in 4 African cities, all ages. CHU = Centre Hospitalier Universitaire.
a) Abidjan 2001
Communes Adjamé & Attécoubé Cocody Yopougon Abobo Plateau Treichville & Marcory Port-Bouét & Koumassi Total % of admission†
Health centers 35,714 55,500 - 71,437 - - 62,607 225,258
CHU No CHU 2,525 - No CHU No CHU 12,375 No CHU 14,900
Total 35,714 58,025 - 71,437 - 12,375 62,607 240,158 40.2
b) Ouagadougou 2001
Sanitary District Kossodo Paul VI Pissy Secteur 30 Total % of admission
Total 16,007 24,527 95,868 67,064 203,466 29.3–41.4
c) Dar es Salaam 2000
District hospitals‡ Ilala Kinondoni Temeke Total % of admission
Total 178,016 498,991 395,566 1,072,573 45.4–53.7‡
d) Cotonou 2002
Sanitary District I II III IV V VI Total % of admission
Total 6,759 9,678 17,339 7,108 29,890 29,483 100,257 32.1–35.9
†Reported number of malaria cases divided by the total number of consultations.
‡Both Ilala and Temeke district hospitals have malaria reported weekly and monthly. The raw dataset of malaria reports of district hospitals in Kinondoni was missing in 2001. Total numbers of consultations were estimated.
The routine weekly or monthly malaria reports provided a baseline on the burden of malaria in public health facilities, as well as an assessment of the scale of malaria treatment. Overall, case detection in the antenatal clinics and public health services was poor and reporting was not systematic and consistent.
In Abidjan, data were collected from the national malaria control programme (Table 4a). Age-specific monthly data were available. The statistics for 2001 from four out of 10 communes were missing. The malaria cases reported from the main hospitals (Centre Hospitalier Universitaire-CHU) in Yopougon, CHU Cocody and CHU Treichville were separated from the commune data. CHU receive many referral patients and the malaria cases may therefore be over-reported. The data from CHU Yopougon were missing for 2001.
In Ouagadougou, the number of malaria-specific cases and the total number of consultations were collected. The raw data were available by season for 1999–2001, but not for 2002. All the data were missing for Paul VI sanitary district from October to December 2001. The reporting of clinical malaria was also inconsistent in Paul VI (Table 4b).
In Dar es Salaam, the weekly malaria reports were collected from the Ilala, Kinondoni and Temeke district health departments. The data were available for 2000-mid 2003, two months before the survey. A discrepancy in records in Kinondoni District was found, as not all health facilities sent their weekly reports to the district municipal office. Moreover, the sums of reported malaria cases in the raw dataset and in the final district reports were not identical. The Kinondoni district health department had lost all of its 2001 weekly reports (Table 4c).
Only Cotonou had complete data sets for 1996–2002, but the raw datasets were unavailable. Hence, it was impossible to review the consistency and accuracy of the data (Table 4d).
Overall, considerable gaps were found in the routine surveillance systems, particularly for remote health services. Often, the data were collected and presented in different formats, making a generalization impossible and this limited their usefulness. Furthermore, the municipal health departments simply summed up the total numbers of reported cases as they lacked the capacity to analyse these data and to extract useful information for management purposes.
Mapping activities (Tables 1 and 3)
As stated above, the mapping activities were only done in Ouagadougou and Dar es Salaam.
a) Public and private health facilities
In Dar es Salaam, the list of existing public and private health facilities was updated and their locations were recorded by a geographic positioning system (GPS). In Ouagadougou, the mapping of health facilities and schools was done in 2002 by the Ecole Inter-Etats d'Ingénieurs de l'Equipement Rural (EIER), Burkina Faso. Both digital city maps were updated and available for public use.
b) Anopheles breeding sites
The malaria risks in Dar es Salaam and Ouagadougou were displayed in relation to the location of health facilities and schools. The mapping of Anopheles breeding sites in Dar es Salaam was done on a city wide-scale in conjunction with another project [36,45]. In Ouagadougou, in the limited time available, the focus was on permanent and semi-permanent breeding sites instead of searching for the numerous temporary breeding sites. The produced maps of breeding sites indicated mosquito productivity and distribution in the city in a given season.
The major drawback of mapping is that ground-truthing is very time-consuming and variable over time. During the rainy season, the city-wide larvae collection, larvae hatching and management of data are difficult tasks. Another disadvantage of this approach is that it tends to be very expensive, unless local Geographic Information Systems (GIS) mapping expertise and/or digital city maps are already available for public use. For future studies, it is recommended focusing on the mapping of health facilities and dropping the breeding sites work as it is difficult to assemble a team with the required expertise within such a short time period.
School parasitaemia surveys (Tables 1 and 3)
It was possible to determine the transmission intensity and gradients in different communities. At each site, parasitaemia and fever prevalence rates were obtained for different schools (Figures 2a, 2b, 2c) and by residential areas of children. Around 10 to 70% of children (from city centre to periphery) attended schools with elevated temperature. Malaria prevalence was always higher than the fever prevalence in Ouagadougou since there were many asymptomatic infections. Different communities in Ouagadougou may be exposed to different patterns of malaria transmission and hence the age at first infection and infection patterns may vary. Certainly, the more exposed areas of Ouagadougou experience hyperendemic (if seasonal) malaria. The association between malaria infections and various risk factors were measured and these results are reported elsewhere [46-49].
Figure 2 Prevalences of parasitaemia and fever detected in schools, in three sites. The vertical bars represent the 95% CI. a) Ouagadougou. b) Dar es Salaam. c) Cotonou
Health facility-based surveys (Tables 1, 3 and 5)
Table 5 Age-specific malaria prevalence rates in cases and controls by each site. Health facility-based surveys.
Study sites Abidjan Cotonou Dar es Salaam Ouagadougou
Age groups/malaria Cases % Control % Cases % Control % Cases % Control % Cases % Control %
Infants <1 year 18/78 (23.1%) 22/169 (13.0%) 0/63 (0%) 2/140 (1.4%) 2/99 (2.0%) 4/116 (3.4%) 7/58 (12.1%) 3/21 (14.3%)
Children 1–5 years 61/142 (43.0%) 16/60 (26.7%) 5/68 (6.8%) 4/137 (2.8%) 15/213 (7.0%) 8/178 (4.5%) 45/174 (25.9%) 15/104 (14.4%)
Children 6–15 years 39/89 (43.8%) 8/35 (22.9%) 0/35 (0%) 1/78 (1.3%) 7/97 (7.2%) 2/56 (3.6%) 23/62 (37.1%) 20/58 (34.5%)
Adults >15 years 31/120 (25.6%) 17/119 (14.3%) 2/213 (0.9%) 11/529 (2.0%) 13/308 (4.2%) 8/423 (1.9%) 48/266 (18.0%) 72/363 (19.8%)
Both the fever and control groups (non-febrile admission) had a medium level of parasitaemia prevalence in the health facilities in Yopougon municipality (Abidjan) and Ouagadougou (Table 5). Some people in the control groups reported self-medication with paracetamol or traditional herbs before visiting the clinics. This could have led some malaria cases to present without fever at the clinic. The overall prevalence of malaria was surprising low in Cotonou and Dar es Salaam. This might have been due to high Insecticide Treated Nets (ITNs) coverage and/or the dry climate at the time of survey [46-49].
The detection of malaria parasites in a febrile case does not necessarily indicate clinical malaria. In an effort to improve the case definition and clinical diagnosis, the method of Smith et al. [43] was used to estimate the probabilities that individual episodes were really due to a malaria infection. The odds ratio (OR) is the proportion of odds of having parasitaemia in fever cases over controls. The formula for the fraction of fever episodes attributable to malaria parasites is: (1-1/Odds Ratio)*P. P is the proportion of fever episodes in which the subjects had parasitaemia. These age-specific malaria attributable fractions were very low: 0.12–0.27, 0–0.04, 0–0.02 and 0–0.13 in Yopougon municipality (Abidjan), Benin, Dar es Salaam and Ouagadougou, respectively. These results indicated substantial over-treatment at all sites [46-49].
The questionnaires (available as a separate file) administered to cases and controls were tailored for local use. They contained four sections: personal information, economic situation of the family, travelling history, clinical signs and malaria history. The information on age, sex, measured axillary temperature, length of febrile illness, types of previous treatment and the reasons for seeking care were obtained. Stay outside the urban area during the previous three months, the type of housing, urban agriculture activities and ITNs usage were also investigated. These data provided indications of disease perception, preventive measures and socio-economic background at community level.
The questionnaires administered to cases and controls in health facilities were similar to the ones used in school surveys. In all settings the two sets of data were comparable, which allowed for an internal consistency check. For example, in Dar es Salaam 43.1% and 40.2% of households reported ITN use in both the health facility surveys and the school parasitaemia surveys. In Cotonou, these figures were 36.6% and 28.4%, in Ouagadougou 7.8% and 11.1%. The similarity of both surveys also made possible a combined planning and implementation strategy. Detailed results are presented elsewhere [46-49], as well as in a series of forthcoming publications.
Brief description of the health care system (Tables 1 and 3)
The administrative structures of the national and municipal health departments were sketched out and the list of health facilities was updated at each site. The total numbers of registered malaria diagnosis or treatment providers were: 1060 in Abidjan, 365 in Cotonou, 1684 in Dar es Salaam and 315 in Ouagadougou. Non-governmental organizations and religious hospitals play an important role in health care delivery in Cotonou and Ouagadougou. The catchment areas of all public and private health facilities were further calculated [46-49]. The city malaria control programmes and WHO offices provided information about current malaria control efforts. In order to assess treatment efficacy, the trend of the susceptibility of P. falciparum to different antimalarials was reviewed at each site [46-49].
This component required few resources and brought strong political commitment because it involved representatives of the Ministry of Health and the Directors of the municipal health department. The extra-budgetary resources from RUMA helped the local governments to better monitor the provision of health care services, which facilitated an effective exchange of information. The health information was updated but the quality of health care delivery was not assessed because of restricted scope and time. The disadvantage of this approach is that effective communication and dissemination of official documents depends on the attitude of senior officers.
Compared costing of RUMA activities
The cost for conducting a RUMA in a SSA city with a population of 0.5–3 million is around 8,500–13,000 USD for a six to ten-week period (Table 6). The cost of human resources in Dar es Salaam and Ouagadougou was highest, mainly because of the additional fieldwork performed there (mapping of breeding sites and health facilities). Indeed, the per diem standard was lower in these cities. The higher savings on transportation, communications and materials in Abidjan and Ouagadougou were made possible by our affiliation with local research institutions. The total expense in Abidjan was much lower because the school survey was not performed (the children did not attend school during a politically troubled time). In Cotonou, the excess of human resource and transportation cost was due to unforeseen supervisory expenses.
Table 6 RUMA expenses by study sites. QA = Quality control, USD = US dollars 1USD = 650 Francs CFA (Communauté Française Africaine), 1 USD = 1,050 Tanzanian Schilling in 2003.
Sites by the order of total expenses Human resources Transport Communication Stationery Lab. materials & drugs Others Admin. Total expenses USD Total with QA*
Cotonou§ 2,375 2,942 500 447 793 0 1,000 8,582 No QA
Dar es Salaam 4,321 2,040 93 775 1,030 236 0 8,495 12,435
Ouagadougou 2,493 1,613 198 886 721 98 400 6,970 6,970¥
Abidjan 958 411 360 569 707 47 1,000 4,577 7,237
§ without GIS mapping and 1st quality control without GIS mapping and school survey
* with second quality control at Swiss Tropical Institute.
¥ the second quality control was free
In general, the difference in the cost of human resources and communications was due to differences in personnel capacity and fluctuations in the amount of work. The costs of stationery and laboratory materials were less variable, because the needs were the same at each site.
Discussion and conclusion
This assessment was accomplished in four countries within a period of six to ten weeks in the field and has proven to be a helpful tool in supporting planning of urban malaria control. An ongoing urban malaria control intervention in Dar es Salaam has been initiated on the knowledge basis provided by RUMA. With the incentive of extra-budgetary resources and technical support from STI, local partners were committed to incorporate RUMA into existing activities at the municipal level. Qualified personnel and opportunities for integration, synergy and co-ordination were identified during the meetings with local partners and the collaborations were always very successful.
The RUMA methodology is a cross-sectional design and the results are likely to change over time due to seasonality, the dynamics of urbanization and the evolution of malaria transmission. In Dar es Salaam, for example, the surveys were carried out during an exceptionally dry period and results could underestimate the true transmission intensity. Many factors such as the size of the city, the fieldwork logistics, the availability of local expertise and the coordination with local senior officers can influence the schedule and planning, as well as the outcome of such surveys.
The study highlighted the need for improved Health Management Information Systems (HMIS) in SSA urban areas. Municipal health departments routinely collect health facility data but information is rarely fed back to the districts and facilities that generate the information. The data are often not available for analysis or accessible due to false registration and under-reporting from health facilities, as well as poor filing and storage of documents at the district or municipal level. In addition, the low number of true malaria cases among fever episodes treated as "malaria" raises the issue of the validity of the collected data even further. Hence, much progress needs to be made in order to estimate more accurately the urban malaria burden and plan relevant control measures.
GIS provides a platform to display health services and geographic features in relation to population settlements. In this experience policy-makers could readily use the presented information for improved planning, re-allocation of resources and for strengthening the networking between the public and private sectors. While the GIS technology has been shown to be very useful in studying health care delivery and distribution of diseases, its application in an entomological assessment was quite difficult and costly and could only be done in conjunction with other ongoing projects. Hence it should be excluded from the process of RUMA. In contrast, the mapping of health facilities with GIS was feasible and cost-effective.
While results from the school surveys gave an indication of the endemicity range and risks in the targeted community, they cannot be considered as being representative without a wider survey. The variations of malaria risk were sometimes related to political divisions or man-made boundaries, but often were due to divergent socio-environmental factors and the degree of urbanization. Because site-specific environmental conditions lead to an aggregated distribution of vectors and different malaria risks, the sampling sites were selected taking into account the population density, the natural environment and urbanization patterns. This should improve the rough categories that previous researchers applied (centre, intermediate and periphery).
Despite a potential attendance bias, the health facility surveys allowed the determination of prevalence of parasitaemia among presenting clinical cases, and the calculation of the fraction of malaria-attributable fevers. This allowed to document clearly the high rate of malaria mis-diagnosis in the health facilities. This information is of great importance for urban malaria control.
Overall, RUMA is a first step towards understanding malaria endemicity and designing control strategies. It has exemplified the concern for mis-diagnosis of clinical malaria in SSA cities [25,50]. A report by the Tanzania-Japan malaria control programme in Dar es Salaam mentioned that drug administration to diagnosed children was one of the essential interventions that reduced the malaria rates between 1988 and 1996 [36]. An in-depth research is now being implemented in Dar es Salaam to assess the malaria burden with a much larger sample size. The application of RUMA methodology is possible and desirable in other SSA urban areas and it should have a special focus on improved diagnosis.
List of abbreviations
CHU Centre Hospitalier Universitaire
CNRFP Centre National de Recherche et de Formation sur la Paludisme, Burkina Faso
CREC Centre de Recherche Entomologique de Cotonou
CSRS Centre Suisse de Recherches Scientifiques, Côte d'Ivoire
EIER Ecole Inter-Etats d'Ingénieurs et de l'Equipement Rural, Burkina Faso
GIS Geographic Information System
HMIS Health Management Information Systems
ITNs Insecticide-Treated Nets
MOH Ministry of Health
RUMA Rapid Urban Malaria Appraisal
SSA Sub-Saharan Africa
STI Swiss Tropical Institute
Authors' contributions
SW participated in the design of the study, conducted the field work, analysed and interpreted data and drafted the manuscript. CL conceived the study, coordinated the field work and revised the manuscript. TS and PV assisted in the design and the statistic analysis. CG, DD, MA and DM were the key local contacts, facilitated the collaboration and supervised the data collection and laboratory works at each site. AT participated in the design of the study. MT participated in the conception of the work, facilitated the overall coordination and revised it critically at all stages.
Supplementary Material
Additional File 1
the questionnaire for health facility-based survey
Click here for file
Additional File 2
the questionnaire for school parasitaemia survey
Click here for file
Acknowledgements
We would like to acknowledge the support and help of the following institutions and persons. In Benin: Francois Holtz; in Burkina Faso: the Ecole Inter-Etats d'Ingénieurs de l'Equipement Rural; in Côte d'Ivoire: Dr. Joseph Niangue; in Tanzania: Ifakara Health Research and Development Centre. We wish also to express our gratitude to Dr. Andrei Chirokolava for editing and reviewing the city reports. RUMA was supported financially by the Roll Back Malaria Partnership and STI.
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Omumbo JA Guerra CA Hay SI Snow RW The influence of urbanisation on measures of Plasmodium falciparum infection prevalence in East Africa Acta Trop 2005 93 11 21 15589793 10.1016/j.actatropica.2004.08.010
Hay SI Guerra CA Tatem AJ Atkinson PM Snow RW Urbanization, malaria transmission and disease burden in Africa Nat Rev Microbiol 2005 3 81 90 15608702 10.1038/nrmicro1069
UN World Urbanization Prospects: The 2003 Revision 2003 New York
Keiser J Utzinger J Caldas de Castro M Smith TA Tanner M Singer BH Urbanization in sub-saharan Africa and implication for malaria control Am J Trop Med Hyg 2004 71 118 127 15331827
Donnelly MJ McCall PJ Lengeler C Bates I D'Alessandro U Barnish G Konradsen F Klinkenberg E Townson H Trape JF Hastings IM Mutero C Malaria and urbanization in sub-Saharan Africa Malar J 2005 4 12 15720713 10.1186/1475-2875-4-12
Trape JF Malaria and urbanization in central Africa: the example of Brazzaville. Part IV. Parasitological and serological surveys in urban and surrounding rural areas Trans R Soc Trop Med Hyg 1987 81 26 33 3332057 10.1016/0035-9203(87)90474-3
Robert V Macintyre K Keating J Trape JF Duchemin JB Warren M Beier JC Malaria transmission in urban sub-Saharan Africa Am J Trop Med Hyg 2003 68 169 76 12641407
Trape JF Zoulani A Malaria and urbanization in central Africa: the example of Brazzaville. Part III: Relationships between urbanization and the intensity of malaria transmission Trans R Soc Trop Med Hyg 1987 81 19 25 3455564 10.1016/0035-9203(87)90473-1
Trape JF Lefebvre-Zante E Legros F Ndiaye G Bouganali H Druilhe P Salem G Vector density gradients and the epidemiology of urban malaria in Dakar, Senegal Am J Trop Med Hyg 1992 47 181 189 1354414
Staedke SG Nottingham EW Cox J Kamya MR Rosenthal PJ Dorsey G Short report: proximity to mosquito breeding sites as a risk factor for clinical malaria episodes in an urban cohort of Ugandan children Am J Trop Med Hyg 2003 69 244 246 14628938
Benyoussef A Cutler JL Baylet R Collomb H Diop S Gaye P Lacombe B Vaugelade J Migrants' health and adjustment to urban life-Senegal Jimlar Mutane 1976 1 105 12 12264763
Afrane YA Klinkenberg E Drechsel P Owusu-Daaku K Garms R Kruppa T Does irrigated urban agriculture influence the transmission of malaria in the city of Kumasi, Ghana? Acta Trop 2004 89 125 134 14732235 10.1016/j.actatropica.2003.06.001
Robert V Awono-Ambene HP Thioulouse J Ecology of larval mosquitoes, with special reference to Anopheles arabiensis (Diptera: Culcidae) in market-garden wells in urban Dakar, Senegal J Med Entomol 1998 35 948 955 9835685
Noor AM Zurovac D Hay SI Ochola SA Snow RW Defining equity in physical access to clinical services using geographical information systems as part of malaria planning and monitoring in Kenya Trop Med Int Health 2003 8 917 926 14516303 10.1046/j.1365-3156.2003.01112.x
Massele A Mpundu M Hamudu N Utilisation of antimalarial drugs by pregnant women attending the antenatal clinic at Muhimbili Medical Centre, Dar es Salaam East Afr Med J 1997 74 28 30 9145573
Sanon VM Etude du cout financier direct de la prise en charge du paludisme grave en milieu pediatrique de Ouagadougou MSc thesis 1990 Ouagadougou: Université de Ouagadougou
Modiano D Sirima BS Sawadogo A Sanou I Pare J Konate A Pagnoni F Severe malaria in Burkina Faso: urban and rural environment Parassitologia 1999 41 251 254 10697864
Trape JF Quinet MC Nzingoula S Senga P Tchichelle F Carme B Candito D Mayanda H Zoulani A Malaria and urbanization in central Africa: the example of Brazzaville. Part V: Pernicious attacks and mortality Trans R Soc Trop Med Hyg 1987 81 34 42 3455565
Watts TE Wray JR Ng'andu NH Draper CC Malaria in an urban and a rural area of Zambia Trans R Soc Trop Med Hyg 1990 84 196 200 2389308 10.1016/0035-9203(90)90251-9
Lindsay SW Campbell H Adiamah JH Greenwood AM Bangali JE Greenwood BM Malaria in a peri-urban area of The Gambia Ann Trop Med Parasitol 1990 84 553 562 2076033
Trape JF Lefebvre-Zante E Legros F Druilhe P Rogier C Bouganali H Salem G Malaria morbidity among children exposed to low seasonal transmission in Dakar, Senegal and its implications for malaria control in tropical Africa Am J Trop Med Hyg 1993 48 748 756 8333568
Sabatinelli G Bosman A Lamizana L Rossi P Prevalence of malaria in Ouagadougou and the surrounding rural environment during the period of maximal transmission Parassitologia 1986 28 17 31 3455530
Kazadi W Sexton JD Bigonsa M W'Okanga B Way M Malaria in primary school children and infants in Kinshasa, democratic republic of the Congo: surveys from the 1980s and 2000 Am J Trop Med Hyg 2004 71 97 102 15331825
Hendrickse RG Aspects of tropical paediatrics Trans R Soc Trop Med Hyg 1976 70 268 273 795103 10.1016/0035-9203(76)90075-4
Olivar M Develoux M Chegou Abari A Loutan L Presumptive diagnosis of malaria results in a significant risk of mistreatment of children in urban Sahel Trans R Soc Trop Med Hyg 1991 85 729 30 1801337 10.1016/0035-9203(91)90432-X
Greenberg AE Ntumbanzondo M Ntula N Mawa L Howell J Davachi F Hospital-based surveillance of malaria-related paediatric morbidity and mortality in Kinshasa, Zaire Bull World Health Organ 1989 67 189 196 2743538
Gaye O Bah IB Bengue E Diallo S Faye O Malaria morbidity in the urban environment. Study of 353 fever attacks Med Trop 1989 49 401 404
Gaye O Faye O Ndir O Feller-Dansokho E Dieng Y Lakh NC Diallo S Malaria in an urban environment: the case of the city of Rufisque in Senegal Dakar Med 1997 42 54 58 9827119
Coulibaly CO Guiguemde TR Lamizana L Ouedraogo JB Dabiret E The role of malaria in febrile diseases in the urban environment of Ouagadougou Ann Soc Belg Med Trop 1991 71 5 10 2043001
Dabire E Morbidité et mortalité palustres au sein de la pathologie fébrile dans le service de pédiatrie de l'hôpital Yalgado Ouédraogo MSc thesis 1990 Ouagadougou: Université de Ouagadougou 110
Akogbeto M Chippaux JP Coluzzi M Coastal urban malaria in Cotonou (Republic of Benin). Entomological study Rev Epidémiol Santé Publique 1992 40 233 239
Sabatinelli G Rossi P Belli A Dispersion of Anopheles gambiae s.l. in an urban zone of Ouagadougou (Burkina Faso) Parassitologia 1986 28 33 39 3455531
Brock B Olanrewaju B Smith Actual and potential contribution of urban agriculture to environmental sanitation: a case study in Cotonou Agriculture urbaine en Afrique ed l'ouest/Urban agriculture in West Africa 1999 Ottawa: The International Development Research Centre 240
Gerstl S The economic costs and impact of home gardening in Ouagadougou PhD thesis 2001 Basel: University of Basel
Knudsen AB Slooff R Vector-borne disease problems in rapid urbanization: new approaches to vector control Bull World Health Organ 1992 70 1 6 1568273
Caldas de Castro M Yamagata Y Mtasiwa D Tanner M Utzinger J Keiser J Singer BH Integrated urban malaria control: a case study in Dar es Salaam, Tanzania Am J Trop Med Hyg 2004 71 103 117 15331826
Daigl M Evaluation des facteurs de risque pour le paludisme et la diarrhée dans les quartiers Yao-séhi, Niangon Sud Sicogi et le village d'Azito – Commune de Yopougon MSc thesis 2002 Basel: University of Basel
Institut National de la Statistique et de la Démographie (INSD) L'enquête Démographique et de Santé du Burkina Faso (EDSBF-II) Ouagadougou 2000
Institut National de la Statistique et de l'Analyse Economique (INSAE) Recensement général de la population et de l'habitation, synthèse des résultats d'analyse, Cotonou, Bénin (RGPH3) Cotonou 2003
Damas MbogoroK Population and Housing Census Results Republic of Tanzania Dar es Salaam 2002
Warren M Billing P Bendahmane D Wijeyaratne P Malaria in urban and peri-urban areas in sub-Sahara Africa. Environmental Health Project, activity report No.71 1999 Washington, DC 44 10358806
WHO Rapid Urban Malaria Appraisal 2001 Geneva
Smith T Schellenberg JA Hayes R Attributable fraction estimates and case definitions for malaria in endemic areas Stat Med 1994 13 2345 2358 7855468
Wang S-J Lengeler C Tanner M Rapid assessment of urban malaria protocol 2002 Basel 26
Sattler MA Mtasiwa D Kiama M Premji Z Tanner M Killeen GF Lengeler C Habitat characterization and spatial distribution of Anopheles sp. mosquito larvae in Dar es Salaam (Tanzania) during an extended dry period Malar J 2005 4 4 15649333 10.1186/1475-2875-4-4
Wang S-J Lengeler C Smith TA Vounatsou P Tanner M Rapid urban malaria appraisal (RUMA), final report for Abidjan, Roll Back Malaria Partnership, WHO 2004 Basel 58
Wang S-J Lengeler C Smith TA Vounatsou P Tanner M Rapid urban malaria appraisal (RUMA), final report for Cotonou, Roll Back Malaria Partnership, WHO 2004 Basel 68
Wang S-J Lengeler C Smith TA Vounatsou P Tanner M Rapid urban malaria appraisal (RUMA), final report for Dar es Salaam, Roll Back Malaria Partnership, WHO 2004 Basel 67
Wang S-J Lengeler C Smith TA Vounatsou P Tanner M Rapid urban malaria appraisal (RUMA), final report for Ouagadougou, Roll Back Malaria Partnership, WHO 2004 Basel 62
Amexo M Tolhurst R Barnish G Bates I Malaria misdiagnosis: effects on the poor and vulnerable Lancet 2004 364 1896 1898 15555670 10.1016/S0140-6736(04)17446-1
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-481617657810.1186/1477-7827-3-48ResearchNeonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis Nwagwu Margaret O [email protected] Helen [email protected] Jeffrey B [email protected] Francis JP [email protected] School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK2 Department of Anatomy and Cell Biology, Monash University, Victoria 3800, Australia2005 21 9 2005 3 48 48 5 8 2005 21 9 2005 Copyright © 2005 Nwagwu et al; licensee BioMed Central Ltd.2005Nwagwu 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
Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages.
Methods
Hpg mice were treated with 100 μg testosterone propionate or vehicle on postnatal day 2. At 35 days of age, subgroups of these mice were treated with silastic implants containing estradiol or cholesterol. Reproductive behavior was scored in tests with steroid-primed female mice, then testicular development was assessed histologically, and measures of pituitary FSH content made at 85 days of age.
Results
The neonatal testosterone propionate treatment successfully defeminized female litter mates, as revealed by impaired vaginal opening and deficiencies in lordosis behavior, and it allowed appropriate male reproductive behavior to be expressed in a proportion of the hpg males when tested at an adult age. However, neonatal androgen supplementation did not block or even reduce the subsequent actions of estradiol in increasing pituitary FSH content, nor did it affect the ability of estradiol to induce qualitatively normal spermatogenesis.
Conclusion
The ability of the hpg male to show a "female" neuroendocrine response to estradiol is not a result of inadequate androgenization during neonatal development, and thus the actions of estradiol revealed in this rodent model are not an artefact of incomplete sexual differentiation, but reflect a physiological role of estradiol occurring during a specific early temporal window of male reproductive development.
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Introduction
Although estradiol has classically been considered a female hormone, recent data from man shows that it plays important physiological roles in the male. For example, estradiol deficiency or resistance results in lack of bone epiphyseal fusion, delayed skeletal maturation and low sperm viability [[1] for review]. These effects can be reproduced in rodent models so that the underlying mechanisms of estrogen action can be investigated. For example, male mice in which estrogen receptor (ER) α has been knocked out become progressively infertile [2,3], and likewise, if production of estradiol is prevented by knockout of the cyp19 aromatase gene, then such mice show impaired spermatogenesis, reduced spermatid numbers and infertility [4].
We have used the hypogonadal (hpg) mouse to study the actions of estradiol in male reproduction. Such mice are unable to produce gonadotrophin releasing hormone (GnRH) due to a truncation in the GnRH gene, and therefore show a profound hypogonadotrophic hypogonadism [[5] for review]. Surprisingly, treatment of hpg males with low levels of estradiol stimulates spermatogenesis, as evident by an increase in testis weight and the presence of elongated spermatids in the seminiferous tubules of the testis [6]. This induction of spermatogenesis is accompanied by increases in pituitary FSH content and in circulating FSH concentrations [6,7]. FSH is an important component of the spermatogenic process; lack of the FSH β subunit or receptor in genetically-modified mice results in decreased testis size and reduced sperm quality [8]. Conversely, treatment of hpg mice with recombinant human FSH has been shown to increase testis size and the number of spermatogonia [9].
In male mammals, estradiol normally provides a negative feedback signal which inhibits FSH synthesis and secretion [6,10]. Thus, the increase in FSH production in response to estradiol in hpg mice ("positive feedback") might be considered to be a "female" neuroendocrine response. One possibility is that the phenomenon of estradiol-induced FSH production in male hpg mice is due to inadequate masculinization or incomplete defeminization of the neonate due to the lack of androgen exposure in the early postnatal period. Appropriate pre- and postnatal testosterone concentrations are known to be necessary for complete masculinization in rodents. Before postnatal day 5, serum testosterone is higher in male mice compared to females [11] and this difference is believed to be important for defeminization of males. Mice and other rodents have a critical period of neural sexual differentiation before postnatal day 10. Experimental studies have demonstrated that administration of testosterone to female mice in early postnatal life suppresses sexual receptivity and increases aggression at later ages [12]. Conversely, neonatal castration of male mice results in a lack of normal male aggressive behavior in adulthood; which is not restored with later testosterone treatment [13]. Hence early androgen exposure serves to differentiate the subsequent propensity to display aggressive behavior and sexual receptivity [13], and sensitizes appropriate neural elements to androgens encountered in later life [12].
There is clear evidence that hpg mice are not adequately masculinized in the neonatal period. Hpg males will show appropriate physiological reproductive development when treated in adulthood with either grafts of fetal hypothalamic tissue releasing GnRH or with appropriate gonadotropins [5], but importantly such hpg males do not show appropriate reproductive behavior despite the induction of steroidogenesis and spermatogenesis. However, if hpg mice are also treated with exogenous androgens on postnatal day 2, and then the reproductive axis is activated by grafts or gonadotropins, they subsequently display mounting, intromission and ejaculation, and can sire litters in later life [14]. Therefore, the aim of this study was to test the hypothesis that the ability of estradiol to increase in FSH production in male hpg mice is due to the lack of defeminization or masculinization resulting from low testosterone exposure of hpg male mice during the early postnatal period.
The experimental approach was to administer testosterone propionate to neonatal male hpg mice. We predicted that if the ability to respond to estradiol is a consequence of an inadequate postnatal androgen environment in hpg mice, then neonatal treatment with testosterone propionate should block the ability of subsequent estradiol treatment to produce a rise in pituitary FSH and to induce spermatogenesis.
Materials and methods
Animals
All animal procedures were approved by the University of Nottingham Local Ethical Review Committee and carried out in accordance with the Animals Scientific Procedures Act (UK) 1986 (project licence PPL 40/2372). Laboratory Animal Science Association (LASA) guidelines were followed for administration of substances [15]. Male and female mice known to both be heterozygous for the hpg mutation as determined by PCR-based genotyping [16] were housed in breeding cages (n = 15 pairs). All male (n = 126) and female (n = 129) pups born to these breeding pairs were treated with testosterone propionate (100 μg sc) or vehicle alone (arachis oil) on postnatal day 2 (Figure 1). On postnatal day 35, male hpg mice (n = 25) were identified by their micropenis and small scrotal sac, and were then given a subcutaneous (sc) implant containing 2% estradiol (n = 18) or cholesterol (n = 7) as previously described [6]. Implants were left in place for 50 days, about 1.5 spermatogenic cycles (Figure 1).
Figure 1 Experimental design. Male hpg mice were treated with either vehicle (VEH) or 100 μg testosterone propionate (TP) on postnatal day 2, then received a subcutaneous silastic capsule containing either cholesterol (chol) or 2% estradiol in cholesterol on day 35 of age.
Sexual behavior: male hpg mice
Sexual behavior was assessed in hpg males a few days prior to the end of the study (Figure 1). The steroid priming and testing procedure was adapted from the method of McGill [17]. Wild-Type adult C3H female mice were brought in to estrous by treatment with 35 μg of estradiol benzoate followed 36–48 hours later by injection of 100 μg progesterone, and testing was conducted 6 hours following progesterone treatment. These females were initially tested with proven male studs in order to confirm that they were sexually receptive. For the test, hpg males or studs were placed in an observation cage 48 hours prior to testing in order to ensure adaptation to the new cages. Males were tested on several occasions with at least two receptive females; test(s) were carried out by placing a sexually receptive female into the male cage for 15 minutes, and the number of mounts and intromissions observed and recorded.
Female sexual behavior
Female C3H mice injected with vehicle or testosterone propionate on postnatal day 2 were weighed weekly after weaning and were scored for vaginal opening every week until day 70 of age. These females were tested on consecutive days with male studs as described above and were observed to see if they were receptive (e.g. exhibiting lordosis) or whether they persistently non-receptive i.e. demonstrating aggressive behavior by attacking or chasing males round the cage.
Hormone measurements and testis collection
On postnatal day 85, the experimental males and three age-matched wild-type litter mates were killed by anesthesia overdose (sodium pentobarbitone, Rhone Merieux, Harlow). The pituitary, testes, epididymides and seminal vesicles were excised, trimmed of fat and connective tissue, and weighed. One testis was placed in Bouin's fixative and the other was placed on ice and frozen at -20°C. The fixed testes were processed into paraffin blocks and 5 μm sections were stained with haematoxylin-eosin for histological analysis. For measurement of testis testosterone content, the frozen testis was sonicated in 1 ml PBS (3 × 10 seconds) and the homogenate extracted twice with 4 ml of diethyl-ether. The supernatant was left to evaporate overnight in a fume hood at 25°C, and samples were reconstituted in 1 ml PBS. Testosterone was measured using a salivary testosterone ELISA assay kit (IDS Ltd., Tyne & Wear, UK). The minimum detection limit was 0.006 ng/well. Pituitary glands were sonicated in 500 μl PBS and FSH content was measured in a single assay using a commercially available Rat FSH IRMA kit (IDS Ltd., UK). The minimum detection limit was 0.2 ng/tube and the intra-assay CV was 1.8%.
Histological examination of fixed testis
Three sections from each of 3–5 fixed testes from each treatment group were examined. The sections were scored for the presence of lumina and elongating spermatids by an observer who was blind to the experimental treatment. Tissue sections with no evidence of lumen formation in any tubules received a score of 0, sections where less than 50% of tubules containing a lumen received a score of 1, sections where less than 50–95% of tubules had lumina received a score of 2, and sections where all tubules contained a lumen received a score of 3. A similar scoring system was used to estimate the prevalence of elongating spermatids. The mean score for each testis was then calculated, and then used to calculate the group mean score.
Statistical analysis
All statistical analysis was performed using Prism v3 (GraphPad Software, San Diego, CA). Results were analysed by t-test, Chi-squared tests, 2 factor ANOVA or Kruskal-Wallis tests as appropriate.
Results
Effect of neonatal androgenization on development and behavior of female littermates
Vaginal opening and sexual behavior were scored in female litter mates to confirm the validity of the androgenization protocol. The mean weight of the uterus in 70 day old female mice treated neonatally with testosterone propionate was significantly (p < 0.05) greater than that of vehicle-treated females (Figure 2). There was no significant difference in the anogenital distance of the two groups (Figure 2, middle panel), but Chi-squared tests confirmed a significant (p < 0.05) reduction in the proportion of females with full vaginal opening (3% after neonatal testosterone propionate treatment compared to 79% after vehicle treatment, Figure 2). In addition, 37% (13 of 35) of female mice treated neonatally with testosterone propionate showed atypical aggressive behavior and attacked stud males, but this behavior was never observed in any vehicle-treated females (Figure 2, bottom panel, P < 0.05; Chi-squared).
Figure 2 Female littermates of experimental male mice received a subcutaneous injection of either vehicle (VEH) or 100 μg testosterone propionate (TP) on postnatal day 2. Panels indicate body weight (top), reproductive tract weight, anogenital distance, proportion showing vaginal opening by day 70 of age and proportion showing aggressive behavior toward stud males in tests of reproductive receptivity. Values are group mean ± SE (upper panels) or numbers of mice (lower panels).
Effect of neonatal androgenization on sexual behavior in adult hpg males
Chi-squared tests revealed a significant (p < 0.05) difference in the proportion of males displaying mounting behavior. No mounting behavior was ever observed in hpg males treated with vehicle in the neonatal period and with either cholesterol or estradiol implants from day 35, or treated with testosterone propionate neonatally and then with cholesterol implants from day 35. However, 2 of 6 (33%) of hpg mice treated neonatally with testosterone propionate and subsequently with estradiol implants from day 35 demonstrated mounting behavior when tested after a further 40 days (Figure 3). All the wild-type studs were also observed to mount steroid-primed females (Figure 3).
Figure 3 Proportions of male hpg mice displaying mounting behavior when paired with a steroid-primed female. Mice were treated with vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35, and tested after 75 days of age. Three wild-type C3H mice were also tested (+/+).
Effect of neonatal androgenization on estrogen-induced spermatogenesis and FSH production in hpg males
In male hpg mice, neonatal androgenization per se had no significant effect on body weight (Figure 4, top), testis weight (Figure 4) or anogenital distance (Figure 4). Treatment with estradiol significantly increased the weights of the testes, epididymides and seminal vesicles (Figure 4) (p < 0.05), regardless of whether the mice had been treated neonatally with testosterone propionate or vehicle. Estradiol treatment also significantly (p < 0.05) increased pituitary FSH content in hpg mice that received either testosterone propionate or vehicle on postnatal day 2 (Figure 5 upper panel). Two-factor ANOVA using the values for organ weights and FSH content revealed no significant interactions between neonatal treatment (testosterone propionate vs vehicle) and subsequent treatment (estradiol vs cholesterol implant), thus neonatal androgenization did not influence the subsequent response to estradiol treatment. Testicular testosterone content was not significantly affected by neonatal androgenization or subsequent estradiol treatment (Figure 5 lower panel).
Figure 4 Body weight (top), paired testis weight, anogenital distance, and wet weight of epididymides and seminal vesicles (bottom)) of male hpg mice receiving vehicle (VEH) or 100 μg testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35. Values are group mean ± SE. **P < 0.001 vs groups treated with cholesterol implants.
Figure 5 Pituitary FSH content (top) and testis testosterone content (bottom) in male hpg mice; receiving either vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35. For comparison, pituitary FSH and testicular testosterone values derived from wild-type litter mates analysed in the same assay are indicated (+/+). Values are group mean ± SE. **P < 0.001 vs groups treated with cholesterol implants.
Histological analysis
The mean scores for the histological examination of testes are shown in Table 1. Testes from hpg mice that only received cholesterol implants in adult life had a characteristic undeveloped appearance, regardless of whether the mice had received testosterone propionate or vehicle during the neonatal period. The seminiferous tubules were of small diameter (Figure 6), generally lacking a lumen (Figure 6, Table 1), with Sertoli cells frequently located medial to the basal lamina (Figure 6). Round and elongating spermatids were not observed, the most mature cells types in testes from hpg mice treated with cholesterol were spermatogonia type A and pre-pachytene primary spermatocytes (Figure 6). In contrast, after estradiol treatment the seminiferous tubules had expanded and developed a lumen, regardless of whether the mice had been treated with testosterone propionate or vehicle neonatally (Figure 6, Table 1). Sertoli cells were observed to be adjacent to the basal lamina, and round and elongating spermatids (ES) were present (Figure 6). Thus, treatment with testosterone propionate in the neonatal period did not affect testicular histology after treatment with estradiol in later life.
Table 1 VEH+chol VEH+E TP+chol TP+E
lumen 0.7 ± 0.3 2.8 ± 0.2 0.3 ± 0.3 3.0 ± 0.0
elongating spermatids 0.0 ± 0.0 2.2 ± 0.5 0.0 ± 0.0 2.3 ± 0.7
Assessment of the presence of lumen and elongating spermatids. Mice were treated with vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35, and testes tested at 85 days of age. Values (from 0 to 3, see text) are group mean scores ± SE of 3–5 testes per treatment group.
Figure 6 Representative examples of testicular histology from 85 day old hpg mice treated with vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then receiving either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35. Note that the most mature cells types in testes from mice treated with cholesterol (VEH+chol, TP+chol) are type A spermatogonia (SA) and primary spermatocytes (PS); there is little evidence of lumen formation, and Sertoli cells are frequently observed adjacent to the basal lamina and more centrally (St). In contrast, after estradiol treatment (VEH+E, TP+E) the seminiferous tubules are expanded and have developed a lumen (L), Sertoli cells are adjacent to the basal lamina and round and elongating spermatids (ES) are present. Treatment with testosterone propionate in the neonatal period did not affect the testicular response to estradiol in later life. Scale bars represent 30 μm.
Discussion
The principal findings of this study were firstly that all the hpg male mice treated with estradiol demonstrated clear increases in pituitary FSH content and in the wet weight of the testes, epididymides and seminal vesicles, reflecting activation of spermatogenesis as evidenced by the presence of elongated spermatids in the seminiferous tubules. This paradoxical stimulatory action of estradiol in the male is in agreement with our previous studies in the hpg mouse [6,7] and confirms the robustness of the response. Secondly, and more importantly, androgenization of hpg males with testosterone propionate in the neonatal period did not affect the subsequent ability of estradiol to activate the reproductive axis in such mice.
Since the estradiol-induced rise in pituitary FSH was not abolished by neonatal androgenization of hpg male mice, the possibility arises that the current neonatal androgenization protocol was ineffective in the induction of masculinization of the hpg males. However, we followed carefully a well-established dosing protocol previously demonstrated to be effective in male hpg mice [14], and our other experimental observations suggest the androgenization protocol was at least partly successful. The neonatal testosterone propionate injections were certainly effective in defeminizing the female litter mates of the hpg males. First, vaginal opening usually occurs around postnatal day 35 in female mice [18], yet the vast majority of our androgenized females (97%) failed to show full vaginal opening by 70 days of age. Second, a significant proportion (36%) of androgen-treated females displayed aggressive behavior when paired with stud male mice, consistent with previous studies demonstrating that increased aggression is a consequence of neonatal androgenization in female mice [13]. Third, the uterine weights from the females that were treated with testosterone propionate in the neonatal period were significantly greater than those from the vehicle-treated females, also consistent with previous studies demonstrating that androgenized female mice have heavier uteri [19]. The observation that vaginal opening and uterine weight were affected in almost all neonatally-androgenized females whereas a lower proportion displayed abnormal female behavior may indicate that a higher androgen threshold must be reached to affect sexual behavior than to disrupt the endocrine control of the female reproductive tract.
There was also evidence of masculinization of behavior in some of the hpg males: two of the six hpg males treated neonatally with testosterone propionate and then with estradiol in later life displayed mounting and intromission when paired with sexually receptive female mice. Such behaviors did not occur in the hpg mice treated with vehicle in the neonatal period, consistent with the previously hypothesised crucial role of early androgen exposure in mice for masculinizing the brain and the sexually dimorphic spinal nucleus of the bulbocavernosus and its target perineal muscles, which are involved in the control of penile copulatory reflexes [14,20,21]. The failure of the other four neonatally-androgenized mice to display copulatory behaviors is most likely to relate to the limiting endocrine milieu at the time of the behavioural tests. Previous studies of the role of neonatal androgens in regulating male reproductive behavior in hpg mice relied upon additional treatment with testosterone propionate in adult life to permit such behaviors to be expressed in an appropriate context [14]. In the current experiment, levels of intratesticular androgens were found to be low even after estradiol treatment, and our previous studies have likewise failed to detect rises in circulating androgen concentrations after estradiol treatment [6]. Thus, the prevailing androgen milieu is very likely to be inadequate for most mice to be primed to display mounting, intromission and ejaculation.
In view of the evidence of successful androgenization in at least some of the hpg males and female siblings, our interpretation of the current studies is that the increase pituitary FSH content in male hpg mice in response to estradiol is not a failure of early masculinization or defeminization. An alternative explanation may be that the positive FSH response of gonadotrophs to estradiol in "adult" male hpg mice may be due to the immaturity of the pituitary gland reflecting the lack of pituitary exposure to GnRH in such mice. A recent study of the expression of the pituitary melatonin receptor 1 (MT1) gene provides good evidence that the pituitary gland in hpg mice is arrested at an immature stage of development [22]. In normally developing mice exposed to increasing amounts of GnRH, there is a decline in MT1 expression in the pituitary gland, but in hpg mice which are not exposed to GnRH, this decline does not occur [22]. Developmental changes in gonadotroph secretory profiles have been described for rats [23] and rhesus monkeys [24], so it might be hypothesized that ability of estradiol to increase synthesis and secretion of FSH (but not LH, 7) in hpg mice is a reflection of an action of estradiol that is physiologically relevant during early postnatal development in mammals. Indeed, stimulatory actions of estradiol on testis function have been demonstrated in several other rodent models where pituitary development is still immature, and provide some evidence that estradiol exerts its stimulatory effects via an interaction with FSH. For example, treatment of rats with estradiol in the second postnatal week of life stimulates germ cell mitosis increasing the abundance of type A spermatogonia [25]. Correspondingly, treatment of neonatal rats with diethylstilbestrol induces small increases in circulating FSH and advances the initiation of spermatogenesis at puberty [26]. However, these effects of estradiol may not be exclusively via action on pituitary gonadotrophs producing FSH, because in a similar experimental paradigm in neonatal rats, estradiol treatment also enhances the actions of FSH within the seminiferous tubule on pre-meiotic differentiation, resulting in increased abundance of pachytene spermatocytes [27].
Conclusion
Treatment of male hpg mice with physiological levels of estradiol promotes production of FSH in the pituitary gland and induces spermatogenesis. We hypothesized that this paradoxical response to estradiol might be the result of inadequate masculinisation or incomplete defeminization of the hpg male, but this seems unlikely since treatment of neonatal hpg mice with testosterone propionate does not abolish these effects of estradiol. The stimulatory responses to estradiol revealed in hpg mice at an adult age probably mimic physiological actions of estradiol in early male development when pituitary maturation is incomplete.
Authors' contributions
MON, HB, JBK and FJPE all participated in the experimental design, MON, HB and FJPE carried out the experimental procedures, MON collated the results and provided a first draft of the manuscript, JBK and FJPE assisted with analysis of the data and completed the writing of the manuscript.
Acknowledgements
This work was supported by an AstraZeneca Collaborative grant and by a BBSRC committee studentship (HB).
==== Refs
Grumbach MM Auchus RJ Estrogen: consequences and implications of human mutations in synthesis and action JCEM 1999 84 4677 4694 10599737
Lubahn DB Moyer JS Golding TS Couse JF Korach KS Smithies O Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene Proc Natl Acad Sci USA 1993 90 11162 11166 8248223
Hess RA Bunick D Lee KH Bahr J Taylor JA Korach KS Lubahn DB A role for oestrogens in the male reproductive system Nature 1997 390 509 512 9393999 10.1038/37352
Robertson KM O'Donnell L Jones MEE Meachem SJ Boon WC Fisher CR Graves KH McLachlan RI Simpson ER Impairment of spermatogenesis in mice lacking a functional aromatase (cyp19) gene Proc Natl Acad Sci USA 1999 96 7986 7991 10393934 10.1073/pnas.96.14.7986
Charlton HM Neural transplantation in hypogonadal (hpg) mice – physiology and neurobiology Reproduction 2004 127 3 12 15056765 10.1530/rep.1.00066
Ebling FJP Brooks AN Cronin AS Ford H Kerr JB Estrogenic induction of spermatogenesis in the hypogonadal (hpg) mouse Endocrinology 2000 141 2861 2869 10919273 10.1210/en.141.8.2861
Baines H Nwagwu M Furneaux ECF Stewart J Kerr JB Mayhew TM Ebling FJP Estrogenic induction of spermatogenesis in the hypogonadal (hpg) mouse: role of androgens Reproduction 2005
Krishnamurthy H Danilovich N Morales CR Sairam MR Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse Biology of Reproduction 2000 62 1146 1159 10775161
Singh J Handelsman DJ Neonatal administration of FSH increases Sertoli cell numbers and spermatogenesis in gonadotropin-deficient (hpg) mice J Endocr 1996 151 37 48 8943767 10.1677/joe.0.1510037
Moguilevsky JA Scacchi P Szwarcfarb B Effect of estrogens on LH- and FSH-levels in prepuberal male and female androgenized rats Experientia 1977 33 1533 1544 923739 10.1007/BF01918855
Pang SF Tang F Sex differences in the serum concentrations of testosterone in mice and hamsters during their critical periods of neural sexual maturation J Endocr 1984 100 7 11 6690645
Davidson JM Levine S Endocrine regulation of behavior Annu Rev Physiol 1972 34 375 408 4334848 10.1146/annurev.ph.34.030172.002111
Edwards DA Neonatal administration of androstenedione, testosterone or testosterone propionate: effects on ovulation, sexual receptivity and aggressive behavior in female mice Physiology and Behavior 1971 6 223 228 5166472 10.1016/0031-9384(71)90030-8
Livne I Silverman AJ Gibson MJ Reversal of reproductive deficiency in the hpg male mouse by neonatal androgenization Biology of Reproduction 1992 47 561 567 1391342
Morton DB Jennings M Buckwell A Ewbank R Godfrey C Holgate B Inglis I James R Page C Sharman I Verschoyle R Westall L Wilson AB Refining procedures for the administration of substances. Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. British Veterinary Association Animal Welfare Foundation/Fund for the Replacement of Animals in Medical Experiments/Royal Society for the Prevention of Cruelty to Animals/Universities Federation for Animal Welfare Laboratory animals 2001 35 1 41 11201285 10.1258/0023677011911345
Lang J Assay for deletion in GnRH (hpg) locus using PCR Mouse Genome 1991 89 857
McGill TE Sexual behavior in three inbred strains of mice Behaviour 1961 19 341 350
Gao WM Lu HM Dong JC Zhang W Zhou X Jenkins LW Dixon CE Postnatal growth, neurobehavioral and neurophysiologic changes of prenatal low-dose beta-radiation from tritiated water in mice Neurotoxicology and Teratology 2002 24 247 254 11943512 10.1016/S0892-0362(02)00202-7
Ventenas J Lopez-Bote CJ Garcia C Gazquez A Burgos J Effects of neonatal androgenization on growth and carcass composition in female mice J Endocr 1984 120 281 285
Sachs BD Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat Journal of Reproduction and Fertility 1982 66 434 443
Wagner CK Clemens LG Perinatal modification of a sexually dimorphic motor nucleus in the spinal cord of the 86D2F1 house mouse Physiology and Behavior 1989 45 831 835 2780856 10.1016/0031-9384(89)90303-X
Johnston JD Messager S Ebling FJP Williams LM Barrett P Hazlerigg DG Gonadotrophin-releasing hormone drives melatonin receptor down-regulation in the developing pituitary gland Proc Natl Acad Sci USA 2003 100 2831 2835 12598657 10.1073/pnas.0436184100
Childs G Ellison D Foster L Ramaley JA Postnatal maturation of gonadotropes in the male rat pituitary Endocrinology 1981 109 1683 1692 6271539
Meeran D Urbanski HF Gregory SJ Townsend J Tortonese DJ Developmental changes in the hormonal identity of gonadotroph cells in the rhesus monkey pituitary gland JCEM 2003 88 2934 2942 12788908
Kula K Induction of precocious maturation of spermatogenesis in infant rats by human menopausal gonadotropin and inhibition by simultaneous administration of gonadotropins and testosterone Endocrinology 1988 122 34 39 3121284
Atanassova N McKinnell C Turner KJ Walker MJ Fisher S Morley M Millar RM Groome NP Sharpe RM Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels Endocrinology 2000 141 3898 3907 11014247 10.1210/en.141.10.3898
Kula K Walczak-Jedrzejowska R Slowikowska-Hilczer J Oszukowska E Estradiol enhances the stimulatory effect of FSH on testicular maturation and contributes to precocious initiation of spermatogenesis Molecular and Cellular Endocrinology 2001 178 89 97 11403898 10.1016/S0303-7207(01)00415-4
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BMC Dev BiolBMC Developmental Biology1471-213XBioMed Central London 1471-213X-5-191615689710.1186/1471-213X-5-19Research ArticleD-glucuronyl C5-epimerase acts in dorso-ventral axis formation in zebrafish Ghiselli Giancarlo [email protected] Steven A [email protected] Department of Pathology and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA2 Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA3 Department of Microbiology and Immunology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA2005 12 9 2005 5 19 19 24 5 2005 12 9 2005 Copyright © 2005 Ghiselli and Farber; licensee BioMed Central Ltd.2005Ghiselli and Farber; 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
Heparan sulfate (HS) is an ubiquitous component of the extracellular matrix that binds and modulates the activity of growth factors, cytokines and proteases. Animals with defective HS biosynthesis display major developmental abnormalities however the processes that are affected remain to be defined. D-glucuronyl-C5-epimerase (Glce) is a key HS chain modifying enzyme that catalyses the conversion of glucuronic acid into iduronic acid, a biosynthetic step that enhances HS biological activity. In this study the role of Glce during early zebrafish development has been investigated.
Results
Two Glce-like proteins (Glce-A and -B) are expressed in zebrafish at all times. They are the products of two distinct genes that, based on chromosomal mapping, are both orthologues of the same single human gene. Transcripts for both proteins were detected in fertilized zebrafish embryos prior to the onset of zygotic transcription indicating their maternal origin. At later developmental stages the epimerases are expressed widely throughout gastrulation and then become restricted to the hindbrain at 24 h post-fertilization. By monitoring the expression of well characterized marker genes during gastrulation, we have found that misexpression of Glce causes a dose-dependent expansion of the ventral structures, whereas protein knockdown using targeted antisense morpholino oligonucleotides promotes axis dorsalization. The ventralizing activity of Bmp2b is enhanced by Glce overexpression whereas Glce knockdown impairs Bmp2b activity.
Conclusion
Glce activity is an important determinant of of dorso-ventral axis formation and patterning in zebrafish. In particular Glce acts during gastrulation by affecting Bmp-mediated cell specification. The results obtained further corroborate the concept that HS encodes information that affect morphogenesis during early vertebrate development.
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Background
Heparan sulfate proteoglycans (HSPG) are macromolecules found in all connective tissues, extracellular matrices and on the surface of cells [1]. Their most prominent feature is the presence of one or more heparan sulfate (HS) chains covalently attached to a core protein [2]. The heterogeneity of the HSPG is due to the variation of the core protein, as well as to the type and size of the HS chains. Configuration variation in the disaccharide bonds and the position of sulfation leads to increased diversity in the chemical and structural properties of these chains. HS is composed of repeating disaccharide units of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) both of which may be 2O-sulfated, and unsubstituted, N-acetylated, or N-, 3O- or 6O-sulfated glucosamine (Glc). Forty-eight different disaccharides are possible but because of constraints in the biosynthetic process, only 23 have been identified in HS as biosynthetic intermediates [3]. Typical HS chains contain relatively short segments of modified sequences represented by IdoA-GlcNS derivatives of different sulfation content dispersed among large sections of unmodified (GlcA-GlcNAc) units.
The biosynthesis of HS occurs in the Golgi and involves the sequential modification of the nascent polysaccharide chain [4,5]. The conversion of GlcA into IdoA is a critical modification mediated in mammals by a single enzyme: D-glucuronyl-C5-epimerase (GLCE) [4,6]. Epimerization of GlcA increases the flexibility of HS chain thereby enhancing its ability to interact with proteins [2,7-9]. IdoA is the preferential substrate of the HS 2-O-sulfotransferase. Disaccharide units containing IdoA-2-O-S are organized in clusters along the HS chain and are specifically recognized by growth factors and morphogens [5,10]. The essential role played by Glce during development is demonstrated by the fact that transgenic mice that are Glce-null generally express highly abnormal HS structures and die neonatally [11,12]. C. elegans expressing mutated Glce, display abnormal neuronal development characterized by specific axonal and cellular guidance defects [13].
Much of the information concerning the role of HS in development has been obtained from studies in D. melanogaster [14]. An important concept arising from those studies is that the establishment of a morphogen gradient necessary for early patterning requires HSPG. This function is likely to involve the polysaccharide chain since morphogens such as Wingless (Wg) [15], Decapentaplegic (the orthologe of the vertebrate bone morphogenetic protein 4, Bmp4) [16,17], Hedgehog (Hh) [18] and several fibroblast growth factors (FGFs) [19] bind to HS. The specific role of HS in vertebrate development however remains conjectural and the developmental mechanisms that are affected have not been clearly identified. In zebrafish, lack of uridine 5'-diphosphate glucose dehydrogenase [20], an enzyme required for the biosynthesis of extracellular matrix glycosaminoglycans including HS, affects bone and heart morphogenesis. In mice [21] and zebrafish [22] the disruption of HS biosynthesis affects the nervous system development that can be ascribed to the effect HS has on the activity of multiple morphogens. In this paper we report that Glce's activity affects the establishment of the embryonic dorso-ventral (D/V) axis through a mechanism involving the bone morphogenetic proteins (Bmps).
Results
Cloning and chromosomal mapping of zebrafish glce
Zebrafish glce-A and glce-B genes both encode proteins of 585 amino acids. The gene products are homologous to the human protein sequence (67% and 73% respectively) (fig. 1a). Compared to the human, mouse and bovine sequences, the zebrafish proteins lack part of the N-terminus. The C-terminal domain is the most conserved region of Glce. Analysis of the hydrophobicity index determined utilizing the SOSUI [23] and the TMPRED [24] algorithms, reveals the presence in both zebrafish proteins of a conserved hydrophobic domain of ~20 amino acids located between residue 10 and 30 at the N-terminus of the proteins (fig. 1b). As the mammalian enzyme, zebrafish epimerase is a "type-two" transmembrane protein with predicted localization in the Golgi apparatus [4]. The glce-A and the glce-B locus mapped to linkage group LN25 between markers Z24369 and Z20832 and to linkage group 7 between markers Z21519 and Z9521, respectively (fig. 1c). Based on the mapping of neighbor genes, both chromosomal regions are synthenic with human chromosome 15q22, i.e. the region harboring the epimerase gene.
Figure 1 Cloning and structural analysis of zebrafish glce. (a) Alignment of Glce from zebrafish, human, mouse, and bovine. Conserved AAs are dotted. (b) Hydrophobicity plot of zebrafish and human Glce. Values above the line represent positive hydrophobicity scores. (c) Chromosomal mapping of zebrafish glce-A and glce-B and homology to the human Glce locus.
Glce is maternally and zygotically derived
glce transcript level was examined at different developmental stages and compared to that of ext2-A, an HS polymerase acting upstream the epimerization step [3]. The expression of shh which is activated during gastrulation, and that of ef-1 which is expressed at similar level throughout development, were also monitored. glce-A, glce-B and ext2-A transcripts were present in fertilized embryos at developmental stages prior to the onset of zygotic transcription, indicating that these messages are maternally derived. The expression of HS biosynthesis enzymes reached a peak at the onset of gastrulation following midblastula transition (fig. 2a). Assay of epimerase activity in embryonic extracts at different developmental stages, was consistent with the level of mRNA encoding these proteins. In particular epimerase activity at 10 hpf was twice that observed at the 64-cells stage (fig. 2b).
Figure 2 glce mRNA expression pattern in developing embryos. (a) RT-PCR analysis of the transcript level. Thirty embryos were collected in Tri-Reagent at different developmental stages as indicated. cDNA was generated from total RNA (1 μg) using Sensiscript reverse transcriptase primed with oligo-dT at 37°C for 2 h. PCR reactions (25 cycles) were performed in duplicate and analyzed on 1% agarose gel. (b) Glce enzymatic activity in embryos at different developmental stage. At each time point 20 embryos were dechorionated and homogenized. For the enzymatic assay, a cell lysate was incubated (2 h at 28°C) with labeled bacterial K5 heparosan substrate and the 3H2O liberated as result of the epimerization of GlcA into IdoA, measured. The bars represent the mean ± SD of the values from three independent determinations. (c-l) whole-mount in-situ hybridization of glce-a in embryos at different stages of development. (c-j) Top row: lateral views. Bottom row: animal pole views. (c,d) blastoderm at 64 cells stage; (e,f) dome stage; (g,h) shield stage; (i,j) 3 somite stage. (k) 24 hpf embryo showing showing intense glce staining at the perimeter of the forth ventricle as indicated by the arrow-heads. (l) enlargment of the embryo brain forth ventricle area.
Temporal and spatial expression of Glce
glce transcripts were localized in embryos at different developmental stages by in-situ hybridization using gene-specific antisense riboprobes (fig. 2c–l). During the early stages a diffuse staining was observed throughout the blastoderm. At the beginning of segmentation, staining was detected along the entire dorsal axis (fig. 2i,j). At 24 hpf, however, glce expression was higher in the newly forming brain (fig. 2k). At this site epimerase transcripts were mostly detected at the perimeter of the forth ventricle (fig. 2l). The staining specificitity was confirmed through the use of sense control riboprobes in which case only background staining was seen. No significant differences were detected in the expression pattern of the two glce genes.
Overexpression of Glce causes ventralization and potentiates Bmp activity
To investigate the functional significance of the epimerase during zebrafish development the protein was ectopically expressed by injecting embryos (1–2 cell stage) with capped in vitro-transcribed mRNA. The majority of injected embryos displayed a ventralizing phenotype whose severity correlated with the dose of mRNA injected (250 to 1000 pg) (table 1). The affected embryos had smaller head size, expanded blood islands, and abnormal tail somites (fig. 3b,c,f). More strongly ventralized embryos also lacked a notochord and developed somites that were not chevron-shaped and were fused in the midline below the neural tube (fig. 3g). Overexpression of glce-B produced an identical spectrum of phenotypes as the overexpression of glce-A. However, the highest frequency of severely affected embryos was observed when 250 or 500 pg of glce-A and glce-B mRNA were administered together in which case most of the animals failed to form an anterior axis (fig. 3d). In this treatment group, epimerase enzymatic activity at 10 hpf was 73% higher the level detected in uninjected embryos (fig 3h).
Table 1 Effect of the ectopic expression of Glce on Bmp2b ventralizing activity. Capped glce and bmp2b mRNAs were generated by reverse transcription from full length cDNA clones. After extraction in phenol/chloroform and precipitation in isopropanol, mRNA was dissolved in Danieau's buffer and the concentration assessed by UV reading (260 nm). Embryos at one or two cell stage were injected with 1–3 nl of mRNAs to achieve the indicated dose. Each injection session consisted of 2–3 treatment groups of 30 embryos each and several experiments were performed to reach the sample number indicated. Embryos with increasing degree of ventralization were ranked according to previously established criterias [40,41]. Ventralized V1 embryos show reduced eye size and shorter body. In addition to these abnormalities V2 embryos display abnormal notocord, reduced anterior somites, and enlarged blood islands. Ventralized V3 embryos have little or no head structures and no notochord. V4 embryos display gross body abnormalities and lack of anterior structures.
Injected mRNA Strength of Ventralization
No. Wild Type V1 V2 V3 V4
(%) (%) (%) (%) (%)
Uninjected 120 100 0 0 0 0
glce-A (250 pg) 60 65 23 10 2 0
glce-B (250 pg) 60 54 16 28 2 0
glce-A (125 pg)
glce-B (125 pg) 30 60 7 23 10 0
glce-A (250 pg)
glce-B (250 pg) 30 37 6 10 47 0
glce-A (500 pg)
glce-B (500 pg) 60 26 0 38 35 0
bmp2b (20 pg) 50 0 26 37 26 11
bmp2b (20 pg)
glce-A (250 pg) 60 0 0 15 45 40
bmp2b (20 pg)
glce-B (250 pg) 30 0 0 26 44 30
Figure 3 Effect of Glce overexpression on embryo morphogenesis and HS composition. Embryos at 1–2 cells stage were injected with glce-A and/or glce-B mRNA and observed at 24 hpf. (a) wild type embryo. (b,c) mild and moderately ventralized embryos showing enlarged blood sac (indicated by an arrow). (d) Severely ventralized embryos displaying dramatically reduced head and trunk. (e-g) High-contrast images of the somites and the notocord (nt) structures in control (e), and in mildly and moderately ventralized embryos (f,g). Note the loss of chevron-like structure of the somites and the narrowing or disappearance of the notocord in embryos overexpressing glce (f,g). (h) Epimerase enzymatic activity at 10 hpf in controls (WT) and in embryos injected with glce-A plus glce-B mRNA (250 pg each). The enzymatic assay was performed as described in Fig. 2b. (i-t) Whole mount in-situ hybridization with D/V markers in embryos during gastrulation and at 5 somite stage. Top row: wild type embryos. Bottom row: embryos injected with 250 pg each of glce-A and glce-B mRNA. (i,j) eve1 staining viewed from the animal pole at shield stage. The arrowheads point to the edges of the expression range of the marker; (k,l) bmp2b, lateral view with the dorsal side to the right at 70% epiboly; (m,n) fkd3, animal pole view at 70% epiboly; (o,p) chordino, dorsal view at 50% epiboly; (q,r) krox-20/opl double staining (head view) and (s,t) myo-D (dorsal view) in embryos at 5 somite stage. Note in (r) the narrow expression domain of krox20 in embryos injected with glce mRNA whereas opl transcript is undetectable.
In order to better characterize the phenotype of embryos overexpressing Glce, the expression of dorsal and ventral markers were analyzed by in situ hybridization [25,26]. The expression domain of eve1, a marker of ventral and lateral regions at early gastrula stages, was expanded at the shield stage (fig. 3i,j). Likewise the range of expression of bmp2b was greatly enlarged in embryos at the 70% epiboly stage (fig. 3k,l). In contrast expression of fkd3, a marker of the presumptive neuroectoderm, was reduced by Glce overexpression (fig. 3m,n). Similarly the expression domain of chordin, a marker of the dorsal organizer, was reduced (fig. 3o,p). The fact that overexpression of the epimerase alters the pattern but does not prevent the expression of dorsal determinants, is consistent with the idea that Glce acts on D/V axis formation downstream the Wnt/β-catenin pathway that regulates chordin gene expression [27]. glce is also a target of the Wnt/β-catenin transactivation pathway [28] raising the possibility that zygotic glce expression is coordinated with that of other D/V determinants.
Because head size is affected following ectopic expression of glce (fig 3b–d) the expression pattern of the neuroectoderm-specific markers krox 20 [29] and opl (zic1) [30] was determined during somitogenesis. The expression of myoD, a transcript specifically localized to somitic mesoderm was also examined [31]. During somitogenesis, krox-20 is normally expressed in hindbrain rhombomeres R3 and R5 both of which are dorsal ecdoderm derivatives. A reduced area of krox-20 expression was detected at the 5 somite stage in most of the embryos injected with 250 pg each of glce-A and glce-B mRNA whereas opl expression was undetectable (fig. 3q,r). myoD expression in the cells adjacent to the axial mesoderm and in the somitic furrows was also reduced implying a role of Glce in establishing mesodermal cell fate (fig 3s,t).
Since both the phenotype and marker gene expression pattern following ectopic expression of Glce is reminiscent of that of chordino [30,32-34], ogon [33,35,36] and radar [37,38] mutants or of embryos misexpressing alk 8 [39] in which the ventralizing activity of Bmps is enhanced, we examined whether Bmp2b activity is potentiated by Glce. For this purpose we titrated the dose of injected bmp2b mRNA to achieve a preponderance of partially ventralized embryos displaying V2 and V3 phenotype severity [40,41] (table 1). This same dose (20 pg) was then administered together with glce-A and/or glce-B mRNA (250 pg). Following the treatment, the majority of embryos exhibited the most severe V4 class phenotype consistent with Glce activity potentiating the effect of Bmp2b.
Glce availability affects Bmp-mediated ventralization
The effect of Glce protein knockdown on D/V axis formation was next examined by administering antisense morpholino oligonucleotides (MO) targeting glce-A and glce-B transcripts. Most of the embryos after injection of 4 ng of antisense MO, displayed a mild dorsalized phenotype with reduced ventral tail fin (fig. 4b and table 2). At higher dose (8 ng) about half of the morphants showed kinked tail, enlarged heart cavity and in some animals the atrioventricular boundaries failed to form (fig. 4c). A dramatic shortening and reduction of the body mass with tail coiling similar to the phenotype associated with mutation of Bmp2b, and Bmp7 [34,41] was observed in the majority of the embryos receiving the highest dose (16 ng) of MO (fig. 4d). In this group of morphants the epimerase enzymatic activity was significantly decreased (34% of the control at 8 hpf) (fig. 4e). The inability of the MOs to completely abolish the Glce activity suggests that at this time residual maternally derived enzyme is still active. In spite of this, the effect of Glce knockdown on ventral cell fate was already detectable in the mild morphants as revealed by the faint staining of gata1 expressing blood islets (fig. 4g), a ventral tissue derivative [42]. In stronger morphants, a broadening of the chordin expression domain wasobserved (fig. 4i–l). In addition, consistent with the axis dorsalization, the expression of bmp2b at shield stage was severely reduced as also evidenced by its undetectable expression in the tail during somitogenesis (fig. 4m–p). The administration of human or zebrafish glce mRNAs to embryos injected with MO rescued the enzymatic activity and prevented the development of the most severe dorsalized phenotypes (table 2).
Figure 4 Effect of Glce knockdown on embryo morphogenesis. (a-d) Embryos at 1–2 cells stage were injected with a mix (8 ng each) of glce-A and glceB MO and the phenotype scored at 48 hpf. (a) wild type embryo. (b,c) mild phenotypes displaying reduced head volume and ventral fin extension. (d) severe phenotype with shortened A/P axis and loss of ventral structures. (e) Epimerase enzymatic activity in 10 hpf embryos and effect of Glce knockdown. The enzymatic assay was performed as described in Fig. 2b. (f-p) Whole mount in-situ hybridization with D/V markers of embryos at different developmental stages. (f) gata1 expression in wild-type embryos, (g) glce morphants, and (h) in embryos overexpressing glce at 24 hpf. (i-l) chordino at 50% epiboly in wild type (top) and morphants (bottom). (i,j) animal pole view, (k,l) dorsal view. (m,n) bmp2b at 70% epiboly and (o,p) at 3 somite stage in wild type (top) and morphants (bottom). Note the absence of bmp2b expression in the presumptive tail of the morphants as indicated by the arrows.
Table 2 Effect on D/V axis formation of antisense targeting of glce. Capped mRNA and glce-MOs were dissolved in Danieau's buffer and injected as 1–3 nl bolus into the yolk of one- to two-cell embryos as described in Table 1. Capped human GLCE-mRNA was generated from a full length cDNA clone. The embryos were classified according to the severity of the observed phenotype at 48 hpf based on a classification of the dorsalization severity observed in embryos that had only received MOs. Mildly dorsalized embryos show reduced ventral tail fin extension. Moderately dorsalized embryos display kinked tail, enlarged heart cavity and in some case the absence of atrioventricular boundary. Severely affected morphants show dramatic shortening and reduction of the body mass with tail coiling. For the rescue experiments, embryos were first injected with MO and after randomization half received the indicated amount of mRNA before reaching the 4-cell stage.
Treatment Phenotype Severity
No. Wild Mild Moderate Severe
(%) (%) (%) (%)
Uninjected 42 100 0 0 0
glce-A MO (4 ng) 62 35 46 19 0
glce-A MO (8 ng) 40 20 25 39 16
glce-A MO (16 ng) 28 12 19 31 38
glce-B MO (16 ng) 30 25 19 30 26
glce-A MO (8 ng)
glce-B MO (8 ng) 30 15 10 30 45
glce-B MO (16 ng)
glce-A mRNA (200 pg) 68 28 38 22 12
glce-B MO (16 ng)
glce-B mRNA (200 pg) 30 32 45 15 8
glce-A MO (16 ng)
Human GLCE mRNA (200 pg) 35 21 41 29 9
In order to assess the dependency of Bmp activity on Glce level, embryos were injected with either 50 pg of bmp2b mRNA or 100 pg of bmp4 to generate a preponderance of V3-V4 ventralized embryos (table 3). Following randomization, half of the injected embryos received a mix of MOs targeting both glce-A and glce-B transcripts. About two-third of the embryos receiving the MOs displayed a normal-to-mild (V1-V2) ventralized phenotype whereas few developed the most severe V4 class phenotype. These results are in stark contrast to the embryos that had only received bmp2b and bmp4 mRNA supporting the concept that Glce is required for Bmp-mediated ventralization.
Table 3 Effect of antisense MO on the ventralizing activity of Bmps. Capped mRNA and glce MOs were injected into the yolk of one- to two-cell embryos as in the experiments of table 1. The resulting phenotypes were scored at 48 hpf. For the rescue experiments, embryos were first injected with bmp2b or bmp4 mRNA and after randomization half received the indicated amount of MO before reaching the 4-cell stage. Phenotype severity was scored as in Table 1.
Treatments Strength of Ventralization
No. Wild V1 V2 V3 V4
(%) (%) (%) (%) (%)
Uninjected 40 100 0 0 0 0
bmp2b mRNA (50 pg) 62 0 0 6 18 76
bmp2b mRNA (50 pg)
glce-A MO (8 ng)
glce-B MO (8 ng) 60 15 15 31 25 16
bmp4 mRNA (100 pg) 60 0 0 0 15 85
bmp4 mRNA (100 pg)
glce-A MO (8 ng)
glce-B MO (8 ng) 60 21 18 23 23 15
Discussion
In mammals, HS plays a crucial role in a variety of important biological processes including the regulation of blood coagulation, cell adhesion and differentiation, angiogenesis, and virus infection [1,3,43]. Most of the information concerning the role of HSPG in development has been obtained in the invertebrate model organism D. melanogaster and support the idea of a major functional role for HS in the morphogen's gradient establishment [3,14,44,45]. The fly mutants Sugarless [46], fringe connection [47], sulfateless [48], and tout-velu [49,50], display cuticle abnormalities that are reminiscent of the phenotypes exhibited by the mutations in Wg, Hh, or FGF and suggest an involvement of HS in the activity of these morphogens. In Drosophila the lack of HS also affects the body axis formation, but this effect is evident only at later stages of development [14]. Compared to the wealth of data generated in invertebrate species, the functional role of HSPG in vertebrate development is still poorly investigated. Transgenic mice carrying null-mutations in genes coding for enzymes implicated in HS chain polymerization [51], glucosamine N- or IdoA 2O-sulfation [52,53], and GlcA epimerization [11] are not viable leading to the conclusion that HS encoding critical structural epitopes is required for normal embryonic development [54]. The developmental mechanisms affected by the lack or by structurally aberrant HS, however remain to be assessed.
In this study the specific role of Glce has been investigated. GlcA epimerization endows the nascent polysaccharide HS with increased biological activity and is necessary to direct further chain sulfation at specific sites [3,6]. In mammals the enzyme is a single gene product whereas in zebrafish two genes have been identified likely arising from gene duplication. Transcripts for the two epimerases are already detected during the early cell divisions indicating a maternal contribution to the zygotic pool. The temporal expression pattern of glce closely resemble that of ext2-A suggesting that HS chain elongation and GlcA epimerization may be activated at the same time. Up to 12 hpf glce expression is detected along the entire axis. At 24 hpf however, the epimerase is highly expressed in the hindbrain, most notably along the perimeter of the fourth ventricle. In the hindbrain, at this developmental stage, Glce may play a specialized role involving axonal guidance as postulated based on observations made in C. elegans with mutated glce [13]. It will be of interest in future studies to compare the pattern of expression of glce and ext2 with that of the other enzymes involved in polysaccharide chain formation and sulfation to test the hypothesis that HS structure is developmentally regulated. For example zebrafish glucosamine 6O-sulfotransferase which act downstream to the biosynthetic step catalyzed by Glce, is not maternally derived [55] suggesting that GlcA epimerization and glucosamine sulfation represents two distinct pathways regulating HS structure during development.
The fact that the expression of Glce is rather ubiquitous throughout gastulation, has given us the opportunity to investigate the role of this enzyme by globally perturbing its level either by injecting capped mRNA or antisense oligonucleotides. When misexpressed the protein produced a ventralized phenotype similar to that observed in null-mutants for genes ogon [33,35,36], radar [37,38] and chordino [25,36] that directly modulate the function of the ventralizing agents Bmps albeit through different mechanisms. This phenotypic similarity lead us to focus on the role of Glce with respect to the activity of Bmps. During development the cell fate in zebrafish depends on the position within the embryo during blastula and gastrula stages. Positional information to cells are provided through the establishment of an activity gradient of Bmp proteins that promotes ventral specification in a dose dependent manner [40,56,57]. The idea that the specification of cells fate along the D/V axis is mediated through Bmp acting as terminal effectors, is supported by the fact that activation of the Bmp signaling pathway is a rather late event during embryogenesis and by the observation that functional inactivation of the zebrafish genes Bmp2b (swirl) [40] and Bmp7 (snailhouse) [34] both result in dramatic suppression of ventral fates and expansion of dorsal structures. Bmp2b and Bmp4 interact with HS through a cluster of positively charged aminoacids located at the N-terminus outside the receptor-binding domain of the protein [17]. Additional studies indicate that the interaction of Bmps with HS has important functional significance in that mutations in the HS/heparin binding domain results in an increase in the long-range activity of the morphogens [58]. HS also potentiates the biological activity of Bmps by serving the ligand to their receptor and/or by stabilizing the biological activity of the morphogen by preventing its proteolytic degradation [59]. Changes in IdoA content affecting HS binding to Bmp can thus change Bmp activity through different mechanisms. A spectrum of D/V mutants ranging from strongly ventralized to dorsalized embryos are generated when Glce activity is modulated suggesting that correct axial patterning requires that the activity of the epimerase be maintained within a critical range. An analysis of the structural domain in HS responsible for binding to Bmps can further elucidate what specific role IdoA residues play in this context.
Because HS ability to interact with proteins generally correlates positively with the IdoA content [2], Glce may be involved in the regulation of the activity of other heparin-binding morphogens involved in D/V axis formation. Fgf-8 has been demonstrated to play a key role is the establishment of D/V axis by acting upstream of Bmp2 and Bmp4 [60] and interacts with IdoA-rich regions in HS [61]. In zebrafish Fgf-8 inhibits the expression of Bmps in the ventral part of the embryo leading to the expansion of dorso-lateral derivatives at the expenses of ventral and posterior domains [60,62]. Based on our results an activation of Fgf-8 mediated pathway following ectopic Glce expression seems unlikely since an expansion rather than a reduction of ventral structures has been observed in embryos overexpressing the enzyme. It is possible that Glce overexpression inhibits Fgf-8 mediated signaling. This would occur if Fgf-8 is sequestered in the extracellular matrix by HS enriched in FGF binding regions or if the FGF receptor dimerization is negatively affected by HS [19]. However Fgf-8 null mutants display rather mild D/V abnormalities and similarly to fgf-8 morphants or mutant embryos with disrupted Ras/MAPK-mediated FGF signaling, do not form a midbrain-hindbrain boundary and do not develop the cerebellum [62]. Both these brain structures are present in the glce morphants and in embryos overexpressing glce unless, as a consequence of marked dorsalization or ventralization, the entire body plan is grossly altered. This finding is consistent with the observation that Glce-null mice have normal brain morphology [11] as it would be expected if Fgf-8 function is not affected [63]. Conceivably Fgf-8 mediated D/V patterning is little influenced by perturbation in Glce activity pointing to a downstream mediator of axis formation as sensitive to changes in HS IdoA content. A similar conclusion can be reached with regard to the D/V patterning effect of Wnt which acts upstream to Fgf-8, since activation of this pathway would result in posteriorization of the neural ectoderm affecting the eyes and the midbrain-hindbrain boundary development [64,65].
Taken together our results identify the stage of D/V patterning controlled by Bmp as sensitive to changes in HS structure. Previously it has been shown that mutants of the HSPG Dally have altered Dpp gradient formation resulting in abnormal patterning of the wing imaginal disc [16]. It was hypothesized that the HS chains of Dally bind Dpp and promote the interaction of the morphogen with the cognate cell surface receptor. Decreased interaction with HS, as may occurs when Glce activity is lowered, may reduce the concentration of Bmp available for interaction with the cognate receptors or the receptor-ligand binding is affected. Conversely, as a result of enhanced GlcA epimerization, HS affinity for Bmp may increase enhancing the concentration of the ligand at the receptor site and prolonging the morphogen activity [58,59,66]. The fact that Bmp is antagonized by proteins such as Chordin, Noggin, and Follistatin that require HS for diffusion and activation [67-69], represents an additional potential mechanism of regulation of Bmp activity that is dependent on HS. Chordin is required to dorsalize surrounding neuroectoderm and mesoderm and its expression pattern is affected when Glce activity is altered. A specific class of HSPG strongly potentiates the antagonism of Bmp signaling by Chordin and is necessary for the retention of Chordin by cells [69]. Likewise the interaction of Noggin with HSPG in vivo regulate its diffusion [67]. Conceivably in tissues rich of HS that binds with high affinity to Chordin and Noggin, the range of action of these Bmp antagonists is reduced and the ventralizing effect of Bmps may prevail. Our results support the hypothesis that correct D/V patterning depends upon the regulated expression of specific structural elements in HS and provide the basis for the interpretation of the functional role of Glce in vivo.
Conclusion
The results obtained corroborate the concept that HS encodes information that directs morphogenesis during early vertebrate development. In particular Glce emerges from this study as an important modulator of vertebrate morphogenesis that acts in a dose-dependent fashion on D/V axis formation. Bmp-dependent cell fate specification is the main target of Glce activity. Glce effect may be mediated by potentiating the effect of Bmps or by restricting the range of action of other HS-binding proteins such as Chordin and Noggin that by antagonizing Bmps act as indispensable dorsalizing agents.
Methods
Zebrafish breeding and phenotype scoring
Embryos were obtained from natural mating of wild-type (Oregon, AB) fish and breed, raised and staged according to established criterias [70].
Cloning of zebrafish Glce cDNA
A query of the zebrafish dEST database identified a number of putative clones whose translated sequence matched the N- and C-terminus of the human protein. Analysis of the predicted protein sequence of these clones indicated that zebrafish have two highly homologous Glce proteins. Cloning of the putative glce cDNAs was performed by reverse transcription of adult male zebrafish mRNA (Qiagen Sensiscript) primed with oligo-dT. cDNAs were amplified by PCR by combining primers matching the different possible cDNA terminal sequences. Forward primers were 5'-ATGCGCTGTCTGGTGGCTCGAATCAATC ACAAGACT-3' and 5'-ATGCGTTGTCTGGCAGCCGGTGTTCACTACAAGACC-3'. Reverse primers were 5'-CTAGTTGTGCTTAGCCCGACCTCCTTTCAGGTAAGT-3' and 5'-TTAATTGTGCTTAGCCCTCCCTCCTTTCAGGTAGCT-3'. This strategy resulted in two products of the expected size (~1.8 kb) from two of the four possible primer combinations. The amplified products were named glce-A (GenBank AY388516) and glce-B (AY388517), cloned into pcDNA3.1-TOPO (Invitrogen) and sequenced.
The 5'-end translated region of the zebrafish homologue of human exostosin 2 (EXT2) gene, was cloned using a similar strategy. The existence of two zebrafish ext2 genes were predicted from alignments of published EST sequences. The 5'-end of the ext2-A coding sequence including part of the UT region was cloned by RT-PCR using forward and reverse primers of sequence 5'-CATTCAACTTAAATATTCACCATA-3' and 5'-GGCGCTCAGCAGGTCATTGTATTC-3' respectively. Sequencing of an expected 528 bp fragment confirmed the identity of the amplified cDNA.
Chromosomal mapping of zebrafish glce
In the human, the glce translational start site is located in a 602 bp exon. Assuming that zebrafish glce genes maintain the same genomic organization as the human, PCR primers were designed to amplify the exon containing the translation initiation site for each each zebrafish orthologue. PCR amplification with primers 5'-ATGCGCTGTCTGGTGGCTCGAATC-3' and 5'-AGATGAAGGGCAGATACACCTCGC-3' for glce-A and 5'-ATGCGTTGTCTGGCAGCCGGTGTTCACTACAAG-3' and 5'-GACCTTTAATGGTGGCATCGTCATTGATCAGGC-3' for glce-B using genomic male zebrafish DNA as template, gave products of the expected size (420 bp and 261 bp for glce-A and glce-B respectively) that were cloned in pGEM-T vector and sequenced to confirm their identity. These sets of primers were then used to determine the chromosomal location of each gene by radiation hybrid panel (LN54) mapping.
Antisense targeting of the transcripts
Antisense Morpholino oligonucleotides (MOs) (Gene Tools LLC) were designed to target the 5'-UT region of the genes of interest. glce-A and glce-B MOs had sequence 5'-AGCCATGAGGAACACGTCAGCAAAC-3' and 5'-TCCCTGCTTACCTGCAATGCAAACA-3', respectively. MOs were dissolved in water at a concentration of 4 mM and diluted in Danieu's buffer before injection.
Generation of capped mRNA
Full-length glce cDNAs were subcloned in pT3TS vector at the BglII and EcoRV sites and in vitro-transcribed capped mRNAs were synthesized (T3 mMESSAGE mMACHINE kit, Ambion). mRNAs (1 μg) were tested prior to injection for protein expression in vitro using a rabbit reticulocyte lysate assay kit and 35S-methionine. Labeled proteins were separated on 9% SDS-PAGE gel followed by autoradiography. Human glce mRNA was generated from a full length cDNA clone in pcDNA 3.1 vector using T7 RNA polymerase. The human clone (AY635582) was obtained by RT-PCR using primers of sequence 5'-CTGCATATGCTGTGCTTGGCA-3' and 5'-CTAGTTGTGCTTTGCCCTGCTGCCTTT-3' based on published coding sequences and on cDNA 5'-end extension experiments we have performed [28]. bmp2b and bmp4 capped mRNA were kindly provided by Dr M. Mullins (U.Penn).
Microinjection
MOs and mRNAs were injected (1–3 nl) into the yolk of 1–2 cell embryos [71]. Post-injection (6 h) embryos were sorted, the unfertile/damaged removed and the rest allowed to grow at 28°C for further observation.
RNA in situ hybridization of zebrafish embryos
Antisense digoxigenin-labeled riboprobes were generated using SP6 or T7 RNA polymerase-based labeling kit (Roche). cDNA clones for glce-a, glce-b, chordino, krox-20, opl, myoD, were generated by RT-PCR [see Additional file 1]. bmp2b, fkd3, and eve1 antisense riboprobes were generated by reverse transcription from cDNA clones (cb670, cb114, and cb872 respectively) obtained from the Zebrafish International Resource Center (University of Oregon). Whole embryo in situ hybridization was performed as previously described [72].
Semi-quantitative RT-PCR
RNA was extracted and cDNA generated by reverse transcriptase using oligo-dT primers. An aliquot of the reaction was used as template for PCR amplification using gene-specific primers (Appendix I). Reactions were performed in duplicate and the product generated after 20 and 30 cycles analyzed by agarose electrophoresis to ensure that the products were quantitated during the exponential phase of the chain reaction.
Epimerase enzymatic activity assay
Embryos were collected at the indicated times, dechorionated and washed in ice-cold 25 mM HEPES, pH 7.0 buffer containing 0.1% Triton X-100. Embryos were then homogenized in 200 μl of the same buffer with a pestle that fits tightly into an Eppendorf tube and stored at -70°C. The substrate for the epimerase enzymatic assay consisted of radiolabeled modified bacterial N-acetylheparosan prepared as described previously [28]. The final N-sulfated heparosan product was purified by ion-exchange chromatography and eluted at higher ionic strength than the starting bacterial polysaccharide (0.66 M vs. 0.40 M NaCl). The epimerase enzymatic assay was performed as described by Crawford et al. [4]. Briefly, reactions were set up by combining the homogenates from 20 embryos with labeled substrate (1 nmole ~30,000 dpm) followed by 2 h incubation at 28°C. Reactions were stopped by addition of DEAE-Sepharose (1 ml) equilibrated in 50 mM Na-acetate, 50 mM NaCl pH 4.0 buffer (1:1 volume) followed by 15 min incubation at 4°C. Tritiated water generated as a result of the epimerization of GlcA into IdoA was recovered in the supernatant following centrifugation at 10,000 rpm. The sample and a 800-μl rinse of the DEAE slurry with buffer, were counted for the associated radioactivity in a Wallac 1219 Rackbeta liquid scintillation counter. Values were corrected for a reaction blank obtained by adding the substrate to the embryo homogenate just prior to the addition of DEAE-Sepharose.
Authors' contributions
GG cloned the zebrafish glce gene and the cDNAs used in the study, generated the capped mRNAs, performed the Glce enzymatic assay, and drafted the manuscript. SAF supervised the zebrafish injection experiments, provided training regarding the whole-mount in situ hybridization and performed some of the microscope analysis.
Supplementary Material
Additional file 1
Oligonucleotide primers Primers used for semiquantitative RT-PCR analysis and for the generation of riboprobes
Click here for file
Acknowledgements
The Authors wish to thank Dr. Mary Mullins for the help provided with the evaluation of the morphants' phenotype, for providing the Bmp capped mRNAs, and for the insightful discussion of the results. Mr Amit Agrawal and Chirag Patel have provided excellent technical support with many of the molecular biology techniques utilized. This work was supported by grant RO1-CA82290 to GG, by a Pew Scholar Award to S.F., and in part by RO1-GM063904 to S.F.
==== Refs
Bernfield M Gotte M Park PW Reizes O Fitzgerald ML Lincecum J Zako M Functions of cell surface heparan sulfate proteoglycans Annu Rev Biochem 1999 68 729 777 10872465 10.1146/annurev.biochem.68.1.729
Casu B Lindahl U Structure and biological interactions of heparin and heparan sulfate Adv Carbohydr Chem Biochem 2001 57 159 206 11836942
Esko JD Selleck SB Order out of chaos: assembly of ligand binding sites in heparan sulfate Annu Rev Biochem 2002 71 435 471 12045103 10.1146/annurev.biochem.71.110601.135458
Crawford BE Olson SK Esko JD Pinhal MA Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase J Biol Chem 2001 276 21538 21543 11279150 10.1074/jbc.M103414200
Lindahl U Kusche-Gullberg M Kjellen L Regulated diversity of heparan sulfate J Biol Chem 1998 273 24979 24982 9737951 10.1074/jbc.273.39.24979
Li JP Gong F El Darwish K Jalkanen M Lindahl U Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate J Biol Chem 2001 276 20069 20077 11274177 10.1074/jbc.M011783200
Mulloy B Forster MJ Conformation and dynamics of heparin and heparan sulfate Glycobiology 2000 10 1147 1156 11087707 10.1093/glycob/10.11.1147
Ferro DR Provasoli A Ragazzi M Casu B Torri G Bossennec V Perly B Sinay P Petitou M Choay J Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences Carbohydr Res 1990 195 157 167 2331699 10.1016/0008-6215(90)84164-P
Inoue Y Inouye Y Nagasawa K Conformational equilibria of the L-iduronate residue in non-sulphated di-, tetra- and hexa-saccharides and their alditols derived from dermatan sulphate Biochem J 1990 265 533 538 2302183
Maccarana M Casu B Lindahl U Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor J Biol Chem 1994 269 3903 8106436
Li JP Gong F Hagner-Mcwhirter A Forsberg E Abrink M Kisilevsky R Zhang X Lindahl U Targeted disruption of a murine glucuronyl C5-epimerase gene results in heparan sulfate lacking L-iduronic acid and in neonatal lethality J Biol Chem 2003 278 28363 28366 12788935 10.1074/jbc.C300219200
Ledin J Staatz W Li JP Gotte M Selleck S Kjellen L Spillmann D Heparan sulfate structure in mice with genetically modified heparan sulfate production J Biol Chem 2004 279 42732 42741 15292174 10.1074/jbc.M405382200
Bulow HE Hobert O Differential sulfations and epimerization define heparan sulfate specificity in nervous system development Neuron 2004 41 723 736 15003172 10.1016/S0896-6273(04)00084-4
Perrimon N Bernfield M Specificities of heparan sulphate proteoglycans in developmental processes Nature 2000 404 725 728 10783877 10.1038/35008000
Reichsman F Smith L Cumberledge S Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction J Cell Biol 1996 135 819 827 8909553 10.1083/jcb.135.3.819
Jackson SM Nakato H Sugiura M Jannuzi A Oakes R Kaluza V Golden C Selleck SB dally, a Drosophila glypican, controls cellular responses to the TGF- beta-related morphogen, Dpp Development 1997 124 4113 4120 9374407
Ruppert R Hoffmann E Sebald W Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity Eur J Biochem 1996 237 295 302 8620887 10.1111/j.1432-1033.1996.0295n.x
Rubin JB Choi Y Segal RA Cerebellar proteoglycans regulate sonic hedgehog responses during development Development 2002 129 2223 2232 11959830
Ornitz DM FGFs, heparan sulfate and FGFRs: complex interactions essential for development Bioessays 2000 22 108 112 10655030 10.1002/(SICI)1521-1878(200002)22:2<108::AID-BIES2>3.0.CO;2-M
Walsh EC Stainier DY UDP-glucose dehydrogenase required for cardiac valve formation in zebrafish Science 2001 293 1670 1673 11533493 10.1126/science.293.5535.1670
Inatani M Irie F Plump AS Tessier-Lavigne M Yamaguchi Y Mammalian brain morphogenesis and midline axon guidance require heparan sulfate Science 2003 302 1044 1046 14605369 10.1126/science.1090497
Lee JS von der HS Rusch MA Stringer SE Stickney HL Talbot WS Geisler R Nusslein-Volhard C Selleck SB Chien CB Roehl H Axon sorting in the optic tract requires HSPG synthesis by ext2 (dackel) and extl3 (boxer) Neuron 2004 44 947 960 15603738 10.1016/j.neuron.2004.11.029
Hirokawa T Boon-Chieng S Mitaku S SOSUI 1998
Hofmann K Stoffel W TMPERD 1993
Mullins MC Hammerschmidt M Kane DA Odenthal J Brand M van Eeden FJ Furutani-Seiki M Granato M Haffter P Heisenberg CP Jiang YJ Kelsh RN Nusslein-Volhard C Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes Development 1996 123 81 93 9007231
Hammerschmidt M Serbedzija GN McMahon AP Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor Genes Dev 1996 10 2452 2461 8843197
Shimizu T Yamanaka Y Ryu SL Hashimoto H Yabe T Hirata T Bae YK Hibi M Hirano T Cooperative roles of Bozozok/Dharma and Nodal-related proteins in the formation of the dorsal organizer in zebrafish Mech Dev 2000 91 293 303 10704853 10.1016/S0925-4773(99)00319-6
Ghiselli G Agrawal A The human D-glucuronyl C5-epimerase gene is transcriptionally activated through the beta-catenin/TCF4 pathway Biochem J 2005 15853773
Oxtoby E Jowett T Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development Nucleic Acids Res 1993 21 1087 1095 8464695
Rohr KB Schulte-Merker S Tautz D Zebrafish zic1 expression in brain and somites is affected by BMP and hedgehog signalling Mech Dev 1999 85 147 159 10415355 10.1016/S0925-4773(99)00044-1
Weinberg ES Allende ML Kelly CS Abdelhamid A Murakami T Andermann P Doerre OG Grunwald DJ Riggleman B Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos Development 1996 122 271 280 8565839
Gonzalez EM Fekany-Lee K Carmany-Rampey A Erter C Topczewski J Wright CV Solnica-Krezel L Head and trunk in zebrafish arise via coinhibition of BMP signaling by bozozok and chordino Genes Dev 2000 14 3087 3092 11124801 10.1101/gad.852400
Miller-Bertoglio V Carmany-Rampey A Furthauer M Gonzalez EM Thisse C Thisse B Halpern ME Solnica-Krezel L Maternal and zygotic activity of the zebrafish ogon locus antagonizes BMP signaling Dev Biol 1999 214 72 86 10491258 10.1006/dbio.1999.9384
Schmid B Furthauer M Connors SA Trout J Thisse B Thisse C Mullins MC Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation Development 2000 127 957 967 10662635
Yabe T Shimizu T Muraoka O Bae YK Hirata T Nojima H Kawakami A Hirano T Hibi M Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling Development 2003 130 2705 2716 12736214 10.1242/dev.00506
Wagner DS Mullins MC Modulation of BMP activity in dorsal-ventral pattern formation by the chordin and ogon antagonists Dev Biol 2002 245 109 123 11969259 10.1006/dbio.2002.0614
Sidi S Goutel C Peyrieras N Rosa FM Maternal induction of ventral fate by zebrafish radar Proc Natl Acad Sci U S A 2003 100 3315 3320 12601179 10.1073/pnas.0530115100
Goutel C Kishimoto Y Schulte-Merker S Rosa F The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish Mech Dev 2000 99 15 27 11091070 10.1016/S0925-4773(00)00470-6
Payne TL Postlethwait JH Yelick PC Functional characterization and genetic mapping of alk8 Mech Dev 2001 100 275 289 11165484 10.1016/S0925-4773(00)00541-4
Nguyen VH Schmid B Trout J Connors SA Ekker M Mullins MC Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes Dev Biol 1998 199 93 110 9676195 10.1006/dbio.1998.8927
Kishimoto Y Lee KH Zon L Hammerschmidt M Schulte-Merker S The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning Development 1997 124 4457 4466 9409664
Detrich HWIII Kieran MW Chan FY Barone LM Yee K Rundstadler JA Pratt S Ransom D Zon LI Intraembryonic hematopoietic cell migration during vertebrate development Proc Natl Acad Sci U S A 1995 92 10713 10717 7479870
Kjellen L Lindahl U Proteoglycans: structures and interactions Annu Rev Biochem 1991 60 443 475 1883201 10.1146/annurev.bi.60.070191.002303
Baeg GH Perrimon N Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila Curr Opin Cell Biol 2000 12 575 580 10978892 10.1016/S0955-0674(00)00134-4
De Cat B David G Developmental roles of the glypicans Semin Cell Dev Biol 2001 12 117 125 11292377 10.1006/scdb.2000.0240
Hacker U Lin X Perrimon N The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis Development 1997 124 3565 3573 9342049
Selva EM Hong K Baeg GH Beverley SM Turco SJ Perrimon N Hacker U Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events Nat Cell Biol 2001 3 809 815 11533660 10.1038/ncb0901-809
Lin X Buff EM Perrimon N Michelson AM Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development Development 1999 126 3715 3723 10433902
The I Bellaiche Y Perrimon N Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan Mol Cell 1999 4 633 639 10549295 10.1016/S1097-2765(00)80214-2
Toyoda H Kinoshita-Toyoda A Selleck SB Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo J Biol Chem 2000 275 2269 2275 10644674 10.1074/jbc.275.4.2269
Lin X Wei G Shi Z Dryer L Esko JD Wells DE Matzuk MM Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice Dev Biol 2000 224 299 311 10926768 10.1006/dbio.2000.9798
Fan G Xiao L Cheng L Wang X Sun B Hu G Targeted disruption of NDST-1 gene leads to pulmonary hypoplasia and neonatal respiratory distress in mice FEBS Lett 2000 467 7 11 10664446 10.1016/S0014-5793(00)01111-X
Merry CL Bullock SL Swan DC Backen AC Lyon M Beddington RS Wilson VA Gallagher JT The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse J Biol Chem 2001 276 35429 35434 11457822 10.1074/jbc.M100379200
Habuchi H Habuchi O Kimata K Sulfation pattern in glycosaminoglycan: does it have a code? Glycoconj J 2004 21 47 52 15467398 10.1023/B:GLYC.0000043747.87325.5e
Bink RJ Habuchi H Lele Z Dolk E Joore J Rauch GJ Geisler R Wilson SW Den Hertog J Kimata K Zivkovic D Heparan sulfate 6-O-sulfotransferase is essential for muscle development in Zebrafish J Biol Chem 2003 12782624
Mullins MC Embryonic axis formation in the zebrafish Methods Cell Biol 1999 59 159 178 9891360
Myers DC Sepich DS Solnica-Krezel L Bmp activity gradient regulates convergent extension during zebrafish gastrulation Dev Biol 2002 243 81 98 11846479 10.1006/dbio.2001.0523
Ohkawara B Iemura S ten DP Ueno N Action range of BMP is defined by its N-terminal basic amino acid core Curr Biol 2002 12 205 209 11839272 10.1016/S0960-9822(01)00684-4
Takada T Katagiri T Ifuku M Morimura N Kobayashi M Hasegawa K Ogamo A Kamijo R Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins J Biol Chem 2003 278 43229 43235 12912996 10.1074/jbc.M300937200
Furthauer M Thisse C Thisse B A role for FGF-8 in the dorsoventral patterning of the zebrafish gastrula Development 1997 124 4253 4264 9334274
Loo BM Salmivirta M Heparin/Heparan sulfate domains in binding and signaling of fibroblast growth factor 8b J Biol Chem 2002 277 32616 32623 12077148 10.1074/jbc.M204961200
Reifers F Bohli H Walsh EC Crossley PH Stainier DY Brand M Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis Development 1998 125 2381 2395 9609821
Chi CL Martinez S Wurst W Martin GR The isthmic organizer signal FGF8 is required for cell survival in the prospective midbrain and cerebellum Development 2003 130 2633 2644 12736208 10.1242/dev.00487
Kudoh T Wilson SW Dawid IB Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm Development 2002 129 4335 4346 12183385
Kelly GM Greenstein P Erezyilmaz DF Moon RT Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways Development 1995 121 1787 1799 7600994
Saksela O Moscatelli D Sommer A Rifkin DB Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation J Cell Biol 1988 107 743 751 2971068 10.1083/jcb.107.2.743
Paine-Saunders S Viviano BL Economides AN Saunders S Heparan sulfate proteoglycans retain Noggin at the cell surface: a potential mechanism for shaping bone morphogenetic protein gradients J Biol Chem 2002 277 2089 2096 11706034 10.1074/jbc.M109151200
Sugino K Kurosawa N Nakamura T Takio K Shimasaki S Ling N Titani K Sugino H Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell J Biol Chem 1993 268 15579 15587 8340384
Jasuja R Allen BL Pappano WN Rapraeger AC Greenspan DS Cell-surface heparan sulfate proteoglycans potentiate Chordin antagonism of bone morphogenetic protein signaling and are necessary for cellular uptake of Chordin J Biol Chem 2004 279 51289 51297 15381701 10.1074/jbc.M408129200
Westerfield M The zebrafish book 1995 Eugene, OR, University of Oregon
Nasevicius A Ekker SC Effective targeted gene 'knockdown' in zebrafish Nat Genet 2000 26 216 220 11017081 10.1038/79951
Thisse C Thisse B Schilling TF Postlethwait JH Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos Development 1993 119 1203 1215 8306883
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1131613893210.1186/1471-2164-6-113Methodology ArticleImproved tagging strategy for protein identification in mammalian cells Bialkowska Agnieszka [email protected] Xian-Yang [email protected] Jakob [email protected] Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA2005 4 9 2005 6 113 113 1 6 2005 4 9 2005 Copyright © 2005 Bialkowska et al; licensee BioMed Central Ltd.2005Bialkowska 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 tagging strategy enables full-length endogenous proteins in mammalian cells to be expressed as green fluorescent fusion proteins from their authentic promoters.
Results
We describe improved genetic tools to facilitate protein tagging in mammalian cells based on a mobile genetic element that harbors an artificial exon encoding a protein tag. Insertion of the artificial exon within introns of cellular genes results in expression of hybrid proteins consisting of the tag sequence fused in-frame to sequences of a cellular protein. We have used lentiviral vectors to stably introduce enhanced green fluorescent protein (EGFP) tags into expressed genes in target cells. The data obtained indicate that this strategy leads to bona fide tripartite fusion proteins and that the EGFP tag did not affect the subcellular localization of such proteins.
Conclusion
The tools presented here have the potential for protein discovery, and subsequent investigation of their subcellular distribution and role(s) under defined physiological conditions, as well as for protein purification and protein-protein interaction studies.
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Background
Technologies for increasingly comprehensive evaluation of RNA expression in mammalian cells yield qualitative, quantitative and temporal information about gene activity at the mRNA level. However, the correlation between mRNA and protein levels is often times poor because the rates of degradation of individual mRNAs and proteins differ [1,2] and because many proteins are modified after, they have been synthesized, so that one mRNA can give rise to more than one protein [3]. Thus, new tools are needed to detect global changes in protein expression patterns and to determine their subcellular localization [4,5]. Improved molecular tools are also needed to detect changes in protein expression and/or localization during differentiation and development allowing a detailed study of protein function at the single cell level.
Fusion of marker proteins such as β-galactosidase (β-Gal) or EGFP with cellular proteins facilitates detection of such proteins and provides information about their intracellular localization and their potential function(s) [6]. Most EGFP-based protein tagging techniques reported to date involved fragments of genomic libraries or individual cDNAs that were fused to the coding region of EGFP. Fusion proteins were subsequently expressed and their subcellular localization in target cells determined by microscopic inspection. Subsequently, the respective cDNAs or genes were rescued from the cells or tissues, cloned and sequenced. EGFP-tagged proteins can be immediately followed in living cells by time-lapse microscopy to determine their cellular dynamics [7]. However, when tagging proteins N-terminally or C-terminally, consideration must be given to the effect of the reporter protein on masking targeting signals contained within the expressed protein. For example, amino-terminal fusions of EGFP to target proteins potentially block signal sequences associated with import into mitochondria or the endoplasmic reticulum. Another disadvantage of the strategies described above is that they rely on exogenous promoters to drive expression of the tagged protein, possibly leading to higher levels of the tagged protein relative to its untagged endogenous counterpart. This again may impact the correct sorting of such proteins.
To bypass these shortcomings, alternative strategies to tag proteins were developed. Morin et al. [8] have presented a novel protein trap approach in which full-length endogenous proteins were expressed in Drosophila as EGFP fusion proteins from their endogenous promoters. They described a transposable artificial exon encoding an EGFP reporter. Devoid of initiation and stop codons and flanked by splice acceptor (SA) and splice donor (SD) sites, its insertion into an intron resulted in the production of a chimeric protein in which EGFP was fused with the trapped protein to yield a tripartite fusion protein. Several hundred independent lines were generated and shown, in the case of known proteins, that the chimera's subcellular distribution reflected that of the unmodified endogenous protein. Furthermore, the use of EGFP allowed a dynamic study of this distribution in live tissues. Jarvik et al. [9] have tested a similar approach in mammalian cells. Several hundred mouse NIH 3T3 cell clones expressing EGFP from Moloney murine leukemia virus (MLV)-based protein tagging vectors were isolated and some 60 of them analyzed. The cellular location of the tagged proteins analyzed corresponded with those of the untagged counterparts, indicating that the EGFP tag did not affect the subcellular sorting of the tagged proteins. Protein tagging approaches involving small epitope tags have also been described [10-12]. The usefulness of this approach in the context of mammalian cells has recently been established [13].
A shortcoming of the EGFP-based tagging strategy reported by Jarvik et al. [9] is that the MLV vectors tend to preferentially integrate within transcriptional start regions [14]. This may lead to a biased distribution of tags within protein coding regions. In this communication, we describe a system that overcomes this shortcoming by using lentiviral vectors to stably introduce EGFP in mammalian cells. The system also employs a removable drug resistance marker that allows for selection of insertion events into expressed genes.
Results
Design of improved protein tagging strategies
The protein tagging strategy is outlined in Figure 1. A drug resistance marker (blasticidin resistance gene, BSD) lacking a translation initiation codon and harboring two consecutive stop codons allows selection of cells expressing fused proteins by drug selection. To facilitate subsequent excision of the drug resistance marker by Cre recombinase [15], loxP sites were placed 5' and 3' of the BSD gene. Upon providing BSD-positive cells with Cre recombinase, the BSD resistance gene is deleted. This leaves behind an EGFP-encoding region, ultimately resulting in a tripartite fusion protein consisting of EGFP flanked by sequences of the tagged cellular protein (Figure 1).
Figure 1 Protein tagging system for mammalian cells. Schematic diagram of the tagging strategy. The strategy involves an artificial exon that is delivered into a target cells where it becomes stably associated with the host cell genome. The artificial exon consists of a BSD drug resistance gene sequence flanked by 34-bp loxP sites and lacking an ATG codon. An EGFP coding region lacking translational start and stop codons was placed in-frame with the BSD coding region. The artificial exon contains a branch point sequence, a polypyrimidine-tract sequence, the mandatory AG dinucleotide of the splice acceptor (SA) site and the mandatory GT of the splice donor (SD) site. The presence of a stop codon in the BSD cassette generates a fusion protein containing a truncated version of an endogenous protein fused to the BSD resistance protein and enables selection of BSD-resistant cell clones. Excision of BSD cassette by Cre-mediated recombination leads to the expression of EGFP fused to an endogenous protein. E1, E2 and E3 refer to cellular exons.
Protein tagging in human osteosarcoma cells
The usefulness of the method was tested in human osteosarcoma (HOS) cells. HOS cells were transduced with a modified lentiviral vector [16] bearing the artificial exon in the long terminal repeat (LTR) sequence (Figure 2). A total of 105 cells were transduced and then subjected to BSD selection (5 μg/ml) for two weeks resulting in a total of 300 BSD-resistant cell clones. Finally, seventy clones were isolated and subsequently transduced with a Cre recombinase-encoding lentiviral vector (NL-Cre). All seventy clones were subjected to FACS analysis. Six of the clones analyzed exhibited a significant increase in the percentage of EGFP-positive cells upon transduction with the NL-Cre vector encoding Cre recombinase as judged by flow cytometry (Figure 3) indicating that excision of BSD sequences and concomitant activation of EGFP expression had taken place.
Figure 2 Lentiviral delivery of protein tag-encoding sequences. The artificial exon sequence was incorporated into the U3 region of the lentiviral 3' LTR. During reverse transcription, a duplicated copy of the 3' LTR region replaces the 5' LTR region. Successful integration of this construct occurs into an intronic region of a cellular gene. Cre-mediated recombination involves two consecutive loxP-to-loxP recombination events and resulting in the excision of vector sequences between the loxP sites present in the 5' and 3' LTRs. This leaves behind the EGFP artificial exon as well as the R and U5 regions of viral 3' LTR integrated. ΔGAG, 5' portion of the gag coding region; RRE, Rev-response element; pSV40, simian virus early promoter; NEO, neomycin phosphotransferase coding region. Genomic DNA sequences are marked as straight black lines while vector sequences are marked orange.
Figure 3 Analysis of cell clones bearing EGFP-tagged proteins by flow cytometry. The panel shows FACS profiles from BSD-resistant cell clones before (gray) and after (green) transduction with a Cre-encoding lentiviral vector. Numbers 1 through 6 indicate individual clones.
Analysis of tagged proteins by confocal microscopy
Clones that exhibited significant EGFP fluorescence were then subjected to confocal microscopy to investigate the subcellular localization of EGFP fusion proteins. As shown in Figure 4, EGFP expression was detected in different subcellular compartments. For example, two of the clones analyzed displayed nuclear staining with EGFP accumulation in the nucleolus and nucleoplasm respectively, while four of the clones exhibited cytoplasmic staining.
Figure 4 Analysis of cell clones bearing EGFP-tagged proteins by confocal microscopy. The panels show the subcellular localization of the corresponding tagged proteins. Images were taken using confocal microscopy without (clones 1, 3, 5 and 6) and with deconvolution (clones 2 and 4). Numbers 1 to 6 correspond to clones displayed in Figure 3.
Identification of tagged proteins
To identify the tagged proteins, RNA was prepared from cell clones and then subjected to Rapid Amplification of cDNAs ends (RACE). The PCR products that were obtained following nested 3' and 5' RACE were sequenced and analyzed by BLAST (Table 1). In all cases, the subcellular localization of the EGFP-tagged proteins agreed with the reported subcellular localization of the untagged proteins indicating that the tag sequence had no adverse effects as far as the subcellular localization of the proteins analyzed is concerned. For example, the Ras-GTPase-activating protein-binding protein G3BP is ubiquitously distributed in the cytoplasm of cells and participates in cell signaling [17]. The nucleophosmin/B23 protein is a nucleolar phosphoprotein that changes its localization depending on the stage of the cell cycle and growth conditions [18]. Kinectin/KTN1 binds to the motor protein kinesin and participates in cellular transport mediated by microtubules. This protein is distributed throughout the cytoplasm. The Bcl-2-associated transcription factor/BTF is found in the nucleus in dot like structures and its translocation depends on apoptotic signals [19]. ST13 is an adaptor protein that mediates the association of the heat shock proteins HSP70 and HSP90 [20], while G3BP2 corresponds to the cytoplasmic Ras_GTPase activating protein SH3 domain-binding protein 2 [21].
Table 1 Examples of tagged proteins.
Clones number Protein name Localization Accession number [GeneBank:] Site of EGFP tag insertion
1 Ras_GTPase activating protein SH3 domain-binding protein (G3BP) cytoplasm AAH06997 -E8b
2 Nucleophosmin/B23 nucleolus NP_954654 E9-E10a
3 Kinectin1/KTN1(kinesin receptor) cytoplasm/membrane NP_891556 -E14b
4 Bcl-2-associated transcription factor (BTF) nucleus NP_055554 E6-E7a
5 Ras_GTPase activating protein SH3 domain-binding protein 2 (G3BP2) cytoplasm NP_036429 E7-E8a
6 Suppression of tumorigenicity 13/ST13 cytoplasm AAH52982 -E5b
aSite of EGFP tag insertion was determined using 5' and 3' RACE; E: Exon
bSite of EGFP tag insertion was determined using 3' RACE
Analysis of sites of proviral integration
Figure 5 shows the distribution of the tagging cassette among the various cell clones analyzed. In all cases, the tagging cassette had integrated either centrally or toward the 3' end of the gene sequence.
Figure 5 Schematic representation of insertion sites of the artificial exon. Sites of insertion were identified using 5' and/or 3' RACE followed by DNA sequencing. Green triangles represent the artificial exon. Red rectangles represent genomic exons, and blue rectangles represent genomic untranslated exons. The exons and introns arrangements were obtained from Human Genomic databases through NCBI.
Functional analysis of tagged proteins
In order to directly confirm that tagged proteins retained their physiological properties, we investigated a HOS cell clone expressing a B23/EGFP fusion protein. A Western blot analysis of extracts prepared from cells expressing the B23/EGFP fusion protein revealed a band around 67 kDa after probing with anti-EGFP antibody (Figure 6A). This finding is consistent with the view that a full-length version of B23 fused to EGFP was present in such extracts. B23 was previously shown to dissociate from nucleoli of cells after treatments with various anticancer drugs including daunomycin, actinomycin D, camptothecin, and toyocamycin [22]. To investigate whether B23/EGFP retained these properties, cells were treated with actinomycin D for 4 h at 37°C. Exposure to this drug provoked translocation of the B23/EGFP fusion protein from nucleoli to the nucleoplasm (Figure 6B). This shows that the fusion protein retained its translocation properties.
Figure 6 Analysis of clone bearing B23/EGFP fusion. A. Western blot analysis was performed as described in Methods section. Panel a: Extract from mock-transduced cells; Panel b: Extract from cells expressing a B23/EGFP fusion protein. B. Localization of B23/EGFP fusion protein after treatment with actinomycin D. Left panels: Control cells untreated. Right panels: Cells treated with 0.01 μg/ml of actinomycin D for 4 h at 37°C. After treatment, cells were washed twice with PBS, fixed for 5 min with formaldehyde, stained for 5 min with DAPI (0.8 μg/ml) and then washed once with PBS. Images were then taken using confocal microscopy with deconvolution.
Discussion
An attractive feature of the protein tagging method described in this study is that it is independent of antibody probes and allows for direct visualization of tagged proteins by confocal microscopy. This is in contrast to a recently described protein trapping method [13] involving oncogenic retroviral vectors encoding a myc epitope tag [11]. This method includes fixation and antibody labeling steps and is more cumbersome and ultimately limited to in vitro applications.
Lentiviral vector-mediated delivery of artificial exons for protein tagging provides a number of advantages over the traditional approaches involving oncoretroviral vectors [9,13]. In contrast to oncogenic retroviral vectors, lentiviral vectors can transduce dividing and non-dividing cells [23,24] and they appear to integrate preferentially into transcriptional units [14,25-27]. The preference for expressed genes for lentiviral vector integration is an attractive feature in the context of protein tagging strategies.
In our strategy, EGFP was used for tagging endogenous proteins and subsequent subcellular protein localization studies. However, insertion of a bulky EGFP moiety into cellular proteins may interfere with their native structure, thus leading to changes in their subcellular localization, stability and function. A report published by Jarvik et al. [9] clearly documented the usefulness of EGFP as a marker for protein tagging. These authors isolated more than 300 EGFP-expressing cell lines and more than 60 of them were analyzed in detail. The abundance and cellular location of the tagged proteins analyzed mirrored that of the untagged counterpart. Our data support this view. However, it remains to be determined on a case-by-case basis whether the tagged protein retains its biological activity.
Our method takes advantage of an initial enrichment step using BSD selection to enrich for cell clones expressing tagged cellular proteins. A subsequent Cre recombinase-mediated excision step removes all the vector sequences except for some 330 base pairs derived from the R and U5 regions of the vector LTR. A related selection/excision strategy for protein trapping events was described by Sineshchekova et al. [13]. In their strategy, the retroviral genome was retained. However, the presence of the complete vector genome could potentially affect the correct expression of the endogenous gene into which the vector genome has integrated.
Conclusion
Genome-wide protein tagging approaches have previously been implemented in Drosophila [8] and in the yeast Saccharomyces cerevisiae [28-31]. Tagged yeast strains have provided an unprecedented view of the yeast proteome in terms of expression levels of defined proteins and their subcellular localization and they have allowed investigating the dynamics of protein abundance and movement in cells in response to chemical and genetic influences. Similar applications are emerging in mammalian cells [9,13]. We expect the improved protein tagging strategy described in this communication to strengthen such approaches. We also believe that the tagging strategy described in this report will allow for the application of approaches akin to tandem affinity purification tagging [32] that can be used to purify and analyze protein complexes. Another potential application of our technique would involve the use of fluorescence resonance energy transfer (FRET) to detect protein-protein interactions between fluorescent tags on interacting proteins [33].
Methods
Plasmid constructs
Plasmid pNL-5.1 was constructed as follows. A mini-exon bearing SD and SA sequences, a myc tag encoding sequence [11] and EcoRI and PstI sites, flanked by 34-bp loxP sites was generated by overlap extension [34] and subcloned between the KpnI and XhoI sites present in pLITMUS 28 (New England BioLabs). A BSD resistance gene cassette without an ATG codon but containing two consecutive stop codons was generated by PCR using pcDNA6 /V5-His A (Invitrogen) and primers BSD-F-EcoRI (5'-tta tgg gaa ttc ctg gcc aag cct t-3') and BSD-R-PstI (5'-agt tat ctg cag tca tta gcc ctc cca cac ata-3'). The resulting PCR fragment was subcloned between the EcoRI and PstI sites present in the mini-exon sequence. The myc tag sequence was then replaced with EGFP. To do this, a PCR was performed using pEGFP-C1 (Clontech) as a template and two primers PstI/loxP/EGFP-F (5'-aac tgc aga taa ctt cgt ata atg tat gct ata cga agt tat ggg tga gca agg gcg agg agc-3') and XhoI/5'SS/EGFP-R (5'-cgg ctc gag cga gat cta ctt acc ttc ttg tac agc tcg tcc atg cc-3'). The resulting PCR fragment was subcloned between the PstI and XhoI sites replacing the myc tag sequence. The mini-exon sequence was released and subcloned into the 3' LTR of the pNL-neo vector [24] between the EcoRV site and an XbaI site placed 28 nucleotides upstream of the R region [35] to generate pNL-5.1. Dr. Alexander Chestkov provided the Cre recombinase-encoding pNL-Cre plasmid. A Cre recombinase-encoding fragment preceded by the CMV-IE promoter was derived from pBS185 (Life Technologies). The VSV-G envelope-encoding pLTR-G plasmid [23] and the pCD/NL-BH*ΔΔΔ helper plasmid [36] were described before. All plasmid sequences are available on-line .
Cell culture
Human embryonic kidney 293T cells [37] and HOS cells (ATCC, CRL-1543) were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10 mM HEPES, 10% heat inactivated FBS (HyClone), 2.5 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.
Virus production
Vector particles were produced in 293T cells by transient co-transfection involving a three-plasmid expression system [24]. Briefly, 293T cells were plated onto 150 mm plates in 25 ml of medium (8 × 106 cells per plate) and 24 h later, pNL-5.1 vector plasmid DNA (21 μg), pCD/NL-BH* ΔΔΔ helper plasmid DNA (14 μg), and pLTR-G DNA (7 μg) were added. Transfection by calcium phosphate in the presence of 25 μM chloroquine was carried out for 12–15 h. The medium was replaced and virus particles released into the medium were harvested 60–65 h after transfection. Vector particles were concentrated by ultracentrifugation as described [38]. Vector titers were determined by real-time PCR as described [36].
Transduction of cells and clone selection
HOS cells (1 × 105) were plated on 100 mm plates. 24 h later, NL-5.1 virus was added in medium containing 8 μg/ml of polybrene (Sigma). A multiplicity of infection (MOI) of 10 was used. Selection of BSD resistance colonies was carried on in medium containing 5 μg/ml of blasticidin (Invitrogen) for two weeks. Colonies were picked, expanded in 6-well plates and transduced with NL-Cre virus (MOI = 10). Transduction was followed by FACS analysis using a Becton-Dickinson FACSCalibur.
Analysis of tagged sequences
Total RNA was isolated using the Trizol reagent (Invitrogen) according to the manufacturer's protocol. 5' and 3' RACE was performed with the GeneRacer Kit (Invitrogen) according to the manufacturer's protocol. The 3' and 5' RACE products were run on a 1% agarose gel and the bands sequenced on a 3100 Genetic Analyzer (Applied Biosystems) using EGFP-specific primers. The sequences obtained were subsequently analyzed by BLAST.
Confocal analysis of cell clones
Cells (5 × 104) were plated onto cover slips in six well plates. After 24 h, the cells were washed twice with PBS, dried and mounted with ProLong Antifade supplied by Molecular Probes. Images were taken on a Nikon TE300 inverted microscope (confocal analysis) using the BioRad Radiance 2000 Laser Scanning Confocal System or a Leica DMRXA microscope and analyzed with Slidebook software 4.0 from Intelligent Imaging Innovations (deconvolution analysis).
Western blot analysis
Nuclear extracts were prepared by resuspending cell pellets in lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% v/v Nonidet P-40, 1 mM PMSF) and incubating for 5 min on ice. After centrifugation at 6,000 rpm, pellets were resuspended in extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% v/v glycerol, 1 mM DTT, 0.05 mM PMSF), incubated on ice for 15 min and the extracts centrifuged at 14,000 rpm for 15 min. Proteins were separated by PAGE using 4–12% NuPAGE Bis-Tris gels (Invitrogen). After electrophoresis, proteins were transferred to a Immobilon-P membrane (Millipore). The membrane was blocked with PBS containing 3% BSA for 2 h at room temperature. Probing was done using rabbit anti-GFP antibody (A-11122, Molecular Probes) diluted 1:200 in PBS containing 3% BSA for 1 h at room temperature followed by alkaline phosphate-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:2000 in PBS. The blot was developed using BCIP/NBT (Sigma-Aldrich).
Authors' contributions
A.B. and X.-Y.Z. performed all experiments. JR conceived the idea, designed and coordinated the study, and wrote major parts of the manuscript.
Acknowledgements
We thank Robert Kutner for vector production. This work was supported by NIH grants ES 012026 and NS 044832.
==== Refs
Greenbaum D Colangelo C Williams K Gerstein M Comparing protein abundance and mRNA expression levels on a genomic scale Genome Biol 2003 4 117 12952525
Tian Q Stepaniants SB Mao M Weng L Feetham MC Doyle MJ Yi EC Dai H Thorsson V Eng J Goodlett D Berger JP Gunter B Linseley PS Stoughton RB Aebersold R Collins SJ Hanlon WA Hood LE Integrated genomic and proteomic analyses of gene expression in Mammalian cells Mol Cell Proteomics 2004 3 960 969 15238602
Roberts GC Smith CW Alternative splicing: combinatorial output from the genome Curr Opin Chem Biol 2002 6 375 383 12023119
Simpson JC Pepperkok R Localizing the proteome Genome Biol 2003 4 240 14659010
Wiemann S Arlt D Huber W Wellenreuther R Schleeger S Mehrle A Bechtel S Sauermann M Korf U Pepperkok R Sultmann H Poustka A From ORFeome to biology: a functional genomics pipeline Genome Res 2004 14 2136 2144 15489336
Gonzalez C Bejarano LA Protein traps: using intracellular localization for cloning Trends Cell Biol 2000 10 162 165 10740271
Weijer CJ Visualizing signals moving in cells Science 2003 300 96 100 12677060
Morin X Daneman R Zavortink M Chia W A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila Proc Natl Acad Sci U S A 2001 98 15050 15055 11742088
Jarvik JW Fisher GW Shi C Hennen L Hauser C Adler S Berget PB In vivo functional proteomics: mammalian genome annotation using CD-tagging Biotechniques 2002 33 852 4, 856, 858-60 passim 12398194
Jarvik JW Adler SA Telmer CA Subramaniam V Lopez AJ CD-tagging: a new approach to gene and protein discovery and analysis Biotechniques 1996 20 896 904 8723939
Smith DJ Mini-exon epitope tagging for analysis of the protein coding potential of genomic sequence BioTechniques 1997 23 116 120 9232241
Telmer CA Berget PB Ballou B Murphy RF Jarvik JW Epitope tagging genomic DNA using a CD-tagging Tn10 minitransposon Biotechniques 2002 32 422 430 11848418
Sineshchekova OO Kawate T Vdovychenko OV Sato TN Protein-trap version 2.1: screening for expressed proteins in mammalian cells based on their localizations BMC Cell Biol 2004 5 8 15018653
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
Branda CS Dymecki SM Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice Dev Cell 2004 6 7 28 14723844
Reiser J Lai Z Zhang XY Brady RO Development of multigene and regulated lentivirus vectors J Virol 2000 74 10589 10599 11044103
Parker F Maurier F Delumeau I Duchesne M Faucher D Debussche L Dugue A Schweighoffer F Tocque B A Ras-GTPase-activating protein SH3-domain-binding protein Mol Cell Biol 1996 16 2561 2569 8649363
Yun JP Chew EC Liew CT Chan JY Jin ML Ding MX Fai YH Li HK Liang XM Wu QL Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix J Cell Biochem 2003 90 1140 1148 14635188
Haraguchi T Holaska JM Yamane M Koujin T Hashiguchi N Mori C Wilson KL Hiraoka Y Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery-Dreifuss muscular dystrophy Eur J Biochem 2004 271 1035 1045 15009215
Prapapanich V Chen S Nair SC Rimerman RA Smith DF Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein Mol Endocrinol 1996 10 420 431 8721986
Prigent M Barlat I Langen H Dargemont C IkappaBalpha and IkappaBalpha /NF-kappa B complexes are retained in the cytoplasm through interaction with a novel partner, RasGAP SH3-binding protein 2 J Biol Chem 2000 275 36441 36449 10969074
Chan PK Bloom DA Hoang TT The N-terminal half of NPM dissociates from nucleoli of HeLa cells after anticancer drug treatments Biochem Biophys Res Commun 1999 264 305 309 10527882
Reiser J Harmison G Kluepfel-Stahl S Brady RO Karlsson S Schubert M Transduction of nondividing cells using pseudotyped defective high- titer HIV type 1 particles Proc Natl Acad Sci U S A 1996 93 15266 15271 8986799
Mochizuki H Schwartz JP Tanaka K Brady RO Reiser J High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells J Virol 1998 72 8873 8883 9765432
Schroder AR Shinn P Chen H Berry C Ecker JR Bushman F HIV-1 integration in the human genome favors active genes and local hotspots Cell 2002 110 521 529 12202041
Mack KD Jin X Yu S Wei R Kapp L Green C Herndier B Abbey NW Elbaggari A Liu Y McGrath MS HIV insertions within and proximal to host cell genes are a common finding in tissues containing high levels of HIV DNA and macrophage-associated p24 antigen expression J Acquir Immune Defic Syndr 2003 33 308 320 12843741
Mitchell RS Beitzel BF Schroder AR Shinn P Chen H Berry CC Ecker JR Bushman FD Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences PLoS Biol 2004 2 E234 15314653
Ross-Macdonald P Coelho PSR Snyder M Large-scale analysis of the yeast genome by transpoon tagging and gene disruption Nature 1999 402 413 10586881
Kumar A Agarwal S Heyman JA Matson S Heidtman M Piccirillo S Umansky L Drawid A Jansen R Liu Y Cheung KH Miller P Gerstein M Roeder GS Snyder M Subcellular localization of the yeast proteome Genes Dev 2002 16 707 719 11914276
Huh WK Falvo JV Gerke LC Carroll AS Howson RW Weissman JS O'Shea EK Global analysis of protein localization in budding yeast Nature 2003 425 686 691 14562095
Ghaemmaghami S Huh WK Bower K Howson RW Belle A Dephoure N O'Shea EK Weissman JS Global analysis of protein expression in yeast Nature 2003 425 737 741 14562106
Rigaut G Shevchenko A Rutz B Wilm M Mann M Seraphin B A generic protein purification method for protein complex characterization and proteome exploration Nat Biotechnol 1999 17 1030 1032 10504710
Wouters FS Verveer PJ Bastiaens PI Imaging biochemistry inside cells Trends Cell Biol 2001 11 203 211 11316609
Horton RM Ho SN Pullen JK Hunt HD Cai Z Pease LR Gene splicing by overlap extension Methods Enzymol 1993 217 270 279 8474334
Chang LJ McNulty E Martin M Human immunodeficiency viruses containing heterologous enhancer/promoters are replication competent and exhibit different lymphocyte tropisms J Virol 1993 67 743 752 8419644
Zhang XY La Russa VF Reiser J Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins J Virol 2004 78 1219 1229 14722277
DuBridge RB Tang P Hsia HC Leong PM Miller JH Calos MP Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system Mol Cell Biol 1987 7 379 387 3031469
Marino MP Luce MJ Reiser J Federico M Small- to large-scale production of lentivirus vectors Lentivirus Gene Engineering Protocols Methods in Molecular Biology 2003 229 Totowa NJ , Humana Press 43 55
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1151615014810.1186/1471-2164-6-115Research ArticleMicroarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide Chang Wook [email protected] David A [email protected] Freshteh [email protected] William E [email protected] Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA2 Microarray Research Laboratory, Biological and Economic Analysis Division, Office of Pesticide Programs, U. S. Environmental Protection Agency, Fort Meade, Maryland 20755, USA2005 8 9 2005 6 115 115 1 3 2005 8 9 2005 Copyright © 2005 Chang et al; licensee BioMed Central Ltd.2005Chang 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
Pseudomonas aeruginosa, a pathogen infecting those with cystic fibrosis, encounters toxicity from phagocyte-derived reactive oxidants including hydrogen peroxide during active infection. P. aeruginosa responds with adaptive and protective strategies against these toxic species to effectively infect humans. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure.
Results
Herein, we performed a genome-wide transcriptome analysis of the cellular responses to hydrogen peroxide in order to determine a more complete picture of how oxidative stress-induced genes are related and regulated.
Our data reinforce the previous conclusion that DNA repair proteins and catalases may be among the most vital antioxidant defense systems of P. aeruginosa. Our results also suggest that sublethal oxidative damage reduces active and/or facilitated transport and that intracellular iron might be a key factor for a relationship between oxidative stress and iron regulation. Perhaps most intriguingly, we revealed that the transcription of all F-, R-, and S-type pyocins was upregulated by oxidative stress and at the same time, a cell immunity protein (pyocin S2 immunity protein) was downregulated, possibly leading to self-killing activity.
Conclusion
This finding proposes that pyocin production might be another novel defensive scheme against oxidative attack by host cells.
==== Body
Background
Many microorganisms continuously face a range of reactive oxygen species (ROS) including hydrogen peroxide, superoxide, and the hydroxyl radical derived from many sources. During the process of active infection, pathogenic bacteria are exposed to exogenous oxidative stress that phagocytes utilize as a host defense mechanism [1]. Even normal cellular metabolism produces cytotoxicity arising from its partially-reduced intermediates [2]. For instance, by reacting with intracellular iron, hydrogen peroxide can form the hydroxyl radical through the Fenton reaction, which damages various cellular molecules including lipids, proteins, and DNA [1,3]. Superoxide is also capable of promoting oxidative damage by increasing the concentration of intracellular iron [2,4,5]. Because of the vast array of stimuli and their sources, it is intuitive to anticipate that organisms have developed complex antioxidant strategies that serve to neutralize and repair oxidative damage.
Pseudomonas aeruginosa PA01 (P. aeruginosa), a Gram-negative pathogen responsible for respiratory infections in individuals with cystic fibrosis and cancer, is also known to possess a multifaceted defense system against reactive oxidants that includes such enzymes as catalase and superoxide dismutase [6-8]. There are many specific defense genes that have been identified and regulatory aspects of their activities have been elucidated in many cases [1,5]. Despite this marked progress, cystic fibrosis remains problematic and our knowledge of P. aeruginosa pathogenicity remains incomplete. A more thorough understanding of this bacterium's defense system might serve to enhance the development and efficacy of therapeutic agents for this disease. In particular, an understanding of the linkage between the cell's ROS defense mechanism and the remainder of the cell's metabolism can lead to more innovative methods for combating this pathogen. For example, better elucidation of the molecular events responsible for establishing and maintaining pathogenicity might improve optimal drug and vaccine design [9]. That is, by using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we might reinforce known relationships between genes with previously identified functions, and also reveal new target genes that give us more insight into P. aeruginosa-host interactions.
To provide a more complete linkage between cell physiology and the well-characterized defense response, we investigated genome-wide changes in P. aeruginosa gene transcription upon exposure to hydrogen peroxide using Affymetrix P. aeruginosa GeneChip arrays. Notably, we made a significant finding that hydrogen peroxide induced the transcription of each and every pyocin (bacteriocins) reported in P. aeruginosa. Moreover, we found that a pyocin immunity gene, which prevents bacterial cell death during pyocin synthesis, was downregulated, possibly leading to self-killing activity. Finally, we've corroborated an anticipated result regarding iron uptake; that oxidative stress in our experimental conditions lead to the repression of iron uptake genes.
Results and discussion
To investigate the effect of sublethal oxidative stress on P. aeruginosa, we performed a transcriptome analysis with microarrays upon 20 min exposure to 1 mM hydrogen peroxide. This concentration successfully induces sublethal oxidative damage in Escherichia coli and P. aeruginosa [10-12]. Besides providing requisite quantities of mRNA for microarray analyses, sublethal doses of antibiotics are receiving increased interest because of their potential for attenuating pathogenicity but with concern for increased rates of mutation and resistance [13,14]. We confirmed that 1 mM hydrogen peroxide caused strong growth inhibition but not cell death for the first 60 min post-treatment (data not shown).
To determine genome-wide transcriptional changes in response to hydrogen peroxide, we conducted four and five independent microarray experiments in the absence (control) and the presence (experimental) of hydrogen peroxide, respectively. Transcriptome analysis with Affymetrix P. aeruginosa GeneChip arrays suggested that mRNA levels of 805 and 827 out of a total of 5,570 genes were increased and decreased (IR <> 1), respectively, after 20 min treatment. We refer to "statistically marked" changes in transcript level for those genes that meet the following criteria: (i) a p-value for a Mann-Whitney test should be less than 0.05, (ii) an absolute fold change in transcript level should be equal to or greater than 2, and (iii) a gene should have a presence or marginal call [15] from 50% or more replicates on both the experimental and control replicate sets. Based on these criteria, we pared the set to 116 and 107 genes (a total of 223) that had statistically marked increases and decreases in transcript level, respectively. This result suggests that these genes might be associated with the defensive response of P. aeruginosa to hydrogen peroxide-induced oxidative stress. Interestingly, this number is comparable to that found in a study of E. coli cells treated with 1 mM hydrogen peroxide (140 genes, = 4-fold) [16], and significantly less than those of a comparable P. aeruginosa study (1,854 genes, ≥ 2-fold) [12]. In Table 1, from among the 223 genes, we list 30 most strongly induced and repressed genes as well as their putative functions, many of which are discussed below. Additionally, Supplementary Table 1 shows the average signals, p-values, and fold changes of all 5,570 predicted P. aeruginosa open reading frames. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [17] and are accessible through GEO Series accession number GSE3090.
Table 1 List of 30 P. aeruginosa genes most strongly induced and repressed in response to hydrogen peroxide
Gene (Name) Fold change p-value Protein (Function)
Induced Genes
PA5530 24.7 0.008 Probable MFS dicarboxylate transporter (Membrane proteins; Transport of small molecules)
PA1466 13.9 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA2288 10.3 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA3414 9.8 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA4763 (recN) 8.3 0.008 DNA repair protein (DNA replication, recombination, modification and repair)
PA0182 6.8 0.024 Probable short-chain dehydrogenase (Putative enzymes)
PA3008 5.7 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0670 5.4 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0612 5.3 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0641 5.0 0.008 Probable bacteriophage protein (Related to phage, transposon, or plasmid)
PA0671 5.0 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA4613 (katB) 4.5 0.024 Catalase (Adaptation, protection)
PA3413 4.4 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0620 4.2 0.008 Probable bacteriophage protein (Related to phage, transposon, or plasmid)
PA0615 4.2 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA5471 4.1 0.024 Hypothetical protein (Hypothetical, unclassified, unknown)
PA5470 4.1 0.024 Probable peptide chain release factor (Translation, post-translational modification, degradation)
PA3007 (lexA) 4.0 0.008 Repressor protein (Adaptation, protection; Translation, post-translational modification, degradation)
PA0922 3.7 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0635 3.6 0.008 Hypothetical protein (Related to phage, transposon, or plasmid)
PA0616 3.5 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0634 3.5 0.008 Hypothetical protein (Related to phage, transposon, or plasmid)
PA0625 3.4 0.008 Hypothetical protein (Related to phage, transposon, or plasmid)
PA0636 3.4 0.008 Hypothetical protein (Related to phage, transposon, or plasmid)
PA0617 3.4 0.008 Probable bacteriophage protein (Hypothetical, unclassified, unknown)
PA0633 3.3 0.008 Hypothetical protein (Related to phage, transposon, or plasmid)
PA0637 3.3 0.008 Conserved hypothetical protein (Related to phage, transposon, or plasmid)
PA3923 3.3 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA4582 3.2 0.008 Conserved hypothetical protein (Hypothetical, unclassified, unknown)
PA0638 3.2 0.008 Probable bacteriophage protein (Hypothetical, unclassified, unknown)
Repressed Genes
PA2398 (fpvA) 5.7 0.008 Ferripyoverdine receptor (Transport of small molecules)
PA4156 5.1 0.008 Probable TonB-dependent receptor (Transport of small molecules)
PA4230 (pchB) 4.3 0.008 Salicylate biosynthesis protein (Transport of small molecules; Secreted Factors)
PA2405 3.9 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA2409 3.7 0.008 Probable permease of ABC transporter (Membrane proteins; Transport of small molecules)
PA5235 (glpT) 3.4 0.008 Glycerol-3-phosphate transporter (Membrane proteins; Transport of small molecules)
PA4225 (pchF) 3.3 0.008 Pyochelin synthetase (Transport of small molecules; Secreted Factors)
PA2407 3.3 0.008 Probable adhesion protein (Motility & Attachment)
PA2403 3.2 0.008 Hypothetical protein (Hypothetical, unclassified, unknown; Membrane proteins)
PA5479 (gltP) 3.2 0.008 Proton-glutamate symporter (Membrane proteins; Transport of small molecules)
PA3009 3.1 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA2426 (pvdS) 3.0 0.024 Sigma factor (Transcriptional regulators)
PA2404 3.0 0.008 Hypothetical protein (Hypothetical, unclassified, unknown; Membrane proteins)
PA2667 3.0 0.008 Conserved hypothetical protein (Transcriptional regulators)
PA3610 (potD) 3.0 0.008 Polyamine transport protein (Transport of small molecules)
PA4555 (pilY2) 3.0 0.008 Type 4 fimbrial biogenesis protein (Motility & Attachment)
PA1414 2.9 0.024 Hypothetical protein (Hypothetical, unclassified, unknown)
PA0280 (cysA) 2.9 0.024 Sulfate transport protein (Transport of small molecules)
PA2408 2.9 0.008 Probable ATP-binding component of ABC transporter (Transport of small molecules)
PA4839 (speA) 2.9 0.008 Biosynthetic arginine decarboxylase (Amino acid biosynthesis and metabolism)
PA4231 (pchA) 2.9 0.008 Salicylate biosynthesis isochorismate synthase (Secreted Factors; Transport of small molecules)
PA0281 (cysW) 2.8 0.008 Sulfate transport protein (Membrane proteins; Transport of small molecules)
PA1228 2.8 0.008 Hypothetical protein (Hypothetical, unclassified, unknown)
PA5049 (rpmE) 2.8 0.008 50S ribosomal protein (Translation, post-translational modification, degradation)
PA2619 (infA) 2.7 0.008 Initiation factor (Translation, post-translational modification, degradation)
PA4221 (fptA) 2.7 0.008 Fe(III)-pyochelin receptor precursor (Transport of small molecules)
PA0976 2.7 0.008 Conserved hypothetical protein (Hypothetical, unclassified, unknown)
PA5446 2.7 0.048 Hypothetical protein (Hypothetical, unclassified, unknown)
PA4229 (pchC) 2.6 0.008 Pyochelin biosynthetic protein (Transport of small molecules; Secreted Factors)
PA4218 2.6 0.008 Probable transporter (Membrane proteins; Transport of small molecules)
To validate the relative transcript levels obtained by the array analysis, we employed quantitative real-time PCR analysis on PA4613 (katB), PA2850 (ohr), PA4763 (recN), and PA5530. These genes were selected since they displayed a range of mRNA level changes (-1- to 24-fold). Moreover, PA0576 (rpoD) was used as a control gene for the relative mRNA level calculation due to the fact that rpoD exhibits stable expression level [18]. As shown in Table 2, our microarray results were corroborated with quantitative real-time PCR analyses of the selected genes.
Table 2 Transcript level comparison of P. aeruginosa genes between real-time PCR analysis and microarray analysis
Gene mRNA level change Sense primer sequence (5'-3') Antisense primer sequence (5'-3')
real-time PCR microarray
PA4613 1.69 (± 0.44) 4.54 GAGCAGAACTTCAAGCAGAC CTCTCGTCGTCGGTGATC
PA2850 -1.64 (± 0.19) -1.37 GAGGTCGAACTGCACATC GGGTAGCGTTGGAGTAGG
PA4763 4.24 (± 0.17) 8.27 GGAGCAGGAGCAGAAGAC GTTGAGGCTGGCATTGAG
PA5530 34.26 (± 7.17) 24.70 AAGAAGGAAGAGCCGAAGG ATGTAGGTGGTGTAGGTGTAG
PA0576 CGTCCTCAGCGGCTATATCG TTCTTCTTCCTCGTCGTCCTTC
Functional classification
To examine how the genes are distributed with regard to their functions, we classified these 223 genes according to the categories described in the Pseudomonas aeruginosa Community Annotation Project [19] (see, Figure 1). Both Figure 1 and Table 1 indicate that the most distinctive feature was the completely uniform upregulation of genes involved in DNA modulation (Phage, transposon, or plasmid and DNA replication and repair); this is discussed further below. It also appeared that genes related to various membrane functions including "transport of small molecules", "secreted factors", and "membrane proteins" were noticeably downregulated, suggesting that hydrogen peroxide alters the regulation of membrane proteins. Even among the 30 most downregulated genes presented in Table 1, 17 genes belonged to these functional classes (PA2398 (fpvA), PA4156, PA4230 (pchB), PA2409, PA5235 (glpT), PA4225 (pchF), PA2403, PA5479 (gltP), PA2404, PA3610 (potD), PA0280 (cysA), PA2408, PA4231 (pchA), PA0281 (cysW), PA4221 (fptA), PA4229 (pchC), and PA4218). This result may reflect attenuation of active and/or facilitated transport through the cell membrane. Notably, many genes associated with these classes were found linked with iron regulation (discussed below). Also, consistent with the sublethal but significant applied stress, as shown in Table 3, several genes involved in primary metabolism exhibited decreased mRNA levels: (i) energy metabolism-related genes, PA1317 (cyoA), PA1319 (cyoC), and PA3621 (fdxA), and PA4133, (ii) polyamine synthesis and uptake genes (related to amino acid synthesis and metabolism), PA0654 (speD), PA1687 (speE), PA4839 (speA), and PA3607-3610 (potABCD), and (iii) ribosomal protein genes, PA4432 (rpsL), PA4563 (rpsT), PA5049 (rpmE), and PA5315 (rpmG). It is also interesting that putative cell division inhibitors such as PA0671 and PA3008, which are similar to E. coli sulA [20], showed increased transcript levels upon exposure to hydrogen peroxide, suggesting that these genes might promote the repression of primary metabolism.
Figure 1 Functional classification of genes with statistically significant increase and decrease in mRNA level (a total of 223 genes). The number in parenthesis represents the percentage of the total number of genes within the genome in each functional class.
Table 3 List of P. aeruginosa genes that were discussed in this report, categorized by their related functions.
Gene (Name) Average signal of experimentals Average signal of controls p-value Fold change a
Primary metabolism-related
PA1317 (cyoA) 70.5 159.9 0.048 -2.3
PA1319 (cyoC) 22.5 46.6 0.008 -2.1
PA3621 (fdxA) 321.7 648.6 0.008 -2.0
PA4133 46.8 106.6 0.024 -2.3
PA0654 (speD) 416.9 804.4 0.008 -1.9 c
PA1687 (speE) 77.8 204.8 0.008 -2.6
PA4839 (speA) 111.7 321.2 0.008 -2.9
PA3607 (potA) 65.8 143.7 0.008 -2.2
PA3608 (potB) 25.8 64.6 0.008 -2.5
PA3609 (potC) 40.0 83.00 0.008 -2.1
PA3610 (potD) 35.4 105.9 0.008 -3.0
PA4432 (rpsL) 493.8 1156.9 0.008 -2.3
PA4563 (rpsT) 1600.9 3648.5 0.008 -2.3
PA5049 (rpmE) 174.9 482.0 0.008 -2.8
PA5315 (rpmG) 340.3 761.5 0.008 -2.2
PA0671 127.5 25.4 0.008 5.0
PA3008 618.6 109.2 0.008 5.7
Cellular protective mechanism-related
PA4236 (katA) 543.8 275.1 0.008 2.0
PA4613 (katB) 152.6 33.6 0.008 4.5
PA4366 (sodB) 1779.4 1923.3 0.484 b -1.1 c
PA4468 (sodM) 27.7 18.8 0.008 1.5 c
PA3007 (lexA) 1267.9 320.9 0.008 4.0
PA3008 618.6 109.2 0.008 5.7
PA3616 289.7 105.2 0.008 2.8
PA3617 (recA) 1917.5 706.1 0.008 2.7
PA0669 59.4 20.9 0.008 2.8
PA3413 766.0 174.7 0.008 4.4
PA3414 459.5 47.0 0.008 9.8
PA4763 (recN) 1270.9 153.6 0.008 8.3
Iron regulation-related
PA2426 (pvdS) 21.0 63.0 0.024 -3.0
PA2398 (fpvA) 99.5 566.1 0.008 -5.7
PA4221 (fptA) 154.6 417.9 0.008 -2.7
PA4225 (pchF) 42.1 139.0 0.008 -3.3
PA4226 (pchE) 103.3 263.0 0.008 -2.6
PA4228 (pchD) 101.1 235.1 0.008 -2.3
PA4229 (pchC) 101.2 267.6 0.008 -2.6
PA4230 (pchB) 49.6 212.1 0.008 -4.3
PA4231 (pchA) 35.6 102.6 0.008 -2.9
PA2403 53.7 173.8 0.008 -3.2
PA2404 45.6 138.1 0.008 -3.0
PA2405 46.9 181.1 0.008 -3.9
PA2406 61.4 113.3 0.008 -1.8 c
PA2407 91.8 300.9 0.008 -3.3
PA2408 35.8 103.7 0.008 -2.9
PA2409 50.9 188.6 0.008 -3.7
PA2410 38.2 94.4 0.008 -2.5
PA4156 3.2 16.3 0.008 -5.1
PA3531 (bfrB) 114.0 227.5 0.008 -2.0
Pyocin system-related
PA0985 86.9 45.2 0.024 1.9 c
PA1150 (pys2) 359.3 123.9 0.008 2.9
PA3866 129.6 58.9 0.008 2.2
PA0612 941.5 177.8 0.008 5.3
PA0613 220.4 96.1 0.008 2.3
PA0614 635.5 220.0 0.008 2.9
PA0615 708.0 169.1 0.008 4.2
PA0616 1177.1 336.0 0.008 3.5
PA0617 708.9 211.5 0.008 3.4
PA0618 1085.4 350.1 0.008 3.1
PA0619 1189.2 392.5 0.008 3.0
PA0620 1328.8 313.4 0.008 4.2
PA0621 1909.6 742.5 0.008 2.6
PA0622 2536.5 833.8 0.008 3.0
PA0623 2053.5 740.5 0.008 2.8
PA0624 960.5 382.8 0.008 2.5
PA0625 537.4 159.5 0.008 3.4
PA0626 633.5 272.6 0.008 2.3
PA0627 630.3 270.0 0.008 2.3
PA0628 652.2 297.4 0.008 2.2
PA0629 407.9 154.8 0.008 2.6
PA0630 416.1 211.9 0.008 2.0
PA0631 294.6 128.3 0.008 2.3
PA0632 270.8 143.8 0.008 1.9 c
PA0633 1979.9 596.6 0.008 3.3
PA0634 589.4 169.8 0.008 3.5
PA0635 836.9 232.3 0.008 3.6
PA0636 1278.8 380.6 0.008 3.4
PA0637 589.8 178.4 0.008 3.3
PA0638 908.6 283.9 0.008 3.2
PA0639 617.9 209.7 0.008 3.0
PA0640 142.0 55.6 0.008 2.6
PA0641 509.7 102.0 0.008 5.0
PA0642 45.1 16.2 0.008 2.8
PA0643 135.5 65.7 0.008 2.1
PA0644 210.3 119.3 0.008 1.8 c
PA0645 222.9 88.8 0.008 2.5
PA0646 158.0 64.2 0.008 2.5
PA0647 50.84 39.95 0.341 b 1.3 c
PA0648 143.4 94.8 0.024 1.5 c
PA1151 (imm2) 34.88 71.58 0.008 -2.1
aThe fold change is a positive number when the expression level in the experiment increased compared to the control and is a negative number when the expression level in the experiment declined.
b The p value is higher than 0.05.
c The fold change is less than 2.
Genes related to cellular protective mechanisms
Figure 1 and Table 3 show that a number of genes primarily in the classes of "adaptation, protection" and "DNA replication, recombination, modification and repair", which are known to be involved in cellular protective mechanisms, were induced in response to hydrogen peroxide. First, two catalase genes, PA4236 (katA), and PA4613 (katB) showed significant increases in mRNA levels. It was reported that the KatB enzyme is essential for optimal resistance to hydrogen peroxide [6,21]. The mere 2-fold upregulation of katA may support a prior hypothesis that katA serves as a primary housekeeping catalase; expressed constitutively throughout the growth cycle [7,21]; as a result, dramatic transcript increases might not occur. Surprisingly, superoxide dismutase genes, PA4366 (sodB) and PA4468 (sodM), did not show significant increases in mRNA levels. However, sodB was among genes that had the highest signal intensities on both control and experimental replicates, whereas sodM exhibited low mRNA levels under both conditions. Again, this result may correspond well to a previous report suggesting that SodB expression may be important even during normal aerobic growth while SodM activity is increased only in response to iron deprivation, which is not the case in our study (discussed below) [22].
Second, DNA repair-related genes were highly induced in the presence of hydrogen peroxide. Indeed, it is known that hydrogen peroxide causes oxidative DNA damage by generating hydroxyl or ferryl radicals [1,2,23]. Palma et al. demonstrated that genes of P. aeruginosa SOS regulon exhibit increases in mRNA level upon exposure to 1 mM hydrogen peroxide [12]. Reportedly, RecA stimulates autodigestion of repressor protein LexA for the activation of the SOS regulon in E. coli [24]. Our study also revealed increased expression of PA3007 (lexA) and PA3617 (recA) (Table 3). Furthermore, PA3008, adjacent to lexA, showed high increase in transcript level; notably, this gene is similar to E. coli sulA, which inhibits cell division until DNA repair is finished [12,25]. Besides, PA3616 (probable recX), a downstream gene of recA, is reportedly involved in regulation of RecA expression [26,27]. Other interesting genes possibly relevant to DNA repair-related functions included PA0669 (a DNA polymerase gene), PA3413-PA3414, and PA4763 (recN, a DNA repair gene). To be specific, a protein encoded by PA3414 exhibited a high homology to YbeG in E. coli, which is inducible by DNA damage [28,29]. Moreover, the fact that DnaE, similar to a product encoded by PA0669, is involved in DNA mismatch correction or DNA repair in Bacillus subtilis suggests that the PA0669 protein might also participate in repairing DNA damage caused by hydrogen peroxide in our study [30,31].
As shown in Figure 2, the complete profile of our response is compared directly to that of Palma et al. [12]. Our results were generally consistent and, overall, this portion of our analysis corroborates previous studies that have associated oxidative stress response genes with hydrogen peroxide and other ROS insults, and reinforces the conclusion that DNA repair proteins and catalases may be among the most central mechanisms that P. aeruginosa employs to counteract lethal effects of reactive oxygen intermediates.
Figure 2 Comparison of 223 genes with statistically significant mRNA level changes in this study with those of Palma et al. [12]. The fold change is a positive number when the expression level in the experiment increased compared to the control and is a negative number when the expression level in the experiment declined.
Genes related to iron regulation
As mentioned above, however, one of the aims of this study was to examine the responses of ROS stress genes to the rest of cellular metabolism, in order to provide new insight on P. aeruginosa regulation in response to varied oxygen conditions as is expected in niche environments of the human lung. Among the more interesting findings was an apparent discrepancy between previous reports of iron-regulated genes. These genes are highlighted in Figure 2. Iron metabolism is important for many reasons: there are many iron catalyzed oxygen reactions that can damage cells; there are many sources of iron, particularly localized in the human lung; and iron-related genes are often coordinately regulated with oxidative stress defenses in E. coli [32]. Notably, this study also revealed that hydrogen peroxide treatment drastically altered iron regulation in P. aeruginosa. In particular, the following genes, all of which are known to be regulated by the ferric uptake regulator (Fur, encoded by PA4764), were repressed in response to hydrogen peroxide (Table 3): (i) iron starvation sigma factor, PA2426 (pvdS), (ii) ferri-siderophore (iron-chelating compound) receptor genes, PA2398 (fpvA) and PA4221 (fptA), (iii) siderophore (pyochelin) biosynthesis genes, PA4225-PA4226 (pchFE) and PA4228-PA4231 (pchDCBA), and (iv) siderophore (pyoverdin) system-related genes, PAPA2403-PA2410 and PA4156 [33,34].
As is widely accepted, in order to overcome a limited supply of iron, P. aeruginosa produces two major siderophores, pyoverdin and pyochelin [34-36] and upon iron deprivation, these siderophores are excreted from the cells, chelate iron and transport it back to the cells through outer membrane receptors, FptA and FpvA, which are specific for the iron-siderophore complexes [37,38]. Moreover, in the absence of iron, PchR, encoded by PA4227, seems to become a repressor of the receptor gene, fptA, and of its own gene, pchR. On the other hand, excess iron represses the biosynthesis of pyoverdin and pyochelin [35,39]; that is, in the presence of abundant iron, the Fur protein, a repressor, binds to the promoters of genes encoding PvdS and PchR proteins, which positively regulate the biosynthesis of pyoverdin and pyochelin, respectively [38]. These phenomena seem congruent with the transcript levels of iron-related genes in this study.
It has been documented that the intracellular iron level could be affected by oxidative stress [3,5,32]. In particular, superoxide, generated during the reduction process of oxygen, releases free iron from iron-sulfur proteins, thus increasing the levels of intracellular iron [2-4]. Consequently, the result that the iron regulation-related genes such as pvdS, fpvA, fptA, and pchABCDEF were repressed in our study could be accounted for by prior reports suggesting that the amount of intracellular iron might be increased as a result of exposure to reactive oxidants [4]. At this point, it should be pointed out that "free iron" was intended to suggest not that the iron is hexa-aqueous, but that the iron is not integrated into enzymes. Iron vigorously binds to most biomolecules so that iron atoms free inside the cell likely exist in a form associated with the surfaces of cellular molecules. As shown in Figure 2, Palma et al. previously reported induction of several iron starvation-inducible genes such as PA0471, PA0472, PA2426 (pvdS), PA2686 (pfeR), PA4221 (fptA), PA4227 (pchR), PA4228 (pchD), PA4230 (pchB), and PA5531 (tonB) upon exposure to hydrogen peroxide, suggesting that cells experience iron starvation and/or a transient loss of Fur repressor function [12]. The reason for this discrepancy is not understood at present; however, it should not be excluded that different growth mediums, exposure times, and/or growth phase (tryptic soy broth, 10 min, optical density 0.5, respectively) as well as a different P. aeruginosa isolates were used in the two studies (discussed in more detail later).
Lastly, it should be pointed out that iron regulation system might be an attractive therapeutic target because it appears to be closely related to the antioxidant mechanism of P. aeruginosa. For instance, siderophore malfunction by potent drugs could lead to inhibition and/or imbalance of iron regulation and thus, could increase the cell's susceptibility to exogenous oxidative stress that phagocytes utilize during active infection.
Genes related to pyocin system
The most striking result of our study, which has not been shown previously in any oxygen-insult studies of any bacterium, was that F-, R- and S- type pyocins, bacteriocins of P. aeruginosa, were strongly induced in response to hydrogen peroxide. As shown in Figure 2, these 41 genes were uniformly upregulated. Many strains are known to adapt bacteriocins so as to preserve the initial predominance of bacteriocinogenic bacteria [40]; however, bacteriocins are effective only against the same or closely related species [41-43]. Surprisingly, our study revealed that PA0985 (probable pyocin S5 gene), PA1150 (pys2, pyocin S2 gene), and PA3866 (probable pyocin S3 gene) were induced upon exposure to hydrogen peroxide (Table 3). Further, as shown in Figure 3 and Table 3, all the genes of a R2/F2 pyocin gene locus (PA0612-PA0648) exhibited increased mRNA levels (Figure 1, the "phage, transposon, and plasmid" class). Thus, R2 pyocin (PA0615 – PA0628), F2 pyocin (PA0633 – PA0648), and lysis enzymes (PA0614, PA0629 – PA0631) were likely expressed with the hydrogen peroxide treatment (Figure 3). Pyocins cause cell death by DNA breakdown and the inhibition of lipid synthesis; their production is inducible by mutagenic agents such as UV and mitomycin C [40,44]. RecA (a DNA repair protein), PrtR (a repressor protein) and PrtN (an activator protein) co-regulate activity of many pyocin genes. As noted above, DNA-damaging agents increase production of RecA, which in turn, cleaves PrtR and releases PrtN [45]. Hence, since DNA repair-related genes including recA were upregulated in our study and regulatory genes such as recA, prtN, and prtR are probably shared by S-, F-, and R-type pyocins [41], we may conclude that DNA damage might also be a reason for the induction of these pyocins, S2, S3, S5, R2, and F2. Moreover, the induction of lysis enzymes (PA0614, PA0629, PA0630, and PA0631), which are likely shared by S-, F-, and R-type pyocins, indicates that these pyocins might have been released from the cells [41].
Figure 3 Genetic organization of the R2/F2 pyocin gene locus according to Michel-Briand and Baysse [40] and Nakayama et al. [41]. Each box represents a gene between PA0609 (trpE) and PA0649 (trpG). Genes inside the dotted line were upregulated upon exposure to hydrogen peroxide (see, Table 3).
These results suggest that host cells are exposed to pyocins that are induced by oxidative stress during active infection. As indicated above, pyocins are known to kill species closely related to P. aeruginosa (e.g. some of Gram-negative bacteria). Interestingly, pyocins display cytotoxic effects on human cancer cells, triggering lethal events [46,47]. At present, it is unclear whether pyocins are causative toxic agents in patients with defective immune systems; however, it should not be excluded that pyocin production might be another defense mechanism that P. aeruginosa employs against oxidative attack by host cells.
Another notable finding was that PA1151 (imm2) encoding the pyocin S2 immunity protein exhibited a significant decrease in transcript level (Table 3). That is, secreted bacteriocins pose a survival problem to host bacteria, so cells produce immunity enzymes, high affinity inhibitors of bacteriocins, during bacteriocin synthesis [42]. P. aeruginosa possesses several pyocin immunity proteins that provide a self-protection function against S-type pyocins [26,40], by binding to the pyocin nuclease domain and neutralizing its activity [40,42]. Thus, our result that the pyocin S2 immunity protein was repressed during pyocin production could render P. aeruginosa vulnerable to this bacteriocin, which might even lead to its own death during pyocin synthesis.
Coincident with this, it was earlier observed that strains of P. aeruginosa produce pyocins active against themselves [48]. The conditions leading to their production were not investigated. The potential for programmed cell death in this system is an intriguing speculation. It may be that self-killing activity by part of a P. aeruginosa population benefits the remainder. For example, microbial population may dissipate damaging effects of extracellular stress such as pH change and oxidative stress by lysing some of the cells, which might be particularly effective for P. aeruginosa biofilms where layers of cells exist. Besides, given that DNA damage might elicit pyocin production, cells with damaged DNA may be the most appropriate targets for cell death, enabling preservation of the remainder of the population.
These kinds of activities apparently require cell-to-cell communication; therefore, it is interesting to question whether the quorum sensing system might be involved in pyocin synthesis. Based on quorum sensing-regulated genes that have been previously reported (163 genes from Hentzer et al. [49]; 315 genes from Schuster et al. [50]; 388 genes from Wagner et al. [51]), we found that 62, 69 and 56 genes, respectively, also showed transcription level changes in response to hydrogen peroxide in our study. While coincident up- or down-regulation does not establish a connection between quorum sensing and pyocin systems, this result might indicate that some genes involved in the quorum sensing system were also activated during cellular response to oxidants. In this light, it is noteworthy that degradation of secreted peptide Staphylococcus aureus autoinducers has been linked to the antioxidant response [14].
The last and perhaps most interesting result is that S-type pyocins become more lethal under iron-limited conditions because they presumably enter cells through siderophore receptors [52]; thus, the repression of siderophore receptors in our study (discussed above) may increase the possibility that the activation of S-type pyocins had larger impact on producer cells. However, in contrast, it might also be possible that the induction of pyocin production might contribute to the repression of iron uptake-related genes of the other population. Further investigation is required to verify the role of the immunity protein repression and the relationship between pyocins and iron-uptake genes.
Co-transcription of genes in operons
In our attempt to further evaluate the transcription levels of genes discussed above, we investigated the co-transcription of their operons. That is, since genes within operons are largely co-regulated, we expected a considerable correlation between these genes and their neighboring genes within the same operon. To test this, we first selected 16 putative operons of the genes described in this report using the "Predicting operons in microbial genome" resource of the Institute for Genomic Research . We then calculated the Pearson's correlation coefficient (R) from the transcript levels of 55 pairs of neighboring genes in these 16 operons. For baseline comparison, we also analyzed 1000 pairs of randomly selected neighboring genes across the genome. Note that each pair has nine (four control and five experimental) array datasets. In Table 4, we observed that the neighboring genes sharing the same operon exhibited significantly high co-transcription levels (the mean and median R values) in this study. This result also provides a high degree of confidence in the transcriptome data we presented herein.
Table 4 Descriptive statistics for the co-transcription level of neighboring gene pairs
Number of gene pairsa Mean R ± standard error Median R
Pairs within operons 55 0.7688 ± 0.0573 0.9449
Random pairs 1000 0.1984 ± 0.0138 0.2154
aEach gene pair has nine microarray datasets.
Comparison of microarray data to other data in the literature
Lastly, in order to provide another assessment on our results, we compared our genes with statistically marked transcript level changes with those published in Palma et al. [12]. As indicated above, our study examined the transcriptional response upon 20 min exposure to 1 mM hydrogen peroxide in Luria-bertani broth, whereas Palma et al. [12] utilized 10 min exposure time in tryptic soy broth. Since the Mann-Whitney test with the cutoff p and fold change values of 0.05 and 2, respectively, was applied to both studies, we performed a comparative analysis between the two transcriptome profiles. Supplementary Table 2 shows that of 223 genes that we previously considered statistically marked in our report, 41 and 46 genes were upregulated and downregulated, respectively, in both studies. Further, 4 genes induced in this report were repressed in Palma et al. [12], while 11 genes displayed the opposite expression pattern. In Supplementary Table 2, 70 and 46 genes that were upregulated and downregulated, respectively, in this study were not among genes with statistically significant mRNA level changes in Palma's report. Interestingly, the fold changes were generally lower under our experimental conditions. This result demonstrates that about 40% of our statistically marked genes were regulated in the same direction in Palma et al. [12].
Next, we focused on genes that we earlier discussed in this report, which were originally listed in Table 3. Supplementary Table 3 shows that most genes in the categories of "primary metabolism-related" and "cellular protective mechanism-related" were concurrently regulated in the two reports. To be particular, genes encoding enzymes that play integral roles in oxidative response, such as catalases and DNA repair proteins, were consistently upregulated. This result reinforces our prior conclusion that the early transcriptional responses of P. aeruginosa to hydrogen peroxide include the repression of primary metabolism and the induction of protective mechanisms. On the other hand, most genes related to iron regulation and pyocin system were discordantly regulated in the two studies (Supplementary Table 3). As previously discussed, while we cannot fully elucidate this disagreement, we should not exclude the possibility that different growth mediums, strain backgrounds, and exposure times account for the divergent microarray data. Indeed, several reports are in line with this speculation: (i) Vasil proposed that dissimilar experimental conditions including strain history and growth mediums bring about discrepancies in the quorum-sensing transcriptional profiles [53], (ii) Spiegeleer et al. demonstrated that oxidative stress resistance of E. coli is dependent on the growth medium [54], (iii) Kang et al. suggested that strain background is one of the factors affecting a microarray-based transcriptome analysis [55], and (iv) our laboratory observed a large difference in the transcriptional profiles of Staphylococcus aureus response to oxidative stress between 10 min and 20 min exposures (unpublished data). Therefore, this result suggests that further investigation on iron regulation and pyocin system in P. aeruginosa under oxidative stress requires careful evaluation of transcriptional effects by experimental variances.
Conclusion
In summary, we demonstrated how oxidative stress-induced genes were related and regulated in P. aeruginosa by utilizing whole-genome microarrays. Our results suggest that DNA repair proteins and catalases be among the most vital antioxidant defense systems of P. aeruginosa for preventing lethal effects of reactive oxygen intermediates. Second, a slowdown of membrane function-related genes was observed, implying that sublethal oxidative damage reduced active and/or facilitated transport through the cell membrane. Third, it was confirmed that oxidative stress could affect iron metabolism in that many of Fur-regulated genes were repressed upon exposure to hydrogen peroxide. In addition, this finding, together with other reports [5,32] suggests that intracellular iron level might be a key factor for the relationship. Most importantly, we provided evidence that reactive oxidant treatment results in the induction of all three types of pyocin genes in P. aeruginosa. Furthermore, it was demonstrated that a pyocin immunity protein was repressed, which if coincident events occur might lead to self-killing activity. To our knowledge, this is the first study reporting pyocin upregulation in response to oxidative stress. Hence, we are currently exploring whether the repression of an immunity protein increases the sensitivity of P. aeruginosa to pyocins and pyocin production benefits its population against toxic oxidants.
Methods
Bacterial strains and growth conditions
In this study, we used Pseudomonas aeruginosa PA01 obtained from Dr. E. Peter Greenberg's laboratory at the University of Iowa. To maintain homogeneous culture samples throughout our experiments, we employed the following three steps: (i) we initiated P. aeruginosa cultures at 37°C with shaking at 250 rpm using sterilized Luria-Bertani (LB) broth (10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride per liter), (ii) after 17 hours, we diluted the overnight cultures 1:100 in pre-warmed LB broth and incubated at 37°C with shaking at 250 rpm until an optical density at 600 nm (OD600) reached the early logarithmic phase (~ 0.8), and (iii) we re-diluted the cells 1:10 in pre-warmed LB broth and incubated at 37°C with shaking at 250 rpm [10,56]. Then, we added 1 mM hydrogen peroxide (Aldrich Chemical Co., St. Louis, MO) immediately after OD600 reached 0.8. Note that culture volumes for all growth conditions were adjusted to be less than 1/10 the total flask volume to maximize aeration.
Affymetrix P. aeruginosa GeneChip arrays
Total RNA was isolated after 20 min incubation using RNeasy Mini kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. As described previously [56], we added 2 mL of RNAprotect Bacteria Reagent (Qiagen, Inc., Valencia, CA) into 1 mL of the culture before the isolation to stabilize our RNA. RNA quality was determined using both Lambda 25 spectrophotometer (PerkinElmer, Inc., MA) and RNA 6000 Nano LabChip with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Next, we used 12 μg of total RNA with random primers and SuperScript II (both from Invitrogen Corp., Carlsbad, CA) for cDNA synthesis, cDNA fragmentation, labeling, hybridization, staining and washing steps were performed according to the manufacturer's protocol for the Affymetrix P. aeruginosa GeneChip arrays (Affymetrix, Inc., Santa Clara, CA). Finally, the arrays were scanned with the Affymetrix GeneChip Scanner 3000.
Data analysis and real-time PCR
To analyze the array data, we utilized Affymetrix GeneChip Operating Software (GCOS) v. 1.0 and Data Mining Tool (DMT) v. 3.1 (Affymetrix, Inc., Santa Clara, CA) with the following parameters: alpha 1, 0.04; alpha 2, 0.06; tau, 0.015; target signal, 150. Alpha 1 and 2 are significance levels that define detection calls (see below), while tau determines analysis sensitivity [15]. Further, the average intensity of arrays was scaled to a target signal. We calculated fold change as the ratio between the signal averages of four untreated (control) and five hydrogen peroxide-treated (experimental) cultures. Gene expression fold changes were identified with statistical significance by the Mann-Whitney test (cutoff p-value, 0.05). The GCOS detection calls of "Present", "Marginal", and "Absent" are determined based on the Affymetrix detection algorithm [15]. This call indicates whether a transcript is reliably detected (present) or not detected (absent). Genes that received absent calls from 50% or more of the replicates in GCOS were excluded from the final list.
Lastly, to determine the validity of the array data, transcript level changes obtained with the microarray analysis were compared with those from quantitative real-time PCR. Genes and primer sequences employed for the real-time PCR analysis are listed in Table 2. We performed the real-time PCR by utilizing iCycler iQ Real-Time PCR Detection System with iScript cDNA Synthesis Kit and One-Step RT-PCR Kit with SYBR Green (BioRad Laboratories, Inc., Hercules, CA). As stated above, RNA samples were treated with DNase I (Qiagen, Inc., Valencia, CA) to preclude DNA contamination, which was confirmed with Agilent 2100 Bioanalyzer LabChip and gel electrophoresis. In this report, relative quantification based on the relative expression of a target gene versus a reference gene was utilized to determine transcript level changes [57]. For each gene, five biological replicates with three technical replicates each were employed. PCR efficiencies were also derived from standard curve slopes in the iCycler software v. 3.1 (BioRad Laboratories, Inc., Hercules, CA). Finally, melt-curve analysis was performed to evaluate PCR specificity and resulted in single primer-specific melting temperatures.
Authors' contributions
WC performed microarray experiments, and data analysis, and drafted the manuscript. DAS performed microarray experiments. FT initiated and supervised the study, and reviewed the manuscript. WEB supervised the study and reviewed the manuscript.
Supplementary Material
Additional File 1
Probe set data (average signals, p-values, and fold changes) for experimental and control samples.
Click here for file
Additional File 2
Comparison of the fold changes and their directions of the significantly marked genes in this study with those in Palma et al. [12](a total of 223 genes). The Mann-Whitney test with the cutoff p and fold change values of 0.05 and 2, respectively, was applied to both studies,
Click here for file
Additional File 3
Comparison of the expression change directions of genes that we discussed in this study with those in Palma et al. [12]. The genes were classified into the categories of "primary metabolism-related", "cellular protective mechanisms-related", "iron regulation-related", and "pyocin system-related", as presented in Table 3.
Click here for file
Acknowledgements
We thank Hyunmin Yi for thoughtful comments on the manuscript. This research is supported by the United States Environmental Protection Agency Grants T- 83100801-0, IPA HQ-514-04-06, and IPA HQ-515-04-06. Although the research described in this paper has been funded wholly by the United States Environmental Protection Agency, it has not been subjected to the Agency's peer and administrative review and therefore may not necessarily reflect the views of the EPA; nor does the mention of trade names or commercial products constitute endorsement of recommendation of use.
==== Refs
Miller RA Britigan BE Role of oxidants in microbial pathophysiology Clin Microbiol Rev 1997 10 1 18 8993856
Keyer K Gort AS Imlay JA Superoxide and the production of oxidative DNA damage J Bacteriol 1995 177 6782 6790 7592468
McCormick ML Buettner GR Britigan BE Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide J Bacteriol 1998 180 622 625 9457866
Keyer K Imlay JA Superoxide accelerates DNA damage by elevating free-iron levels Proc Natl Acad Sci 1996 93 13635 13640 8942986 10.1073/pnas.93.24.13635
Imlay JA Pathways of oxidative damage Annu Rev Microbiol 2003 57 395 418 14527285 10.1146/annurev.micro.57.030502.090938
Brown SM Howell ML Vasil ML Anderson AJ Hassett DJ Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide J Bacteriol 1995 177 6536 6544 7592431
Ochsner UA Vasil ML Alsabbagh E Parvatiyar K Hassett DJ Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF J Bacteriol 2000 182 4533 4544 10913087 10.1128/JB.182.16.4533-4544.2000
Goodman AL Lory S Analysis of regulatory networks in Pseudomonas aeruginosa by genomewide transcriptional profiling Curr Opin Microbiol 2004 7 39 44 15036138 10.1016/j.mib.2003.12.009
Talaat A Lyons R Howard S Johnston S The temporal expression profile of Mycobacterium tuberculosis infection in mice Proc Natl Acad Sci 2004 101 4602 4607 15070764 10.1073/pnas.0306023101
Ma JF Ochsner UA Klotz MG Nanayakkara VK Howell ML Johnson Z Posey JE Vasil ML Monaco JJ Hassett DJ Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa J Bacteriol 1999 181 3730 3742 10368148
Hassett DJ Alsabbagh E Parvatiyar K Howell ML Wilmott RW Ochsner UA A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities J Bacteriol 2000 182 4557 4563 10913089 10.1128/JB.182.16.4557-4563.2000
Palma M DeLuca D Worgall S Quadri LE Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide J Bacteriol 2004 186 248 252 14679246 10.1128/JB.186.1.248-252.2004
Wu CL Domenico P Hassett DJ Beveridge TJ Hauser AR Kazzaz JA Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa Am J Respir Cell Mol Biol 2002 26 731 738 12034573
Rothfork JM Timmins GS Harris MN Chen X Lusis AJ Otto M Cheung AL Gresham HD Inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: new role for the NADPH oxidase in host defense Proc Natl Acad Sci 2004 101 13867 13872 15353593 10.1073/pnas.0402996101
Affymetrix GeneChip® expression analysis technical manual
Zheng M Wang X Templeton LJ Smulski DR LaRossa RA Storz G DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide J Bacteriol 2001 183 4562 4570 11443091 10.1128/JB.183.15.4562-4570.2001
Gene Expression Omnibus (GEO)
Savli H Karadenizli A Kolayli F Gundes S Ozbek U Vahaboglu H Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR J Med Microbiol 2003 52 403 408 12721316 10.1099/jmm.0.05132-0
The Pseudomonas aeruginosa Community Annotation Project
Freudl R Braun G Honore N Cole ST Evolution of the enterobacterial sulA gene: a component of the SOS system encoding an inhibitor of cell division Gene 1987 52 31 40 3297925 10.1016/0378-1119(87)90392-1
Elkins JG Hassett DJ Stewart PS Schweizer HP McDermott TR Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide Appl Environ Microbiol 1999 65 4594 4600 10508094
Hassett DJ Schweizer HP Ohman DE Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism J Bacteriol 1995 177 6330 6337 7592406
Martinez A Kolter R Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps J Bacteriol 1997 179 5188 5194 9260963
Little JW Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease Biochimie 1991 73 411 421 1911941 10.1016/0300-9084(91)90108-D
Huisman O D'Ari R Gottesman S Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation Proc Natl Acad Sci 1984 81 4490 4494 6087326
Sano Y Role of the recA-related gene adjacent to the recA gene in Pseudomonas aeruginosa J Bacteriol 1993 175 2451 2454 8468303
Papavinasasundaram KG Colston MJ Davis EO Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function Mol Microbiol 1998 30 525 534 9822818 10.1046/j.1365-2958.1998.01083.x
Lomba MR Vasconcelos AT Pacheco AB de Almeida DF Identification of yebG as a DNA damage-inducible Escherichia coli gene FEMS Microbiol Lett 1997 156 119 122 9368369 10.1016/S0378-1097(97)00412-6
Oh TJ Kim IG Identification of genetic factors altering the SOS induction of DNA damage-inducible yebG gene in Escherichia coli FEMS Microbiol Lett 1999 177 271 277 10474193 10.1016/S0378-1097(99)00326-2
Dervyn E Suski C Daniel R Bruand C Chapuis J Errington J Janniere L Ehrlich SD Two essential DNA polymerases at the bacterial replication fork Science 2001 294 1716 1719 11721055 10.1126/science.1066351
Le Chatelier E Becherel OJ d'Alencon E Canceill D Ehrlich SD Fuchs RP Janniere L Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis J Biol Chem 2004 279 1757 1767 14593098 10.1074/jbc.M310719200
Zheng M Doan B Schneider TD Storz G OxyR and SoxRS regulation of fur J Bacteriol 1999 181 4639 4643 10419964
Visca P Leoni L Wilson MJ Lamont IL Iron transport and regulation, cell signaling and genomics: lessons from Escherichia coli and Pseudomonas Mol Microbiol 2002 45 1177 1190 12207687 10.1046/j.1365-2958.2002.03088.x
Palma M Worgall S Quadri LE Transcriptome analysis of the Pseudomonas aeruginosa response to iron Arch Microbiol 2003 180 374 379 14513207 10.1007/s00203-003-0602-z
Reimmann C Serino L Beyeler M Haas D Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa Microbiology 1998 144 3135 3148 9846750
Vasil ML Ochsner UA The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence Mol Microbiol 1999 34 399 413 10564483 10.1046/j.1365-2958.1999.01586.x
Poole K Neshat S Krebes K Heinrichs DE Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa J Bacteriol 1993 175 4597 4604 8335619
Heinrichs DE Poole K PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor J Bacteriol 1996 178 2586 2592 8626326
Serino L Reimmann C Visca P Beyeler M Chiesa VD Haas D Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa J Bacteriol 1997 179 248 257 8982005
Michel-Briand Y Baysse C The pyocins of Pseudomonas aeruginosa Biochimie 2002 84 499 510 12423794 10.1016/S0300-9084(02)01422-0
Nakayama K Takashima K Ishihara H Shinomiya T Kageyama M Kanaya S Ohnishi M Murata T Mori H Hayashi T The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage Mol Microbiol 2000 38 213 231 11069649 10.1046/j.1365-2958.2000.02135.x
Kleanthous C Walker D Immunity proteins: enzyme inhibitors that avoid the active site Trends Biochem Sci 2001 26 624 631 11590016 10.1016/S0968-0004(01)01941-7
de Zamaroczy M Buckingham RH Importation of nuclease colicins into E. coli cells: endoproteolytic cleavage and its prevention by the immunity protein Biochimie 2002 84 423 432 12423785 10.1016/S0300-9084(02)01426-8
Sano Y Matsui H Kobayashi M Kageyama M Molecular structures and functions of pyocins S1 and S2 in Pseudomonas aeruginosa J Bacteriol 1993 175 2907 2916 8491711
Matsui H Sano Y Ishihara H Shinomiya T Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes J Bacteriol 1993 175 1257 1263 8444788
Farkas-Himsley H Hill R Rosen B Arab S Lingwood CA The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1 Proc Natl Acad Sci 1995 92 6996 7000 7624357
Abdi-Ali A Worobec EA Deezagi A Malekzadeh F Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines Can J Microbiol 2004 50 375 381 15213746 10.1139/w04-019
Goodwin K Levin RE Doggett RG Autosensitivity of Pseudomonas aeruginosa to its own pyocin Infect Immun 1972 6 889 892 4629211
Hentzer M Wu H Andersen J Riedel K Rasmussen T Bagge N Kumar N Schembri M Song Z Kristoffersen P Manefield M Costerton J Molin S Eberl L Steinberg P Kjelleberg S Hoiby N Givskov M Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors EMBO J 2003 22 3803 3815 12881415 10.1093/emboj/cdg366
Schuster M Lostroh C Ogi T Greenberg E Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis J Bacteriol 2003 185 2066 2079 12644476 10.1128/JB.185.7.2066-2079.2003
Wagner V Bushnell D Passador L Brooks A Iglewski B Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment J Bacteriol 2003 185 2080 2095 12644477 10.1128/JB.185.7.2080-2095.2003
Baysse C Meyer JM Plesiat P Geoffroy V Michel-Briand Y Cornelis P Uptake of pyocin S3 occurs through the outer membrane ferripyoverdine type II receptor of Pseudomonas aeruginosa J Bacteriol 1999 181 3849 3851 10368165
Vasil ML DNA microarrays in analysis of quorum sensing: strengths and limitations J Bacteriol 2003 185 2061 2065 12644475 10.1128/JB.185.7.2061-2065.2003
de Spiegeleer P Sermon J Lietaert A Aertsen A Michiels CW Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli J Appl Microbiol 2004 97 124 133 15186449 10.1111/j.1365-2672.2004.02285.x
Kang Y Weber KD Qiu Y Kiley PJ Blattner FR Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function J Bacteriol 2005 187 1135 1160 15659690 10.1128/JB.187.3.1135-1160.2005
Chang W Small DA Toghrol F Bentley WE Microarray analysis of toxicogenomic effects of peracetic acid on Pseudomonas aeruginosa Environ Sci Technol 2005 39 5893 5899 16124331 10.1021/es0503534
Pfaffl MW A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res 2001 29 e45 11328886 10.1093/nar/29.9.e45
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BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-5-311615014910.1186/1472-6947-5-31Research ArticleVariation in the psychosocial determinants of the intention to prescribe hormone therapy prior to the release of the Women's Health Initiative trial: a survey of general practitioners and gynaecologists in France and Quebec Legare France [email protected] Gaston [email protected] Virginie [email protected] Sylvie [email protected] Lucile [email protected] Joanna [email protected] CHUQ, St-François d'Assise Hospital Research Center, 10 rue de l'Espinay, Quebec, QC, Canada, G1L 3L52 Canada Research Chair on Behaviour and Health, Faculty of Nursing, Laval University, Quebec, QC, Canada, G1K 7P43 INSERM National Institute for Medical Research U149, Epidemiological Research Unit on Perinatal Health and Women's Health, 16, ave Paul Vaillant Couturier, 94807 Villejuif cedex, France2005 8 9 2005 5 31 31 5 4 2005 8 9 2005 Copyright © 2005 Legare et al; licensee BioMed Central Ltd.2005Legare 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
Theory-based approaches are advocated to improve our understanding of prescription behaviour. This study is an application of the theory of planned behaviour (TPB) with additional variables. It was designed to assess which variables were associated with the intention to prescribe hormone therapy (HT). In addition, variations in the measures across medical specialities (GPs and gynaecologists) and across countries (France and Quebec) were investigated.
Methods
A survey among 2,000 doctors from France and 1,044 doctors from Quebec was conducted. Data were collected by means of a self-administered questionnaire. A clinical vignette was used to elicit doctors' opinions. The following TPB variables were assessed: attitude, subjective norm, perceived behavioural control, attitudinal beliefs, normative beliefs and power of control beliefs. Additional variables (role belief, moral norm and practice pattern-related factors) were also assessed. A stepwise logistic regression was used to assess which variables were associated with the intention to prescribe HT. GPs and gynaecologists were compared to each other within countries and the two countries were compared within the specialties.
Results
Overall, 1,085 doctors from France returned their questionnaire and 516 doctors from Quebec (response rate = 54% and 49%, respectively). In the overall regression model, power of control beliefs, moral norm and role belief were significantly associated with intention (all at p < 0.0001). The models by specialty and country were: for GPs in Quebec, power of control beliefs (p < 0.0001), moral norm (p < 0.01) and cytology and hormonal dosage (both at p < 0.05); for GPs in France, power of control beliefs and role belief (both at p < 0.0001) and perception of behavioural control (p < 0.05) and cessation of menses (p < 0.01); for gynaecologists in Quebec, moral norm and power of control beliefs (both at p = 0.01); and for gynaecologists in France, power of control beliefs (p < 0.0001), and moral norm, role belief and lipid profile (all at p < 0.05).
Conclusion
In both countries, compared with GPs, intention to prescribe HT was higher for gynaecologists. Psychosocial determinants of doctors' intention to prescribe HT varied according to the specialty and the country thus, suggesting an influence of contextual factors on these determinants.
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Background
Until the results of the Women's Health Initiative trial [1] were known, hormone therapy (HT) was promoted for preventing osteoporosis and was thought to protect from cardiovascular diseases [2]. Although physicians agreed that menopause was not a disease, they considered it a serious health problem [3]. Hot flashes, night sweats and osteoporosis were reasons for gynaecologists and general practitioners (GPs) to prescribe HT [4]. Notwithstanding these benefits, breast cancer was a major concern [5,6]. Moreover, many women objected to adopting HT at menopause from fear of medicalization of an otherwise natural process [7]. Regardless of these conflicting evidences, in Canada, at the time this study was conducted, Premarin®, a known estrogenic compound, was the most prescribed drug by gynaecologists and the third most prescribed drug by GPs [8].
Following the Women's Health Initiative trial [1], there was a decrease in the number of prescriptions of standard-dose HT but a slight increase in the number of prescriptions of low-dose HT in the United States [9]. In fact, HT remains the most effective option to alleviate severe perimenopausal symptoms such as hot flashes [10]. Moreover, although HT is not a first-line treatment recommended for preventing osteoporosis [11], its use is associated with a decrease in fractures in current users [12]. Based on the results of the Women's Health Initiative trial, HT use for 1 year in 10 000 healthy postmenopausal women is associated with 7 more cardiovascular disease events, 8 more invasive breast cancers, 8 more strokes, 8 more pulmonary emboli, 6 fewer colorectal cancers and 5 fewer hip fractures [1]. Weighing the benefits with the risks associated with HT reminds women and their doctor that deciding about HT requires a careful and individualised assessment of their personal situation.
It is in this context that decisions about HT is said to be representative of clinical decision-making in the face of scientific uncertainty [13,14]. Thus, it is likely that many factors play a role in a doctor decision-making process leading to a prescription of HT. Attitude towards HT is associated with the medical specialty [15,16], and age [17] as well as gender of the provider [15,18]. The country in which a doctor practices medicine also appeared to play a role [19].
Conceptual underpinnings of this study
In studies of doctors' decision-making and related prescription behaviours, more attention needs to be given to the use and combination of different theories [20]. The lack of use of theories restrains effective implementation of change in patients' care because it restrains our understanding of the pathways through which a given implementation strategy is effective. The theory of planned behaviour [21] is well known through its previous applications to the study of doctors' behaviours [22-25]. This theory provides a theoretical account of the way in which attitude, subjective norm and perceived behavioural control combine to predict a given behavioural intention (decision-making) [26] and in turn, a given behaviour [27]. Thus, it provides direction for elaborating effective strategies that will influence the decision-making process leading to change in doctors' behaviours.
This theory postulates that under a controlled situation, intention is the immediate determinant of behaviour [21]. In turn, this intention is under the influence of three main factors: attitude, subjective norm and perceived behavioural control. Each can be assessed directly or indirectly (beliefs-based measure). Attitude is conceptualized as a personal evaluation of the action. It is the product of a set of salient beliefs about the consequences of performing the behaviour, each weighted by an evaluation of the importance of the respective consequences. Subjective norm refers to a perceived social pressure to perform the behaviour in question. The belief-based measure requires that the individual's normative beliefs be multiplied by the individual's motivation to comply with these socially-normative referents. Perceived behavioural control is a measure of the amount of control the individual has over the behaviour in question. It refers to the individual's perception of barriers or facilitating factors likely to influence the adoption of the behaviour. It can also be measured directly or indirectly. For the indirect measurements, the individual's control beliefs must be weighted by the corresponding perceived evaluation of how much each of these control beliefs will impact on the adoption of the behaviour. According to the authors of this theory, sociodemographics and other variables will influence behaviour through their influence on the attitude, the subjective norm and the perceived behavioural control [21]. Successful behavioural change will occur only if the underlying determinants of intention change.
Although the theory of planned behaviour has proven useful when studying health related behaviours, two systematic reviews found that components of this theory explain on average 41% of the variance in intention and 28% to 31% of the variance in behaviour thus, suggesting that other variables must play a direct role on the behavioural intention and possibly, on the behaviour itself [28,29]. Consequently, some authors have expanded the theory of planned behaviour to include other relevant psychosocial constructs [28]. For example, the measure of moral norm [30] was found to be useful in understanding women's intention to use HT [31,32]. Moral norm takes into account feelings of personal responsibility regarding adoption of a specific behaviour, that is, the individual's perception of the moral correctness or incorrectness of performing the behaviour. Because of the potential influence of the medical specialty, in this study, role belief was also taken into consideration [30]. Role belief refers to the perception by the individual that members of a specific group would perform the behaviour under study. The initial theoretical model adopted as a basis for examining the psychosocial determinants associated with doctors' intention to prescribe HT is presented in Figure 1.
Figure 1 Initial theoretical model.
To our knowledge, at the time this study was planned, we were not aware of any studies that had applied the theory of planned behaviour to study variations of doctors' intention to prescribe HT across countries. Physicians in France and the province of Quebec, Canada, share the same language (French) but use it in very different geographical and cultural settings. Therefore, studies of their intention to prescribe HT offered a unique opportunity to provide more insight in the cultural variation in the psychosocial determinants associated with this behaviour without the difficulties associated with having questionnaires in two different languages. Therefore, this study is an application of the theory of planned behaviour with additional variables. It was designed to assess which variables were associated with the intention to prescribe HT. Hypotheses refer mainly to the assumption underlying the theoretical framework. It could nonetheless be added that we hypothesized that variations in the intention to prescribe HT between gynaecologists and GPs would be observed as well as variations in the intention to prescribe HT between French and French Canadians physicians would be observed.
Methods
Population and sampling strategy
The study was conducted in 1997 in France and in the province of Quebec, Canada, where French is the prominent language. In France, questionnaires were mailed to a representative sample of 1,000 GPs and 1,000 gynaecologists, randomly drawn from an exhaustive list of 65,000 GPs and 7,000 gynaecologists. This daily updated list was provided by Fournier Pharma, a French pharmaceutical company that is known to have one of the most exhaustive lists of physicians. This list is used for visiting physicians in France. In Quebec, questionnaires were mailed to all gynaecologists (n = 244) and to a representative sample of 800 GPs from an estimated pool of 7000 GPs. This second list was provided by the medical licensing body of this Canadian province.
Data collection procedure and development of the questionnaire
Data were collected by means of a mailed self-administered questionnaire using a modified Dillman strategy [33]. The development of the questionnaire was performed in both France and Quebec to provide only one questionnaire. The self-administered questionnaire comprised two sections. The first section addressed sociodemographics as well as self-reported practice patterns in the field of menopausal health. The second section assessed the behavioural intention of physician to prescribe HT when faced with a clinical vignette. This clinical vignette was developed in a series of two iterative consultations and tested with a total of 22 GPs and 22 gynaecologists in both countries. It presented a 55 years old menopausal woman who had been menopausal for the past three years. She had no specific opinion about HT and did not complain about hot flashes. This woman had no contra-indication to HT. She had no known risk factors for cardiovascular disease or for osteoporosis. She had a 5-year risk of breast cancer of 2% because her maternal grandmother and her own mother suffered from breast cancer (average risk = 1.5%) [34]. This clinical vignette was purposely designed to maximize the variance in the intention of physicians to prescribe HT.
Measures
Direct measures
In line with the theory of planned behaviour, the behaviour under study was defined as followed: to prescribe (action) HT (target) to a menopausal woman who is consulting for a routine periodical medical exam and who is presented in the clinical vignette (context) [21]. The time frame was not specified. Intention to prescribe HT was assessed by means of two items. After the general comment "If you were the physician of Mrs. X, what would you do?", physicians were asked to answer the following questions on a bipolar 7-point scale: "My intention would be to prescribe her HT." ('unlikely' to 'likely'); "I would prescribe her HT." ('disagreeing' to 'agreeing'). The mean of the composite score was computed (Pearson r = 0.84). Attitude was assessed by means of four items using a semantic differential bipolar 7-point scale. The four pairs of adjectives used were: "not gratifying/gratifying", "not satisfying/satisfying", harmful/harmless" and "not useful/useful". Each pair of adjectives appeared after the sentence: "For me, prescribing HT for Mrs. X would be ...". The mean composite score of the four items was taken as the attitude value (Cronbach α = 0.90). Subjective norm was assessed by means of two items, each assessed on a bipolar 7-point scale. Physicians were invited to indicate their level of agreement with the following statements: "Most of the persons who are important for me in the profession would recommend that I prescribe HT to Mrs. X ", ('disagree' to 'agree'); "The proportion of my colleagues who would prescribe HT to Mrs. X is ..." ('low' to 'high'). These two items were used to compute a mean composite score (Pearson r = 0.84). Two items were included to assess perceived behavioural control, each on a bipolar 7-point response scale. The items were: "I see no barriers to prescribing HT to Mrs. X " ('disagree' to 'agree'); "For me, prescribing HT to Mrs. X would be..." ('difficult' to 'easy'). These two items were used to compute a mean composite score (Pearson r = 0.79). Moral norm was obtained by means of three items, each on a bipolar 7-point response scale: "Given my personal convictions, I would prescribe HT to Mrs. X" ('disagree' to 'agree'); "If I were to prescribe HT to Mrs. X, I would feel guilty" ('agree' to 'disagree'); and "I think that this is totally ethical that I prescribe HT to Mrs. X" ('disagree' to 'agree'). A mean composite score for moral norm was computed (Cronbach α = 0.87). Role belief was obtained by means of two items, each on a bipolar 7-point response scale: "In a situation like the one of Mrs. X, I think that a physician who has the same education as me would prescribe HT" ('disagree' to 'agree'); and "In a situation like the one of Mrs. X, I think that a physician of the same gender as me would prescribe HT" ('disagree' to 'agree'). The mean composite score reliability value for moral norm was 0.87 (Pearson r).
Indirect measures
In line with methodological developments in the use of the theory of planned behaviour, only one arm of each indirect measure of the main constructs (attitude, social norm and perception of control) was assessed, that is attitudinal beliefs, normative beliefs and power of control beliefs [35]. Therefore, no multiplicative procedure was applied [36]. Attitudinal beliefs were assessed by means of six items, each on a bipolar 7-point response scale ('disagree' to 'agree'). Following the statement, "If I were to prescribe HT to Mrs. X", physicians were asked to answer if HT would: "reduce bone mass loss", "protect from cardiovascular diseases", "not raise significantly her risk of breast cancer", "reduce completely her hot flushes", "prevent ageing of the urogenital tract" and "improve her breast exam follow-up". These six items were used to compute a mean composite score for attitudinal beliefs (Cronbach α = 0.74). Normative beliefs were assessed with six items, each on a bipolar 7-point response scale ('would not approve' to 'would approve'). Individuals or groups evaluated were: specialists of breast cancer, family members of Mrs. X, rheumatologists, gynaecologists, cardiologists and physicians in general (Cronbach α = 0.89). Power of control beliefs were assessed with six items presented as follows: "Even if ..., ("Mrs. X has no opinion on HT", "results from the bone mass densitometry are not available", "HT is medicalising menopause", "Mrs. X has not many hot flushes", "Mrs. X has a family history of breast cancer" and "Mrs. X has no risk factors for cardiovascular diseases") I would prescribe HT". These items were assessed on a bipolar 7-point scale ('disagree' to 'agree'). These six items were used to compute a mean composite score for power of control beliefs (Cronbach α = 0.96).
For above variables, the bipolar 7-point scale was rated numerically from -3 to +3. Therefore, a positive score indicated that the physician expressed a positive evaluation of the belief-based construct. Finally, sociodemographics and practice patterns-related variables were assessed with closed-ended questions [37]. Examinations performed variables were assessed with a check list and included elements of the physical examination (for example, breast and pelvic examination) and laboratory (for example, lipid profile) as well as imaging investigations (for example, mammography) that were relevant for a middle-aged woman undergoing a routine periodical medical exam.
Data analysis
Descriptive analyses to assess the distribution of all the explanatory variables were performed. They all showed a near normal to normal distribution. Some elements in the theory of planned behaviour constructs had missing information. If less than one third of the responses were missing for a given construct, then the mean of the other responses within the construct was used to impute these missing answers, otherwise the subject was considered as having too many missing data and was not included in the analysis. The means and SD of psychosocial variables were analyzed by medical specialty and by country using Student's t-test with the Bonferronni correction for multiple group comparisons. A U-shaped distribution of the dependant variable that is, the intention to prescribe HT was observed (Figure 2). This type of distribution was not amenable to a transformation. For further analysis, in line with the theory of planned behaviour, the intention to prescribe HT was dichotomized as follows: mean scores of 1 and more were classified as high intention, whereas mean scores of less than 1 were classified as low intention. Multiple logistic regression analysis was used to determine factors associated with high intention.
Figure 2 Distribution of the intention to prescribe HT.
First, an overall model (all doctors) was produced. Then, doctors were grouped according to their specialty and country of origin. Intention to prescribe HT was first regressed on the direct measure of the main constructs of the theory of planned behaviour (attitude, subjective norm and perceived behavioural control). Second, role belief, moral norm and power of control beliefs were added to the variables retained in the model. Third, attitudinal beliefs and normative beliefs were added. Last, only if they were significant at a p of 0.20 or less in a bivariate analysis with intention to prescribe HT, variables that were external to the initial theoretical model (sociodemographics and practice patterns-related variables) were tested. Adjusted odds ratio and their 95% confidence intervals were computed. All reported p values were two-sided. No interaction term was tested. The Statistical Analysis System (SAS Institute, Cary, NC) was used for data analysis. This study was approved by the Ethics Committee of the University Centre where it was conducted.
Results
Characteristics of respondents
Overall, 1,085 French doctors returned their questionnaire (response rate = 54%) and 516 doctors from Quebec (response rate = 49%). However, due to missing data, 1011 questionnaires from France (425 GPs and 586 gynaecologists) and 464 questionnaires from Quebec (334 GPs and 130 gynaecologists) were analyzed. A larger proportion of the respondents were gynaecologists in France when compared with respondents in Quebec (58% versus 28%). This difference was due to the different GPs/gynaecologists ratio in the two samples invited to participate in each country: half of the doctors in the France sample were gynaecologists compared with 23% in the Quebec sample. In France, as in Quebec, respondents were significantly more likely to be women (in France 41% of the respondents and 33% of the nonrespondents, p < 0.01; in Quebec 37% of the respondents and 31% of the nonrespondents, p < 0.05). Characteristics of the participants are presented in Table 1.
Table 1 Characteristics of participating doctors by country and medical specialty
Quebec France
General Practitioners n = 334 (%) Gynaecologists n = 130 (%) General Practitioners n = 425 (%) Gynaecologists n = 586 (%)
Women 140 (42%) 35 (27%) 98 (23%) 316 (54%)
Age (years) 41.5 ± 8.6 47.4 ± 10.8 43.7 ± 7.6 45.4 ± 8.3
Number of years in practice
<8 years 82 (25%) 31 (24%) 109 (26%) 107 (18%)
9 – 15 years 92 (27%) 20 (15%) 116 (27%) 236 (40%)
16 – 21 years 90 (27%) 28 (21%) 125 (29%) 124 (21%)
>22 years 70 (21%) 51 (39%) 75 (18%) 119 (21%)
Rural place of practice 88 (27%) 8 (6%) 162 (38%) 55 (9%)
Number of patients/day*
<10 19 (6%) 6 (5%) 15 (4%) 42 (7%)
10 – 20 95 (29%) 20 (15%) 170 (40%) 299 (52%)
20 – 40 191 (58%) 83 (64%) 221 (53%) 224 (39%)
>40 24 (7%) 21 (16%) 14 (3%) 15 (2%)
% of menopausal patients in clientele 29.1 ± 25.0 56.0 ± 24.8 21.4 ± 20.2 50.5 ± 27.3
% who use menopausal diagnostic criteria**
Symptoms 118 (35%) 64 (49%) 174 (41%) 290 (49%)
Hormonal dosages 101 (30%) 23 (18%) 126 (30%) 173 (30%)
Cessation of menses 227 (68%) 78 (60%) 268 (63%) 328 (56%)
Discuss HT with all menopausal patients 122 (38%) 66 (51%) 101 (24%) 355 (61%)
Examinations performed**
Cytology (PAP smear) 292 (87%) 129 (87%) 363 (85%) 519 (89%)
Mammography 288 (86%) 120 (92%) 381 (90%) 578 (99%)
Breast examination 306 (92%) 127 (98%) 377 (89%) 580 (99%)
Hormonal dosages 113 (34%) 17 (13%) 139 (33%) 152 (26%)
Lipid profile 245 (73%) 59 (45%) 349 (82%) 387 (66%)
Endometrial biopsy 3 (1%) 16 (12%) 11 (3%) 40 (7%)
Bone mass density 30 (9%) 16 (12%) 84 (20%) 77 (13%)
Vaginal ultrasound 1 (0,3%) 4 (3%) 21 (5%) 158 (27%)
Thyroid gland examination 251 (75%) 61 (47%) 187 (44%) 156 (27%)
Pelvic examination 270 (81%) 126 (97%) 307 (72%) 569 (97%)
Speculum 272 (81%) 126 (97%) 276 (65%) 570 (97%)
Blood pressure 308 (92%) 119 (92%) 391 (92%) 569 (97%)
Cardiac auscultation 285 (85%) 29 (22%) 363 (85%) 148 (25%)
Weight 257 (77%) 58 (45%) 341 (80%) 505 (86%)
HT prescription patterns > 70% of cases 106 (34%) 86 (67%) 36 (9%) 309 (55%)
* Total number of doctors within the subgroup might differ from total number of participating doctors because of missing data.
** Categories are not mutually exclusive.
Means and standard deviation (SD) of the psychosocial variables by medical specialty and by country
Table 2 summarizes the means and standard deviation (SD) of the psychosocial variables. In France, compared with GPs, measures of all the psychosocial variables were higher for gynaecologists (all at p < 0.01). In Quebec, compared with GPs, measures of the following psychosocial variables were higher for gynaecologists: intention to prescribe HT (p < 0.05) and attitude, subjective norm, perceived behavioural control and moral norm (all at p < 0.01). Compared with gynaecologists from Quebec, those from France showed a higher score for attitudinal beliefs (p < 0.01). There were no differences between GPs from France and those from Quebec.
Table 2 Means and standard deviation (SD) of the psychosocial variables by medical specialty and by country
Quebec France
Psychosocial constructs General Practitioners n = 334 Gynaecologists n = 130 General Practitioners n = 425 Gynaecologists n = 586
Intention 0.030 ± 2.121 0.642 ± 2.086* -0.195 ± 2.216 0.556 ± 2.083**
Attitude 0.450 ± 1.456 0.951 ± 1.456** 0.265 ± 1.678 0.847 ± 1.492**
Subjective norm 0.026 ± 1.626 0.594 ± 1.641** -0.220 ± 1.621 0.316 ± 1.665**
Perceived behavioural control 0.053 ± 1.800 0.740 ± 1.721** -0.254 ± 1.929 0.560 ± 1.921**
Role belief 0.180 ± 1.809 0.535 ± 1.706 0.102 ± 1.821 0.521 ± 1.729**
Moral norm 0.585 ± 1.771 1.269 ± 1.671** 0.281 ± 2.037 0.963 ± 1.784**
Power of control beliefs 0.195 ± 1.960 0.614 ± 1.974 -0.041 ± 2.143 0.607 ± 2.057**
Behavioural beliefs 1.301 ± 0.975 1.525 ± 0.965 1.374 ± 1.061 1.963 ± 0.819**¶
Normative beliefs 0.438 ± 1.396 0.506 ± 1.223 0.287 ± 1.445 0.615 ± 1.270**
Statistical significance given after Bonferroni correction for multiple comparisons:
* p < 0.05 between General practitioners and Gynaecologists for each country
** p < 0.01 between General practitioners and Gynaecologists for each country
¶ p < 0.01 between Gynaecologists between countries
Psychosocial factors associated with the intention to prescribe HT
The results of the regression analysis are presented in Table 3. In the overall model (all doctors), three psychosocial variables were associated with high intention: power of control beliefs, moral norm and role belief (all at p < 0.0001). Since neither the country of origin nor the specialty was found statistically significant in this overall model, and because of the difference in distribution of these factors, separate models were prepared to assess the variables associated with intention to prescribe HT for each of the four combinations. For GPs from Quebec, two psychosocial variables were associated with high intention: power of control beliefs (p < 0.0001) and moral norm (p < 0.01). Two variables that were external to the initial theoretical model were also retained in the final model: cytology and using hormonal dosage as a menopausal criterion (both at p < 0.05). For GPs from France, three psychosocial variables were associated with high intention: power of control beliefs (p < 0.0001), role belief (p < 0.0001) and perceived behavioural control (p < 0.05). One variable that was external to the initial theoretical model was added to the final model: using cessation of menses as a menopausal criterion (p < 0.01). For gynaecologists from Quebec (n = 129), two psychosocial variables were associated with high intention: moral norm (p < 0.01) and power of control beliefs (p < 0.01). No variables that were external to the initial theoretical model were added to this final model. For gynaecologists from France (n = 577), three psychosocial variables were kept in the final model: power of control beliefs (p < 0.0001), moral norm (p < 0.05), role belief (p < 0.05). One variable that was external to the initial theoretical model was added to the final model: performing a lipid profile (p < 0.05).
Table 3 Psychosocial factors and other variables predicting intention to prescribe HT
Quebec and France Quebec France
All doctors N = 1472 General Practitioners N = 333 Gynaecologists N = 129 General Practitioners N = 419 Gynaecologists N = 577
OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
TPB Constructs
RB 2.08**** (1.61 – 2.69) - - 4.60**** (2.17 – 9.73) 1.47* (1.03 – 2.11)
MN 2.38**** (1.70 – 3.34) 3.42** (1.52 – 7.66) 84.58** (3.64 – 4.00) - 1.75* (1.11 – 2.77)
PBC - - - 1.65* (1.07 – 2.55) -
Power of control beliefs (POC's) 4.92**** (3.51 – 6.90) 21.92**** (6.86 – 70.05) 15.86** (1.97 – 128.23) 5.80**** (3.05 – 11.03) 6.20**** (3.63 – 10.59)
Other Variables
Cytology (PAP smear) - 11.05* (1.01 – 123.08) - - -
Hormonal dosage as menopausal criteria - 4.36* (1.15 – 16.50) - - -
Cessation of menses as menopausal criteria - - - 4.44*** (1.45 – 13.59) -
Lipid profile - - - - 2.64* (1.23 – 2.68)
Likelihood Ration X2 = 1580; degrees of freedom = 3; p < 0.0001 Likelihood Ration X2 = 384; degrees of freedom = 4; p < 0.0001 Likelihood Ration X2 = 153; degrees of freedom = 2; p < 0.0001 Likelihood Ration X2 = 477; degrees of freedom = 4; p < 0.0001 Likelihood Ration X2 = 584; degrees of freedom = 4; p < 0.0001
Note: Due to incomplete information in some sections of the questionnaire, 996 questionnaires from France (419 general practitioners and 577 gynaecologists) and 462 questionnaires from Quebec (333 general practitioners and 129 gynaecologists) were used in the regression analysis. The differences in the total numbers of subjects in the regressions are due to missing information for some of the explanatory variables in the subgroup models. The outcome variable in the regressions is a dichotomous variable representing high vs. low intention to prescribe.
OR: Odds ratio; 95% CI: 95% Confidence Interval
* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Discussion
To our knowledge, this is the first study that adopted the theory of planned behaviour as a theoretical basis for assessing and comparing the psychosocial determinants of GPs and gynaecologists' intention to prescribe HT in two countries, France and in Quebec. To our knowledge, this is also one of the few studies to adopt the theory of planned behaviour for identifying factors that influence doctors' behavioural intention to prescribe a medication in the context of preference-sensitive care.
The results of this study indicated that doctors' intention to prescribe HT varied according to their specialty. In both countries, gynaecologists showed higher intention to prescribe HT than GPs. This is in line with the results reported by Hovi and colleagues who assessed Estonian and Finnish GPs and gynaecologist opinions and prescribing practices in HT [19]. However, this intention did not vary significantly between GPs in France and those in Quebec, nor did it between gynaecologists in France and those in Quebec.
In the overall model (for all physicians in both countries), perceived barriers to prescription of HT was found to be the most important determinant of the intention to prescribe HT. This means that the more confident a doctor was regarding his/her ability to counter the perceived barriers that were assessed in this study, the more likely he/she was to express a high intention to prescribe HT. This is congruent with the observation made previously that a significant proportion of physicians considered menopause to be a serious health problem that needed to be treated [3]. In other words, even in the absence of a clear indication to prescribe HT (for example, severe hot flashes) or a clear indication by a woman that she would like to use HT, at that the time this study was conducted, some physicians had high intention to do so.
Moral norm was found to be the second most important determinant. In this study, moral norm was assessed with three items referring to the physician's personal convictions, sense of guilt and ethics. In the past, moral norm was found to be the strongest predictor of women's intention to adopt HT over a period of one year [32]. However, to our knowledge, the present study is one of the first to document that moral norm is associated with a doctor's prescription behaviour of HT. This means that, in this context, doctors' most profound personal values might overcome those of the women. As reported before by Hoffmann and colleagues, this suggests that doctors might be inclined to promote their own decision [38]. On one hand, this is in line with the literature that points to the contribution of doctors' preferences on the unexplained practice variation observed in the medical community [39]. On the other hand, this is contrary to the growing social trends in regards with shared decision-making [40]. Shared decision-making is grounded in ethics in which a deliberative model of decision-making is advocated [41]. A deliberative model of decision-making recognizes that both protagonists are competent individuals. It refers to the concepts of autonomy and self determination which underline the obligation of health care providers to provide information [41]. Shared decision-making emphasizes the importance of agreement on the treatment option by the patient and the physicians as a "the test of a shared decision" because "mutual acceptance ... remains a necessary prerequisite for shared decision-making" [42]. Shared decision-making rests on the best evidence of the risks and benefits of all the available options and takes into account the establishment of a context in which the values and preferences of the patient are sought and his opinions valued [40]. Therefore, these results suggest a need for interventions that will facilitate the inclusion of the specific characteristics and values of the patient in the decision. In the overall model, the intention to prescribe HT was also under the influence of role belief. This means that intention to prescribe HT was determined by a sense of belonging to a specific group and how one ought to act accordingly. This is different from the influence of subjective norm, which refers to perceived social pressure to perform a specific behaviour. In other words, doctors' intention to prescribe HT was dependent on what they believe someone like them should do. Doctors attach a high value to their clinical autonomy, which is viewed by certain critics as a major barrier to changing doctors' behaviour [43]. The positive influence of role belief in predicting doctors' behaviour had been reported before in the area of technology of information [24]. It appears that in the context of HT prescription, doctors might not be influenced by what others would want them to do (including a specific patient), but rather by what they believe a doctor like themselves ought to do.
When specific regression models were built by medical specialty and country, power of control beliefs was kept in all models. Moral norm was kept in three out of the four models: GPs in Quebec and gynaecologists in France and in Quebec. Role belief was kept in both models from France: GPs and gynaecologists, but not in the models of doctors from Quebec (GPs and gynaecologists). This suggests that, notwithstanding the medical specialty, there might be differences between doctors from France and those from Quebec regarding this particular behaviour.
In three out of the four models, variables that were external to the initial theoretical model made a statistically significant contribution. All of these variables pertained to diagnostic testing. For example, in each of the regression models pertaining to GPs' behavioural intention, variables associated with diagnosing menopause were found. This suggests that GPs in Quebec and in France might need reassurance in the final objective diagnosis of menopause before embarking on a prescription of HT.
Interestingly, attitude and attitudinal beliefs were not retained in any of the regression models. This means that the intention of doctors to prescribe HT was not under the influence of those doctors' personal evaluation of this action (for example, the consequences of prescribing HT on the woman's health outcomes). This suggests that, when controlled for other psychosocial variables, the prescription of HT was not under the influence of what doctors believe HT would have had as potential benefits or risks to this woman.
Another interesting finding of the present study was the U-shaped distribution of the doctors' intention to prescribe HT. Indeed, given the recommendation regarding HT by some of the professional societies at the time this study was conducted [44-47], one could have expected a skewed distribution towards a more positive physicians' intention to prescribe HT. This U-shaped distribution of the doctors' intention to prescribe HT is in line with the results of a previous study published in 1986, long before HT was thought to be benefiting the cardiovascular system. Interestingly, in this study published in 1986, doctors were shown to act decisively when facing decisions about HT [14]. Facing twelve clinical vignettes, most of the fifty doctors enrolled in this study tended to act decisively (prescribe HT or not) although the decision analysis model had recommended a "toss-up" strategy (uncertain decision) 60% of the time. The authors concluded that "doctors ought to carefully re-examine their practice patterns in light of their beliefs and opinions." [14]. This conclusion would still hold true two decades later. It suggests that it is possible that physicians' intention to prescribe HT and its determinants could be stable overtime.
This U-shaped distribution of the doctors' behavioural intention regarding HT was in sharp contrast with the results from two other studies that applied the theory of planned behaviour to predict middle-aged women intention to use HT at menopause in Québec. These two studies were conducted in the same period as the present study was. In both these studies, the distribution of the intention to use HT was normal. In other words, while most women appeared to be undecided about adopting HT, physicians from the same geographic area appeared to be decided about either prescribing HT or not. Again, these results emphasise the need for future interventions that will facilitate the active participation of women in the decision-making leading to a prescription of HT.
Strengths and limitations of the study results
A major strength of this study is its use of theory to improve understanding of the decision-making of doctors leading to a prescription of HT. Therefore, the use of an existing theoretical framework helped standardize the presentation of the many factors that are likely to influence this prescription behaviour [48]. It has the potential to: 1) facilitate the comparison between similar studies, 2) make it possible to carry out a systematic review in this area, and 3) contribute to the elaboration of a theoretical base for understanding the decision-making leading to a prescription behaviour. Other researchers might want to follow this process.
In spite of these strengths, this study has limitations. First, given that this study was conducted before the results of the Women's Health Initiative trial were publicized [1], we can not guarantee that its results would still be applicable. The decline in postmenopausal HT use was most marked for standard-dose of the estrogenic agent used in the Women's Health Initiative trial [9]. However, contrary to this trend, prescription for lower-dose formulations increased modestly [9]. Moreover, in France, results from the Women's Health Initiative trial have been criticized by French professional societies based on the fact that treatments used were different in France – mainly transdermal estrogens – and that French postmenopausal women were at lower vascular risk than those of the Women's Health Initiative trial [49]. In United States, studies of women younger than those enrolled in this trial and lower HT doses with intermediate endpoints are beginning [50]. Therefore, given the clinical vignette that was used in this study (there was no explicit dosage and formulae of HT), we believe that this study results might still be applicable to the current situation.
Second, doctors were asked to indicate their intention to prescribe HT to a specific but hypothetical woman. The clinical vignette that was used was succinct and did not provide all the clinical information that some doctors might have desired. However, a large proportion of doctors who participated in this study expressed a specific intention to prescribe HT or not, perhaps indicating that they felt they had enough information to make a decision. Moreover, clinical vignettes are known to be a valid and comprehensive method to asses the process of care provided in actual clinical practice [51]. Therefore, we believe our results can improve our understanding of the prescription of HT in this specific clinical situation.
Last, although this study was designed to draw a representative sample of GPs and gynaecologists from each country, we can not guarantee that our results apply to the whole population of GPs and gynaecologists from which our sample was drawn. The response rate that was obtained in both countries compared well with other studies that have applied the theory of planned behaviour to study doctors' behaviour [24,25,52]. Nonetheless, this response rate as well as the ratio of GPs and gynaecologists limits the inference of our results to the whole population of doctors. However, it does not limit the inference of the overall regression model for this group of respondents. Thus, the results of this study provide a valuable contribution to the theory base of doctors' prescription behaviour.
Conclusion
Despite its limitations, the present study still has clinical and research implications. Geographic variations in health care delivery remain a challenge [39]. This study provides empirical data on the significant contribution of moral norm in the decision-making leading to a prescription of HT by doctors and possibly in other contexts of preference-sensitive care [39]. This is preoccupying because it is contrary to on-going efforts to incorporate the best available scientific evidences as well as the patients' views in health [53], one of the cornerstones of improving the quality of health care [54]. Therefore, it would be important in this type of clinical situation to provide guidance to doctors on how to support active participation of patients in the decision-making process.
This study should be useful to those interested in the application of social cognitive models in studying doctors' behaviour. Given the lack of theorization in the area of health care professional practice [20,55], this study results improve the knowledge base in translation of evidence as well as the knowledge base of physicians' decision-making in context of preference-sensitive care [39]. It provides insight about the contribution of moral norm and role beliefs above variables provided by the theory of planned behaviour to explain doctors' behaviour and perhaps, variation in health care delivery. In future studies applying the theory of planned behaviour, these constructs should be tested with other type of doctors' behaviours. This study should also be useful in improving our understanding of cross-cultural variations in medical practices by focusing on theory-based psychosocial variables. Lastly, this study points to possible pathways through which a given implementation strategy is likely to be effective in modifying doctors' prescription of HT. Researchers interested in elaborating interventions to modify this prescription behaviour could use this study results accordingly.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
France Legare, Gaston Godin, Sylvie Dodin and Virginie Ringa designed the study with assistance from Joanna Norton. Data were collected and managed by Lucile Turcot and France Legare. Data were analyzed by France Legare, Gaston Godin and Lucile Turcot. France Legare wrote the report. All authors contributed to the revision of various drafts and approved the final version of the submitted manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We are indebted to the doctors from France and Quebec who participated to this study. This research was supported by an unrestricted grant from Fournier Pharma and peer-reviewed by a scientific committee from the Fonds de la recherche du Quebec (FRSQ). Dr. Legare is a new clinical scientist who is supported by the FRSQ. Dr. Godin is Tier 1 Canada Research Chair in Health Related Behaviour, Université Laval. Dr Dodin holds the Chaire Andree et Lucie Chagnon pour une approche integrée en santé, Université Laval.
==== Refs
Rossouw JE Anderson GL Prentice RL LaCroix AZ Kooperberg C Stefanick ML Jackson RD Beresford SAA Howard BV Johnson KC Morley Kotchen J Ockene J Risks and Benefits of Estrogen Plus Progestin in Healthy Postmenopausal Women. Principal Results From the Women's Health Initiative Randomized Controlled Trial. JAMA-Express 2002 288 321 333
Wilkes HC Meade TW Hormone replacement therapy in general practice: a survey of doctors in the MRC's general practice research framework BMJ 1991 302 1317 1320 2059689
Ghali WA Freund KM Boss RD Ryan CA Moskowitz MA Menopausal Hormone Therappy: Physician Awareness of Patient Attitudes. Am J Med 1997 103 3 10 9236479
Andersson K Tonnes Pedersen A Mattsson LA Milsom I Swedish gynecologists' and general practitioners' views on the climacteric period: knowledge, attitudes and management strategies Acta Obstet Gynecol Scand 1998 77 909 916 9808379
Collaborative Group on Hormonal Factors in Breast Cancer Breast cancer and hormone replacement therapy: collaborative reanalysis of data from 51 epidemiological studies of 52,705 women with breast cancer and 108,411 women without breast cancer. Collaborative Group on Hormonal Factors in Breast Cancer Lancet 1997 350 1047 1059 10213546
Beral V Breast cancer and hormone-replacement therapy in the Million Women Study Lancet 2003 362 419 427 12927427
Kaufert PA Lock M Medicalization of women's third age J Psychosom Obstet Gynaecol 1997 18 81 86 9219103
IMS Health Canada Canadian Compuscript Audit [database]
Majumdar SR Almasi EA Stafford RS Promotion and prescribing of hormone therapy after report of harm by the Women's Health Initiative Jama 2004 292 1983 1988 15507584
Maclennan AH Broadbent JL Lester S Moore V Oral oestrogen and combined oestrogen/progestogen therapy versus placebo for hot flushes Cochrane Database Syst Rev 2004 CD002978 15495039
Brown JP Josse RG 2002 clinical practice guidelines for the diagnosis and management of osteoporosis in Canada Cmaj 2002 167 S1 34 12427685
Banks E Beral V Reeves G Balkwill A Barnes I Fracture incidence in relation to the pattern of use of hormone therapy in postmenopausal women Jama 2004 291 2212 2220 15138243
Mort EA Clinical decision-making in the face of scientific uncertainty: hormone replacement therapy as an example J Fam Pract 1996 42 147 151 8606304
Elstein AS Holzman GB Ravitch MM Metheny WA Holmes MM Hoppe RB Rothert ML Rovner DR Comparison of Physicians' Decisions Regarding Estrogen Replacement Therapy for Menopausal Women and Decisions Derived from aDecision Analytic Model The American Journal of Medicine 1986 80 246 258 3946438
Obermeyer CM Sahel A Hajji N Schulein M Physicians' perceptions of menopause and prescribing practices in Morocco Int J Gynaecol Obstet 2001 73 47 55 11336721
Levy BT Ritchie JM Smith E Gray T Zhang W Physician specialty is significantly associated with hormone replacement therapy use Obstet Gynecol 2003 101 114 122 12517655
Nilsen ST Pedersen AT Moen MH Milsom I Mattsson LA Iversen OE Larsen PM Andersson K Knowledge, attitudes and management strategies in Scandinavia concerning hormone replacement therapy: a comparison between gynecologists in Denmark, Norway and Sweden Maturitas 2001 39 83 90 11451625
Newton KM LaCroix AZ Buist DS Anderson LA Delaney K What factors account for hormone replacement therapy prescribing frequency? Maturitas 2001 39 1 10 11451615
Hovi SL Karttunen T Karro H Hemminki E Comparison of Estonian and Finnish physicians' opinions of menopause and hormone therapy Maturitas 2004 49 107 113 15474754
Grol R Grimshaw J From best evidence to best practice: effective implementation of change in patients' care Lancet 2003 362 1225 1230 14568747
Ajzen I Keynes M Attitudes, personality and behavior Mapping social psychology 1988 , Open University Press xiv, 175 p.
Millstein SG Utility of the theories of reasoned action and planned behavior for predicting physician behavior: a prospective analysis Health Psychology 1996 15 398 402 8891719
Walker AE Grimshaw JM Armstrong EM Salient beliefs and intentions to prescribe antibiotics for patients with a sore throat Br J Health Psychol 2001 6 347 360 12614509
Gagnon MP Godin G Gagne C Fortin JP Lamothe L Reinharz D Cloutier A An adaptation of the theory of interpersonal behaviour to the study of telemedicine adoption by physicians Int J Med Inform 2003 71 103 115 14519403
Liabsuetrakul T Chongsuvivatwong V Lumbiganon P Lindmark G Obstetricians' attitudes, subjective norms, perceived controls, and intentions on antibiotic prophylaxis in caesarean section Soc Sci Med 2003 57 1665 1674 12948575
Gaither CA Bagozzi RP Ascione FJ Kirking DM The determinants of physician attitudes and subjective norms toward drug information sources: modification and test of the theory of reasoned action Pharm Res 1997 14 1298 1308 9358540
Ajzen I Higgins ET and Kruglanski AW The Social Psychology of Decision Making Social Psychology: handbook of basic principles 1996 Chapter II New York (NY), Guilford Press 297 325
Godin G Kok G The theory of planned behavior: a review of its applications to health-related behaviors American Journal of Health Promotion 1996 11 87 98 10163601
Sheeran P Stroebe W and Hewstone M Intention-behaviour relations: A conceptual and empirical review European review of social psychology 2002 12 Chichester, UK, WIley 1 36
Triandis HC Daigen V Interpersonal Behavior 1977 Monterey, Ca., Brooks/Cole Publishing Company A Division of Wadsworth Publishing Company, Inc. 329
Légaré F Godin G Guilbert Laperrière L Dodin S Determinants of the intention to adopt hormone replacement therapy among premenopausal women Maturitas 2000 34 211 218 10717486
Légaré F Godin G Dodin S Turcot-Lemay L Laperrière L Adherence to hormone replacement therapy: a longitudinal study using the theory of planned behaviour Psychology and Health 2003 18 351 371
Dillman DA Mail and Telephone Surveys: The Total Design Method. 1978 New York, Wiley-Interscience 375
National Cancer Institute Breast Cancer Risk Assessment Tool
Gagné C Godin G The Theory of Planned Behavior: Some Measurement Issues Concerning Belief-Based Variables Journal of Applied Social Psychology 2000 30 2173 2193
French DP Hankins M The expectancy-value muddle in the theory of planned behaviour - and some proposed solutions Br J Health Psychol 2003 8 37 55 12643815
Ringa V Legare F Dodin S Norton J Godin G Breart G Hormone therapy prescription among physicians in France and Quebec. Menopause 2004 11 89 97 14716188
Hoffmann M Lindh-Astrand L Ahlner J Hammar M Kjellgren KI Hormone replacement therapy in the menopause. Structure and content of risk talk Maturitas 2005 50 8 18 15590209
Wennberg JE Unwarranted variations in healthcare delivery: implications for academic medical centres Bmj 2002 325 961 964 12399352
Towle A Godolphin W Framework for teaching and learning informed shared decision making [see comments] Bmj 1999 319 766 771 10488010
Durand G Introduction générale à la bioéthique. Histoire, concepts et outils 1999 Première , Editions Fides Cerf 565
Charles C Gafni A Whelan T Shared decision-making in the medical encounter: what does it mean? (or it takes at least two to tango) Soc Sci Med 1997 44 681 692 9032835
Armstrong D Clinical autonomy, individual and collective: the problem of changing doctors' behaviour Soc Sci Med 2002 55 1771 1777 12383461
Schiff I Rebar RW Cramer JA Gerbino PP Hammond CB Jeffe DB Motheral BR Rothert M Sarrel PM Achieving Long-Term Continuance of Menopausal ERT/HRT: Consensus Opinion of The North American Menopause Society Menopause; The Journal of The North American Menopause Society 1998 5 69 76
NAMS A decision tree for the use of estrogen replacement therapy or hormone replacement therapy in postmenopausal women: consensus opinion of the North American Menopause Society. Menopause 2000 76 86 10746889
NAMS Clinical challenges of perimenopause: consensus opinion of the North American Menopause Society Menopause 2000 7 5 13 10646698
SOGC The Canadian Consensus Conference on Menopause and Osteoporosis. 1998 Toronto, Ontario, Ribosome Communications Inc. 64
Saillour-Glenisson F Michel P [Individual and collective facilitators and barriers to the use of clinical guidelines by physicians: a literature review Revue Épidémiologique de Santé Publique 2003 51 65 80
Gayet-Ageron A Amamra N Ringa V Tainturier V Berr C Clavel-Chapelon F Delcourt C Delmas PD Ducimetiere P Schott AM Estimated numbers of postmenopausal women treated by hormone therapy in France Maturitas 2005 15955641
Barrett-Connor E Grady D Stefanick ML The rise and fall of menopausal hormone therapy Annu Rev Public Health 2005 26 115 140 15760283
Peabody JW Luck J Glassman P Dresselhaus TR Lee M Comparison of vignettes, standardized patients, and chart abstraction: a prospective validation study of 3 methods for measuring quality Jama 2000 283 1715 1722 10755498
Hu PJH Chau PYK Physician Acceptance of Telemedicine Technology: An Empirical Investigation Top Health Inform Manage 1999 19 20 35
Walter FM Britten N Patients' understanding of risk: a qualitative study of decision-making about the menopause and hormone replacement therapy in general practice Fam Pract 2002 19 579 586 12429658
Wensing M Elwyn G Methods for incorporating patients' views in health care Bmj 2003 326 877 879 12702627
Eccles M Grimshaw J Walker A Johnston M Pitts N Changing the behavior of healthcare professionals: the use of theory in promoting the uptake of research findings J Clin Epidemiol 2005 58 107 112 15680740
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Microb Cell FactMicrobial Cell Factories1475-2859BioMed Central London 1475-2859-4-211599846910.1186/1475-2859-4-21ResearchStructural genomics of human proteins – target selection and generation of a public catalogue of expression clones Büssow Konrad [email protected] Christoph [email protected] Volker [email protected] Ulrich [email protected] Jörg [email protected] Bernd [email protected] Peer [email protected] Hans [email protected] Udo [email protected] Protein Structure Factory, Heubnerweg 6, 14059 Berlin, Germany2 Max-Planck-Institut für Molekulare Genetik, Ihnestr. 73, 14195 Berlin, Germany3 EMBL Heidelberg, Meyerhofstr. 1, 69117 Heidelberg, Germany4 RZPD German Resource Center for Genome Research GmbH, Heubnerweg 6, 14059 Berlin, Germany5 DIFE, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany6 Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany7 Institut für Chemie/Kristallographie, Freie Universität, Takustr. 6, 14195 Berlin, Germany8 Department of Bioinformatics, University of Würzburg, Biocenter, Am Hubland, 97074 Würzburg, Germany2005 5 7 2005 4 21 21 23 5 2005 5 7 2005 Copyright © 2005 Büssow et al; licensee BioMed Central Ltd.2005Büssow 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 availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells.
Results and Conclusion
Protein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins.
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Background
The Protein Structure Factory
The Protein Structure Factory (PSF) is a joint endeavour of universities, research institutes and companies from the Berlin area [1,2]. It takes part in the international structural genomics initiative [3,4] and aims at the determination of human protein structures by X-ray diffraction methods and NMR spectroscopy using standardised high-throughput procedures. A complete pipeline has been established for this purpose that comprises cloning, protein expression in small and large scale, biophysical protein characterisation, crystallisation, X-ray diffraction and structure calculation.
It is known that eukaryotic proteins are often difficult to express in Escherichia coli [5]. Only a certain fraction of these proteins can be overproduced in E. coli in sufficient yield without formation of inclusion body aggregates or proteolytic degradation. Alternative expression systems include cell cultures of various eukaryotic organisms and cell-free, in vitro protein expression. These systems have been greatly improved since 1999, when the PSF project was initiated. In the meantime, E. coli [5-7] and wheat germ [8]in vitro protein synthesis is routinely used by structural genomics projects. At the PSF, yeast expression hosts, Saccharomyces cerevisiae and Pichia pastoris, were successfully established as alternative systems to E. coli, as described in detail previously [9-11]. We will focus here on the results obtained with the E. coli expression system.
E. coli strains and vectors
The T7 RNA polymerase-dependent E. coli expression vector system (pET-vectors) is a universal system to generate recombinant protein for structural analysis [12]. pET vectors are usually combined with the E. coli B strain BL21 and derivatives that are engineered to carry the T7 RNA polymerase gene. These strains, however, have limitations in cloning and stable propagation of the expression constructs. Expression vectors which are regulated by the lac operator are independent of the host strain. Recombination-deficient E. coli K-12 strains are suitable for cloning because of their high transformation rates and because they allow for stable propagation of recombinant constructs. The strain SCS1 (Stratagene; hsdR17(rK- mK+) recA1 endA1 gyrA96 thi-1 relA1 supE44) was found to perform well at the PSF in cloning experiments. It grows relatively fast and allows for robust protein expression.
Affinity tags allow for standardised protein purification procedures. The first vector that was used routinely in the PSF, pQStrep2 (GenBank AY028642, Figure 1), is based on pQE-30 (Qiagen) and adds an N-terminal His-tag [13] for metal chelate affinity chromatography (IMAC) and a C-terminal Strep-tag II [14,15] to the expression product. pQStrep2 allows for an efficient two-step affinity purification of the encoded protein, as demonstrated in a study of an SH3 domain [16]. The eluate of the initial IMAC is directly loaded onto a Streptactin column. Thereby, only full-length expression products are purified and degradation products are removed. However, the two tags, which are flexible unfolded peptides, remain on the protein and may interfere with protein crystallisation, although we could show that crystal growth may be possible in their presence even for small proteins [16]. To exclude any negative influence by the affinity tags, another vector, pQTEV (GenBank AY243506, Figure 1), was constructed. pQTEV allows for expression of N-terminal His-tag fusion proteins that contain a recognition site of the tobacco etch virus (TEV) protease for proteolytic removal of the tag.
Figure 1 Vector maps. Vector maps of pQStrep2, pQTEV and pSE111
Codon usage has a major influence on protein expression levels in E. coli [17], and eukaryotic sequences often contain codons that are rare in E. coli. Especially the arginine codons AGA and AGG lead to low protein yield [18]. This can be alleviated by introducing genes for overexpression of the corresponding tRNAs into the E. coli host cells. We have used the plasmid pSE111 (Figure 1) carrying the argU gene for this purpose. pSE111 is compatible with pQTEV and other common expression vectors. It carries the lacIQ gene for overexpression of the Lac repressor, which is required when using promoters regulated by lac operators. pSE111 was used at the PSF in combination with the expression vectors pQStrep2 and pGEX-6P-1. Strains for overexpression of rare tRNAs are available from Invitrogen (BL21 Codon Plus) and Novagen (Rosetta). The Rosetta strain contains the chloramphenicol-resistant pRARE plasmid that supplies tRNAs for the codons AUA, AGG, AGA, CUA, CCC, GGA [19]. This plasmid is used at the PSF in combination with pQTEV and pGEX-6P-2.
The Additional file 1 psfClones.xml lists the vector and helper plasmid for overexpression of rare tRNAs that was used for each individual clone.
Selection of target proteins
We selected target proteins with higher-than-average chances of successful expression in E. coli and crystallisation [1]. Proteins were excluded for which sequence analysis predicted that structure determination would be difficult. Starting from the complete set of known human proteins, potentially difficult target proteins and proteins of known structure were excluded according to the following criteria:
• Membrane proteins are known to be complicated targets for structure determination and were excluded. Membrane proteins were identified with the program TMHMM [20,21].
• Since very large proteins are often difficult to express, the maximal length of target proteins was set to 500 amino acids.
• Protein regions that are unstructured or only partially structured [22] may lead to difficulties during protein expression and purification. Unstructured regions are susceptible to proteolyic attack, and represent an obstacle to protein crystallisation. A large proportion of intrinsically unstructured protein sequences are characterised by sequence stretches of low complexity and tandem repeats [23]. Proteins with low complexity regions of more than 20 amino acids length, detected by the SEG program, or with more than one region were excluded [24,25].
• Coiled-coil proteins were excluded from our target list, since this fold is not novel, and structural analysis of coiled coils requires special attention. Many coiled coil proteins form hetero-complexes with other coiled-coil proteins and cannot be studied without their binding partner. Coiled-coils domains are long, extended structures which can usually only be crystallised as domains, i.e. expression constructs lacking other domains have to be prepared. To identify coiled-coil proteins, the program COILS was used [26-28].
• The cellular localisation of target proteins was assigned with the Meta_A(nnotator) [29,30]. Target proteins annotated to be localised in the extracellular space, endoplasmic reticulum, Golgi stack, peroxisome or mitochondria were excluded from expression in E. coli. Many of these proteins require formation of disulphide bonds for correct folding, but these are generally not formed in the reducing environment of the E. coli cytoplasm. Therefore, these proteins were allocated only for extracellular expression by yeast host cells. Proteins with predicted intracellular localisation or which were not assigned with a localisation by Meta_A(nnotator) were expressed in the cytosol of E. coli.
• Potential target proteins were matched to the sequences of proteins with known structure at the Protein Data Bank (PDB) [31]. PSI-BLAST [32] was used to detect even very distinct homologies to PDB entries to rule out proteins with known folds. This filter was later replaced by a less stringent one, which considers the sequence identity and 'coverage' of matches to PDB sequences. The coverage is the length of the sequence match divided by the protein length. According to the less stringent filter, proteins with 60% or more sequence identity over 90% or more of the sequence length were excluded. Thereby target proteins could be included of which only a part, e.g. a single domain, has a known structure.
Results
Target lists
The first target list was generated in 1999, when the PSF project started, from the set of all human proteins known at that time. This set was filtered as described above. PSI-BLAST was used to match potential target proteins to the PDB and to include only proteins of presumably novel folds. To enable high-throughput cloning, we selected target proteins for which full-length cDNA clones were available. In 1999, cDNA clones of the IMAGE consortium represented the main public source of sequenced cDNA clones [33]. However, only partial sequence information existed for these clones – the EST sequences of the dbEST database [34] – and only a small proportion contained complete open reading frames (full-ORF clones). 490 proteins were selected which met the selection criteria, had no match to PDB detected by PSI-BLAST and for which full-ORF IMAGE cDNA clones were available.
In 2003, a second target list was compiled from novel full-length cDNAs discovered by the German cDNA consortium [35,36]. The same filter criteria as for the first target list were applied, except that cellular localisation was not taken into account. Proteins with a PDB match of 60% or more sequence identity and 90% sequence coverage were excluded, resulting in a target list of 259 proteins.
A third set of target proteins was selected from a human cDNA expression library (hEx1), which was cloned in a bacterial expression vector. This library was screened for expression clones on high density arrays and by high-throughput protein expression and purification experiments [37-40]. This identified 2,700 clones expressing soluble His-tag fusion proteins that could be purified by affinity chromatography [39]. The cDNA inserts of these clones were sequenced and assigned to sequences of the Ensembl database [41]. 141 proteins represented by clones of the expression library where selected as targets for structural analysis [39]. These clones express soluble, full-length proteins, of which the three-dimensional structure was unknown.
The numbers of targets and success rates grouped by the type of target cDNA clone are summarised in Table 1.
Table 1 Origin of template cDNA clones Numbers of targets grouped by type of template cDNA clone
Target list Number of targets Number of successfully cloned cDNA Number of proteins with soluble expression Number of structures
1 – IMAGE clones 490 264 54 3
2 – cDNA consortium 259 185 34 2
3 – hEx1 library 141 88 51 3
all 890 537 139 8
Generation and characterisation of expression clones
We established a common cloning strategy that allows for easy shuttling of cDNA fragments between different E. coli and yeast vectors. We adopted a cloning system that adds only a minimal number of extra amino acids to the protein of interest and therefore decided to clone with restriction enzymes instead of using alternative systems, such as Invitrogen's Gateway system or ligation independent cloning [42].
The PSF has been working with more than a thousand target proteins to date. Suitable cDNA clones were selected and subcloned into the E. coli expression vectors pQTEV GenBank AY243506 and pQStrep2 AY028642[16]. These vectors provide for an N-terminal His-tag; pQStrep2 also encodes an C-terminal Strep-tag-II. Some proteins have also been expressed as GST fusion proteins using the vectors pGEX-4T2 or pGEX-6P1 (Amersham Biosciences).
Expression of protein coding genes from multiple transformants per target was tested under multiple conditions. Standardisation and automation was introduced to achieve this throughput. Expression clones were characterised by small scale protein synthesis at different temperatures, 37°C, 30°C and 25°C, in 1 ml volumes in deep-96-well microplates. Proteins were purified in parallel by a pipetting robot, as described previously [43]. 10% of the purified protein eluate from a 1 ml culture was analysed by SDS-PAGE. For each protein expression experiment, the size of the expression product was recorded and the amount of protein was classified into four categories: none, weak, moderate and strong expression. This classification is arbitrary to a certain degree, however, we found it sufficient to select suitable clones for protein production scale-up.
1414 clones for 537 target proteins were successfully cloned in E. coli expression vectors, with 473, 191 and 94 target proteins corresponding to target lists one (IMAGE clones), two (DKFZ clones) and three (hEx1 clones), respectively. Clones for 139 different target proteins were found to be expressed in soluble form by E. coli. Figure 2 and Table 2 show the result of small scale expression and purification of these proteins. The yield varied significantly among different target proteins. The Additional file 1 psfClones.xml contains further details on the expression clones, such as vector, strain and helper plasmid for overexpression of rare tRNAs.
Figure 2 SDS-PAGE of purified human proteins. 15% SDS-PAGE (Coomassie-stained) of proteins expressed in small scale in E. coli and purified by automated immobilised metal chelate affinity chromatography as described in [43]. The identities of the purified proteins are indicated in Table 2. Protein expression was induced at the temperature that is optimal for the individual clone. These temperatures are listed in the supplementary file psfClones.xml. M: Molecular weight marker.
Table 2 PSF E. coli expression clones with soluble expression products. The table corresponds to the proteins shown in Figure 2. More detailed information is available in the supplementary XML file, Additional file 1.
NCBI Entrez gene ID Gene symbol, name Protein accession RZPD clone ID Sequence verified, non-silent mutations Clone accession Protein size [Da] Protein gel, lane
39 ACAT2, acetyl-Coenzyme A acetyltransferase 2 AAM00223 PSFEp250C082 partly, none 44,177 D, 24
203 AK1, adenylate kinase 1 BAA78534 PSFEp250B112 yes, none DQ000549 24,558 C, 21
689 BTF3, basic transcription factor 3 CAA37376 PSFEp758D0224 no 20,622 A, 27
830 CAPZA2, capping protein (actin filament) muscle Z-line, alpha 2 AAC60382 PSFEp758H1226 no 59,649 A, 4
1036 CDO1, cysteine dioxygenase, type I BAA12872 PSFEp758D0124 yes, none DQ000531 25,895 E, 16
1428 CRYM, crystallin, mu AAC16914 PSFEp758B0810 yes, none DQ000499 36,275 D, 4
1460 CSNK2B, casein kinase 2, beta polypeptide CAA34379 PSFEp758H0422 no 26,209 B, 12
1606 DGKA, diacylglycerol kinase, alpha 80kDa AAC34806 PSFEp758F0324 yes, none DQ000529 16,408 A, 23
1627 DBN1, drebrin 1 AAH07567 PSFEp250C052 partly, none 74,354 C, 31
1635 DCTD, dCMP deaminase AAC37579 PSFEp250B121 yes, none DQ000535 22,938 E, 25
1937 EEF1G, eukaryotic translation elongation factor 1 gamma AAH15813 PSFEp250H061 partly, none 53,045 C, 11
1974 EIF4A2, eukaryotic translation initiation factor 4A, isoform 2 AAH48105 PSFEp250H052 partly, none 49,301 D, 20
2963 GTF2F2, general transcription factor IIF, polypeptide 2, 30 kDa CAA42419 PSFEp250B012 yes, none DQ000547 31,304 C, 17
2992 GYG, glycogenin AAB00114 PSFEp758H1122 yes, none DQ000517 40,403 B, 4
3151 HMGN2, high-mobility group nucleosomal binding domain 2 AAA52678 PSFEp250E102 yes, none DQ000559 12,314 D, 27
3312 HSPA8, heat shock 70 kDa protein 8 BAB18615 PSFEp250E022 partly, none 56,444 D, 31
3735 KARS, lysyl-tRNA synthetase AAH04132 PSFEp250D032 partly, A116G 70,976 D, 16
3925 STMN1, stathmin 1/oncoprotein 18 CAC16020 PSFEp250F072 yes, none DQ000556 20,225 D, 19
4043 LRPAP1, low density lipoprotein receptor-related protein associated protein 1 AAC67373 PSFEp250G031 yes, none DQ000540 44,391 C, 22
4695 NDUFA2, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2 AAD27762 PSFEp250D091 no 13,843 C, 13
4698 NDUFA5, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 AAD21526 PSFEp250A0510 no 16,381 A, 13
5184 PEPD, peptidase D AAH28295 PSFEp250H122 yes, none DQ000560 D, 29
5202 PFDN2, prefoldin 2 AAF17218 PSFEp250D112 yes, none DQ000555 19,570 D, 17
5412 UBL3, ubiquitin-like 3 AAD02323 PSFEp758A0510 yes, none DQ000501 15,657
5502 PPP1R1A, protein phosphatase 1, regulatory (inhibitor) subunit 1A AAB02402 PSFEp758G0124 yes, none DQ000526 21,862 A, 15
5716 PSMD10, proteasome (prosome, macropain) 26S subunit, non-ATPase, 10 (gankyrin) AAH11960 PSFEp250A062 yes, none DQ000544 27,351 C, 9
5717 PSMD11, proteasome (prosome, macropain) 26S subunit, non-ATPase, 11 AAH04430 PSFEp250B122 yes, none DQ000548 50,390 C, 19
5877 RABIF, RAB interacting factor AAB18264 PSFEp250C021 no 16,761 E, 23
6133 RPL9, ribosomal protein L9 BAA03401 PSFEp758E0124 yes, none DQ000524 24,786 A, 28
6156 RPL30, ribosomal protein L30 CAA55820 PSFEp758A1113 yes, none DQ000503 15,284 D, 6
6191 RPS4X, ribosomal protein S4, X-linked BC007308 PSFEp758A0923 no 28,670
6342 SCP2, sterol carrier protein 2 AAA03559 PSFEp758C0723 yes, none DQ000515 16,668 B, 1
6451 SH3BGRL, SH3 domain binding glutamic acid-rich protein like AAC27445 PSFEp758C0713 no 15,274 D, 2
6728 SRP19, signal recognition particle 19 kDa CAA31280 PSFEp758E0317 no 41,156
6888 TALDO1, transaldolase 1 AAB53943 PSFEp758B0711 partly, none 40,040 D, 1
6990 TCTE1L, t-complex-associated-testis-expressed 1-like AAA57444 PSFEp758D0814 no 38,062 A, 3
6993 TCTEL1, t-complex-associated-testis-expressed 1-like 1 BAA09317 PSFEp758F1123 yes, none DQ000521 13,719 A, 14
7001 PRDX2, peroxiredoxin 2 AAH03022 PSFEp250A042 yes, none DQ000546 24,815 C, 30
7178 TPT1, tumor protein, translationally-controlled 1 CAA34200 PSFEp250G011 yes, none DQ000539 22,518 C, 14
7247 TSN, translin CAA55341 PSFEp250B111 yes, none DQ000538 29,106 C, 12
7353 UFD1L, ubiquitin fusion degradation 1-like N P_005650 PSFEp758G0123 yes, none DQ000519 41,777 D, 9
7390 UROS, uroporphyrinogen III synthase AAA60273 PSFEp758G0323 no 29,894 D, 11
7518 XRCC4, X-ray repair complementing defective repair in Chinese hamster cells 4 AAH16314 PSFEp250A033 partly, none 41,211 D, 26
7531 YWHAE, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon AAH01440 PSFEp250H041 yes, A146G DQ000541 32,097 C, 24
8125 ANP32A, acidic (leucine-rich) nuclear phosphoprotein 32 family, member A AAB91548 PSFEp250D092 yes, none DQ000553 31,509 D, 22
8214 DGCR6, DiGeorge syndrome critical region gene 6 CAA65339 PSFEp758G0324 yes, none DQ000533 12,111 E, 19
8407 TAGLN2, transgelin 2 BAA04802 PSFEp250F062 yes, none DQ000550 25,314 C, 27
8544 PIR, pirin CAA69195 PSFEp758A0213 yes, none DQ000498 34,613 C, 1
8575 PRKRA, protein kinase, interferon-inducible double stranded RNA dependent activator CAB66550 PSFEp250D014 yes, none DQ000578 37,328 E, 9
8677 STX10, syntaxin 10 AAC05087 PSFEp250D102 yes, none DQ000552 25,369 D, 18
8724 SNX3, sorting nexin 3 AAC16040 PSFEp758A0323 yes, none DQ000514 20,029 B, 15
8896 G10, maternal G10 transcript AAH22821 PSFEp250F011 yes, A394G DQ000536 19,766 C, 16
8926 SNURF, SNRPN upstream reading frame AAD31391 PSFEp250A0610 no 11,334 C, 25
9049 AIP, aryl hydrocarbon receptor interacting protein AAB59004 PSFEp758F1024 yes, none DQ000528 40,589 A, 21
9158 FIBP, fibroblast growth factor (acidic) intracellular binding protein CAG33030 PSFEp250B022 partly, T602C 44,803 C, 20
9168 TMSB10, thymosin, beta 10 AAB25225 PSFEp758A0124 yes, none DQ000522 6,292 A, 18
9337 CNOT8, CCR4-NOT transcription complex, subunit 8 AAH17366 PSFEp758E0524 yes, none DQ000523 36,354 A, 24
9453 GGPS1, geranylgeranyl diphosphate synthase 1 AAH05252 PSFEp250E052 partly, none 37,795 D, 23
9465 AKAP7, A kinase (PRKA) anchor protein 7 AAC39715 PSFEp758E0415 no 34,000 A, 1
9796 PHYHIP, phytanoyl-CoA hydroxylase interacting protein BAA13402 PSFEp758C1124 yes, T806C DQ000530 40,497 A, 29
10000 AKT3, v-akt murine thymoma viral oncogene homolog 3 CAB55977 PSFEp250B063 yes, none 56,516 E, 20
10063 COX17, COX17 homolog, cytochrome c oxidase assembly protein (yeast) AAA98114 PSFEp758D0723 yes, none DQ000516 8,182 B, 3
10094 ARPC3, actin related protein 2/3 complex, subunit 3 AAB64191 PSFEp758G0811 yes, none DQ000507 23,047 D, 7
10169 SERF2, small EDRK-rich factor 2 AAC63516 PSFEp250A057 no 9,821 C, 23
10228 STX6, syntaxin 6 AAH09944 PSFEp758G0526 yes, none DQ000509 32,100 B, 7
10247 HRSP12, heat-responsive protein 12 (14.5 kDa translational inhibitor protein, p14.5) CAA64670 PSFEp758H0822 yes, none DQ000518 15,760 D, 12
10290 APEG1, aortic preferentially expressed protein 1 AAH06346 PSFEp250B082 yes, none DQ000543 15,614 E, 18
10539 TXNL2, thioredoxin-like 2 AAH05289 PSFEp250A022 partly, none 40,357 C, 26
10588 MTHFS, 5,10-methenyltetrahydrofolate synthetase AAC41945 PSFEp758B0824 no 24,522 D, 15
10589 DRAP1, DR1-associated protein 1 (negative cofactor 2 alpha) AAH10025 PSFEp250C042 yes, none DQ000542 25,273 E, 3
10598 AHSA1, AHA1, activator of heat shock 90 kDa protein ATPase homolog 1 (yeast) AAD09623 PSFEp250B091 no 41,199 C, 15
10606 PAICS, phosphoribosylaminoimidazole carboxylase AAH19255 PSFEp758G0226 partly, none 50,005 A, 5
10842 C7orf16, chromosome 7 open reading frame 16 AAF03537 PSFEp758F0923 no 19,132 B, 2
10856 RUVBL2, RuvB-like 2 (E. coli) AAH04531 PSFEp250C072 partly, none 54,083 D, 25
10912 GADD45G, growth arrest and DNA-damage-inducible, gamma AAC83329 PSFEp758F0111 yes, none DQ000504 19,621 C, 4
10933 MORF4L1, mortality factor 4 like 1 AAH22845 PSFEp250G051 partly, none 40,155 C, 10
10963 STIP1, stress-induced-phosphoprotein 1 AAA58682 PSFEp250A012 partly, A86C 65,566 C, 28
11140 CDC37, CDC37 cell division cycle 37 homolog (S. cerevisiae) AAH08793 PSFEp250E042 partly, none 47,394 D, 28
11316 COPE, coatomer protein complex, subunit epsilon AAH07250 PSFEp758G1126 yes, none DQ000510 37,406 B, 9
11333 PDAP1, PDGFA associated protein 1 AAH07873 PSFEp250A073 yes, none DQ000554 23,553 D, 30
11337 GABARAP, GABA(A) receptor-associated protein AAD02337 PSFEp758H0922 no 15,185 D, 10
11344 PTK9L, PTK9L protein tyrosine kinase 9-like (A6-related protein) N P_009215 PSFEp758A0127 yes, none DQ000525 42,473 E, 13
22818 COPZ1, coatomer protein complex, subunit zeta 1 AAD34115 PSFEp758A0812 yes, none DQ000497 22,698 D, 3
22919 MAPRE1, microtubule-associated protein, RP/EB family, member 1 CAB53072 PSFEp250C112 yes, none DQ000557 32,923 D, 21
22931 RAB18, RAB18, member RAS oncogene family CAB66668 PSFEp250C118 no 25,900 B, 27
23589 CARHSP1, calcium regulated heat stable protein 1 AAD25021 PSFEp778D072 no 40,934
25842 ASF1A, ASF1 anti-silencing function 1 homolog A (S. cerevisiae) AAD34093 PSFEp758E1224 yes, none DQ000532 25,891 E, 17
25843 PREI3, preimplantation protein 3 AAD34090 PSFEp758B0812 yes, none DQ000500 28,532 C, 2
25996 DKFZP566E144, small fragment nuclease AAD34109 PSFEp758G0423 yes, none DQ000520 25,021 D, 13
26289 AK5, adenylate kinase 5 AAH12467 PSFEp250G042 yes, 3 bp insertion at 3' end DQ000558 24,897 E, 14
26353 HSPB8, heat shock 22 kDa protein 8 CAB66870 PSFEp250G125 yes, none DQ000565 24,527 B, 16
27095 TRAPPC3, trafficking protein particle complex 3 AAB96936 PSFEp758A0513 yes, none DQ000502 22,774 C, 3
27249 C2orf25, chromosome 2 open reading frame 25 AAD20048 PSFEp758D1124 no 35,864 A, 22
27335 eIF3k, eukaryotic translation initiation factor 3 subunit 12 BAA76626 PSFEp758D0923 no 26,326 D, 8
27430 MAT2B, methionine adenosyltransferase II, beta CAB66599 PSFEp250A114 yes, none DQ000575 40,476 B, 21
28970 PTD012, PTD012 CAB66540 PSFEp250B014 yes, none DQ000586 38,042 E, 24
29789 PTD004, GTP-binding protein PTD004 CAB66481 PSFEp250H073 yes, none DQ000564 47,609 B, 18
30819 KCNIP2, Kv channel interacting protein 2 CAB66656 PSFEp250G064 yes, none DQ000562 28,964 E, 5
51001 CGI-12, CGI-12 AAH12995 PSFEp250B092 yes, none DQ000545 40,832 C, 8
51076 CUTC, cutC copper transporter homolog (E. coli), CGI-32 AAH21105 PSFEp758E1112 yes, none DQ000505 31,811 C, 5
51078 THAP4, THAP domain containing 4 AAD27745 PSFEp250H116 no 21,550 C, 7
51155 HN1, hematological and neurological expressed 1 AAH01420 PSFEp758G0126 yes, 3 bp deletion at 5' end, G457A DQ000508 18,937 C, 6
51160 VPS28, vacuolar protein sorting 28 (yeast) AAF00499 PSFEp758C0323 yes, none DQ000513 26,692 B, 14
51335 NEUGRIN, mesenchymal stem cell protein DSC92 CAD39160 PSFEp250B084 yes, none DQ000566 27,342 B, 28
51397 COMMD10, COMM domain containing 10 AAD44489 PSFEp758H0811 yes, none DQ000506 25,466 D, 5
51433 ANAPC5, anaphase promoting complex subunit 5 BAA76629 PSFEp758E0214 no 47,174 B, 8
51451 LCMT1, leucine carboxyl methyltransferase 1 AAH01214 PSFEp758H0126 partly, none 41,303 B, 11
51534 C6orf55, chromosome 6 open reading frame 55 AAF76210 PSFEp758H0426 partly, none 41,303 B, 13
51629 CGI-69, CGI-69 AAD34064 PSFEp758D0316 no 63,564 B, 6
51678 MPP6, membrane protein, palmitoylated 6 CAB66770 PSFEp250E023 yes, none DQ000583 64,044 E, 27
54816 SUHW4, suppressor of hairy wing homolog 4 (Drosophila) CAB66569 PSFEp250G074 yes, none DQ000587 27,032 E, 22
55255 WDR41, WD repeat domain 41 CAD38853 PSFEp250E105 yes, none DQ000563 26,313
55276 PGM2, phosphoglucomutase 2 CAB66640 PSFEp250B113 partly, none 71,239 B, 26
56681 SARA1, SAR1a gene homolog 1 (S. cerevisiae) CAB66658 PSFEp250G115 yes, none DQ000570 25,290 B, 17
56911 C21orf7, chromosome 21 open reading frame 7 CAD28500 PSFEp250B086 yes, none DQ000571 19,032 E, 6
57019 LOC57019, cytokine induced apoptosis inhibitor 1 AAC24311 PSFEp758E0424 yes, none DQ000534 36,506 E, 21
58485 TRAPPC1, trafficking protein particle complex 1 AAD44697 PSFEp250D071 yes, T350C DQ000537 19,754 E, 1
64284 RAB17, RAB17, member RAS oncogene family CAB66580 PSFEp250E095 yes, none DQ000572 26,398 A, 6
79632 C6orf60, chromosome 6 open reading frame 60 CAB66701 PSFEp250B044 yes, none DQ000568 34,217 B, 20
79666 PLEKHF2, pleckstrin homology domain containing, family F member 2 CAD39132 PSFEp250D054 yes, A271G DQ000581 30,721 A, 9
79791 FBXO31, F-box protein 31 CAB66696 PSFEp250F085 yes, none DQ000585 18,588 E, 12
80347 COASY, Coenzyme A synthase AAF87955 PSFEp250A053 yes, none DQ000551 33,147 C, 29
80895 ILKAP, integrin-linked kinase-associated serine/threonine phosphatase 2C CAB66784 PSFEp250D083 yes, none DQ000569 45,832 B, 19
81876 RAB1B, RAB1B, member RAS oncogene family CAB66570 PSFEp250C128 no 25,094 A, 12
81889 DKFZP566J2046, fumarylacetoacetate hydrolase domain containing 1 CAB66654 PSFEp250B074 yes, none DQ000567 27,766 B, 24
83538 DKFZP434H0115, hypothetical protein DKFZp434H0115 CAB66694 PSFEp250G093 partly, none 79,584 E, 11
83543 C9orf58, chromosome 9 open reading frame 58 CAB66501 PSFEp250B085 yes, none DQ000573 19,990 B, 25
83667 SESN2, sestrin 2 CAB66486 PSFEp250F043 yes, none DQ000576 57,420 E, 7
84072 NOHMA, HORMA domain containing protein CAB66689 PSFEp250F083 yes, none DQ000561 47,343 E, 4
84324 CIP29, cytokine induced protein 29 kDa CAC37950 PSFEp758H1026 yes, none DQ000512 26,594 A, 2
84457 PHYHIPL, phytanoyl-CoA hydroxylase interacting protein-like CAD39006 PSFEp250A084 yes, none DQ000579 45,425 E, 10
84557 MAP1LC3A, microtubule-associated protein 1 light chain 3 alpha CAD38714 PSFEp250E106 yes, none DQ000584 17,194 E, 26
91603 MGC20398, hypothetical protein MGC20398 BAB70992 PSFEp758H0526 yes, none DQ000511 44,915 B, 10
94240 EPSTI1, epithelial stromal interaction 1 (breast) CAD38599 PSFEp250C015 yes, none DQ000580 25,527 A, 11
112611 RWDD2, RWD domain containing 2 CAB52345 PSFEp758A1024 partly, T709C 35,159 B, 5
118812 C10orf83, chromosome 10 open reading frame 83 CAD38849 PSFEp250H085 yes, none DQ000574 19,158 B, 23
122060 FLJ30046, hypothetical protein FLJ30046 CAD38891 PSFEp250E015 yes, none DQ000577 23,468 E, 8
136319 MTPN, myotrophin CAD38909 PSFEp250D016 yes, none DQ000582 15,817 A, 7
140856 C20orf79, chromosome 20 open reading frame 79 CAB56175 PSFEp758G0224 yes, G258T DQ000527 20,585 A, 19
Biophysical properties of proteins which could be expressed in soluble form in E. coli were compared against all tested proteins. We found no significant correlation between expression success and either protein length or mean net charge (data not shown). However, when analysing the mean hydrophobicity, we found that hydrophobic proteins are less likely to be expressed in soluble form. Only one of 139 well expressed proteins has a mean hydrophobicity of more than 0.2, while 8% of the other proteins are above this value. This group of proteins does not contain transmembrane helices according to TMHMM, and therefore may represent peripheral membrane proteins with hydrophobic surface regions.
The E. coli expression clones of the PSF are publicly available from the RZPD German Resource Center [44]. The Additional file 1 is an XML list of these clones. It can be viewed in a web browser (Figure 3). The Additional file contains, for each clone:
Figure 3 The supplementary XML Additional file 1 file displayed in a web browser.
• Gene ID and name,
• Accession number,
• Cloning details,
• Strain and vector,
• Expected sequence,
• Protein expression results,
• Sequence verification.
Solved structures
As a result of the target selection and cloning described in this paper, ten novel X-ray structures of human proteins were determined (Table 3). The structure of one protein, TRAPPC3/BET-3, was determined after protein expression in S. cerevisae, while the other proteins were produced in E. coli.
Table 3 Novel human protein structures The structures of full length proteins solved by the Protein Structure Factory.
NCBI Entrez gene ID Name GenBank protein accession PDB ID Reference
5716 Gankyrin AAH11960 1QYM [55]
10290 APEG1, aortic preferentially expressed protein 1 AAH06346 1U2H
81889 Fumarylacetoacetate hydrolase family member FLJ36880 CAB66654 1SAW [56]
28970 PTD012 CAB66540 1XCR
10247 14.5 kDa translational inhibitor protein, p14.5 CAA64670 1ONI [57]
27095 BET3, trafficking protein particle subunit AAB96936 1SZ7 [58]
5184 Peptidase D AAH28295
51076 CutC copper transporter homolog, CGI-32 AAH21105
122553 TPC6 CAI46185 2BJN [59]
6449 Nicotinamide mononucleotide adenylyltransferase NP_003012 1GZU [60]
Discussion
We describe here the strategies and experiments of our structural genomics project on human proteins. In addition to the expression of full length proteins, the Protein Structure Factory has also studied protein domains by NMR spectroscopy, which has been described elsewhere [45-47]. Our selection of full length target proteins was mainly determined by the availability of full length cDNA clones. In addition, biophysical and bioinformatical criteria were applied, leading to a biased selection of target proteins from the human proteome. Therefore, we expect that the percentage of proteins that we could express and purify in soluble form, 18%, is higher than it would be in a randomly selected set. The low proportion of successfully expressed proteins indicates that E. coli is not the appropriate expression host for many full length human proteins. High throughput protein expression in alternative system such as yeast [9-11] or insect cells/baculovirus [48] has been established and will lead to better success rates in future projects.
Generally, clones that did express a soluble protein were verified by DNA sequencing, while clones that did not express or expressed an insoluble product were usually not sequence verified. It cannot be ruled out that some of the unsuccessful clones contain sequence errors introduced during cloning. Since template cDNA clones of the IMAGE consortium with only partial sequence information were used for most cloning experiments, expression clones that were not sequence verified might represent splice variants or isoforms of the original target. The distribution of mean net charge and length was similar among successfully expressed and all proteins, while very hydrophobic proteins were generally not expressed well in our E. coli expression system.
Future efforts in structural genomics of mammalian proteins will benefit from a much better supply of full length cDNA clones. Clones prepared for protein expression by resource centres and commercial suppliers are becoming available now. With such resources, alternative target selection strategies will become feasible that will not be restricted by the availability of cDNA clones. Instead, all potential target proteins, including splice variants, could be clustered by similarity and the most suitable members of each cluster could be selected by appropriate criteria as outlined in the Background section.
In our approach, we have excluded certain types of proteins such as membrane proteins and very large proteins. A structural genomics approach that includes membrane proteins would require standard protocols to optimise expression conditions and detergents [49]. The best strategy to study large proteins is to divide them into domains and smaller regions. However, such smaller constructs usually have to be designed manually.
All clones listed in the supplementary file (Additional file 1) and Table 2 are available to the research community. Thereby we hope to facilitate further functional characterisation of this set of human proteins.
Materials and Methods
Cloning with restriction enzymes
cDNA inserts were amplified by PCR primers carrying tails with BamHI and NotI sites and cloned into the respective sites of one of the expression vectors pQTEV, pQStrep2 or pGEX-6p1. This had the drawback that the restriction sites chosen for cloning might occur in the insert. In such cases, compatible overhangs were produced by alternative enzymes or by the hetero-stagger cloning method [50]. Alternative enzymes are BglII for BamHI and the type IIs enzymes BpiI, Eco31I, Esp3I, which can replace both BamHI and NotI. Type IIs enzymes cut outside their recognition sequence and can produce arbitrary overhangs.
PCR Primer design
PCR primers with tails carrying restriction enzyme cleavage sites were designed automatically by a Perl program. The primer design program adjusts the length of the primers to achieve a melting temperature close to a common default. Then, restriction enzymes that do not cut within the respective cDNA sequence are selected by the program and restriction enzyme sites are attached to the primer sequences. Finally, since restriction enzymes do not cut well at the very end of a DNA molecule, an additional short nucleotide tail is automatically attached to the primers. The sequence of this tail is optimised to minimise formation of secondary structure, hairpins or dimerisation. A Java version of the primer design software, 'ORFprimer', is publicly available [51].
Automated high-throughput cloning, protein expression and purification
PCR primers and cDNA clones were delivered in 96-well microplate format. Upon delivery, plates with PCR primers and template clones were reformatted to obtain corresponding plate positions by a Zinsser Speedy pipetting robot. The PCR master mix (Roche Expand) and cDNA primers (10 μM stocks) were pipetted into a PCR microplate with a multichannel pipet. Template clone bacteria were added with a 96-pin steel replicating device from overnight cultures in microtitre plates. PCR product size and yield was determined by agarose gel electrophoresis and the software Phoretix 1D Quantifier (Nonlinear dynamics). PCR products were purified with magnetic beads on the pipetting robot with a system that has been developed at the Max Planck Institute of Molecular Genetics in collaboration with Bruker Daltonics (Bruker genopure kit). The correct restriction enzyme master mixes were automatically added and the digested fragments were purified again, analysed by agarose gel electrophoresis and quantified. The robot then adjusted DNA concentrations to a common default by dilution. Ligations were set up manually with a multichannel pipet, and SCS1 E. coli cells carrying pRARE were transformed in a PCR microplate by chemical transformation on a PCR machine [52]. Transformed cells were manually plated on individual agar plates. Four clones were picked per transformation and were checked by PCR using vector primers. E. coli expression clones were ready for protein expression at this stage.
Primer sequences and template clones for cloning of the target cDNAs are listed in the supplementary XML file, Additional file 1.
The characterisation of expression clones by parallel expression and protein purification is described in reference [43].
Sequence analysis
Sequence analysis software was run with default settings unless indicated otherwise. The mean charge of a protein was calculated as the difference of the number of positive and negatively charged amino acids (Lys, Arg and Glu, Asp, respectively) divided by the protein length. The mean hydrophobicity was calculated with the Kyte and Doolittle hydropathy index [53], obtained from the EMBOSS package [54]. The index values were added up for a given protein and divided by the protein length.
Authors' contributions
• Study design and coordination: KB, HL, UHe
• Bioinformatics: UHa, BS, JS, PB, VS, KB
• E. coli cloning: VS, KB
• E. coli protein work: CS, KB
• Manuscript, figures, XML file preparation: KB
Supplementary Material
Additional File 1
A ZIP archive that contains the XML list psfClones.xml of PSF clones available at the RZPD and the XSL stylesheet psfToHtml.xsl to display the XML file in a web browser. The two files should be extracted from the ZIP archive into a local directory. Then psfClones.xml can be opened with a current web browser like Mozilla or Internet Explorer. 1414 Clones for 537 target proteins are described in the XML file. It lists the gene, cloning details, expected sequence, protein expression results and the degree of sequence verification for each clone.
Click here for file
Acknowledgements
We wish to thank Ulf Lenski for help with sequence analysis and Janett Tischer for technical assistance. This work was funded by the German Federal Ministry of Education and Research (BMBF) through the Leitprojektverbund Proteinstrukturfabrik and the National Genome Network NGFN (FKZ 01GR0471, 01GR0472) and with support by the Fonds der Chemischen Industrie to U.H. Support by the Berlin Senate and the European Fund for Regional Development (EFRE) is also gratefully acknowledged.
==== Refs
Heinemann U Büssow K Mueller U Umbach P Facilities and methods for the high-throughput crystal structure analysis of human proteins Accounts of Chemical Research 2003 36 157 163 12641472 10.1021/ar010129t
Protein Structure Factory
Zhang C Kim SH Overview of structural genomics: from structure to function Current Opinion in Chemical Biology 2003 7 28 32 12547423 10.1016/S1367-5931(02)00015-7
Todd AE Marsden RL Thornton JM Orengo CA Progress of structural genomics initiatives: an analysis of solved target structures Journal of Molecular Biology 2005 348 1235 1260 15854658 10.1016/j.jmb.2005.03.037
Gilbert M Albala Joanna S Accelerating code to function: Sizing up the protein production line Current Opinion in Chemical Biology 2002 6 102 105 11827832 10.1016/S1367-5931(01)00291-5
Yokoyama S Protein expression systems for structural genomics and proteomics Current Opinion in Chemical Biology 2003 7 39 43 12547425 10.1016/S1367-5931(02)00019-4
Kigawa T Yabuki T Yoshida Y Tsutsui M Ito Y Shibata T Yokoyama S Cell-free production and stable-isotope labeling of milligram quantities of proteins FEBS Letters 1999 442 15 19 9923595 10.1016/S0014-5793(98)01620-2
Tyler RC Aceti DJ Bingman CA Cornilescu CC Fox BG Frederick RO Jeon WB Lee MS Newman CS Peterson FC Phillips GNJ Shahan MN Singh S Song J Sreenath HK Tyler EM Ulrich EL Vinarov DA Vojtik FC Volkman BF Wrobel RL Zhao Q Markley JL Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies Proteins 2005 59 633 643 15789406 10.1002/prot.20436
Holz C Hesse O Bolotina N Stahl U Lang C A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae Protein Expression & Purification 2002 25 372 378 12182816 10.1016/S1046-5928(02)00029-3
Boettner M Prinz B Holz C Stahl U Lang C High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris Journal of Biotechnology 2002 99 51 62 12204557 10.1016/S0168-1656(02)00157-8
Holz C Prinz B Bolotina N Sievert V Büssow K Simon B Stahl U Lang C Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale Journal of Structural and Functional Genomics 2003 4 97 108 14649293 10.1023/A:1026226429429
Studier FW Rosenberg AH Dunn JJ Dubendorff JW Use of T7 RNA polymerase to direct expression of cloned genes Methods in Enzymology 1990 185 60 89 2199796
Hochuli E Dobeli H Schacher A New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues Journal of Chromatography 1987 411 177 184 3443622 10.1016/S0021-9673(00)93969-4
Schmidt TG Skerra A The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment Protein Engineering 1993 6 109 122 8433964
Schmidt TGM Koepke J Frank R Skerra A Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin Journal of Molecular Biology 1996 255 753 766 8636976 10.1006/jmbi.1996.0061
Mueller U Büssow K Diehl A Bartl FJ Niesen FH Nyarsik L Heinemann U Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags Journal of Structural and Functional Genomics 2004 4 217 225 15185962 10.1023/B:JSFG.0000016119.50040.a3
Kane JF Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli Current Opinion in Biotechnology 1995 6 494 500 7579660 10.1016/0958-1669(95)80082-4
Brinkmann U Mattes RE Buckel P High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the DNAY gene product Gene 1989 85 109 114 2515992 10.1016/0378-1119(89)90470-8
Novy R Drott D Yaeger K Mierendorf R Overcoming the codon bias of E.coli for enhanced protein expression inNovations 2001 12 1 3
Krogh A Larsson B von Heijne G Sonnhammer ELL Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes Journal of Molecular Biology 2001 305 567 580 11152613 10.1006/jmbi.2000.4315
TMHMM
Dyson HJ Wright PE Coupling of folding and binding for unstructured proteins Current Opinion in Structural Biology 2002 12 54 60 11839490 10.1016/S0959-440X(02)00289-0
Wootton JC Federhen S Analysis of compositionally biased regions in sequence databases Methods in Enzymology 1996 266 554 571 8743706
Wootton JC Federhen S Statistics of local complexity in amino acid sequences and sequence databases Computers & Chemistry 1993 17 149 163 10.1016/0097-8485(93)85006-X
Wootton JC Non-globular domains in protein sequences: automated segmentation using complexity measures Computers & Chemistry 1994 18 269 285 7952898 10.1016/0097-8485(94)85023-2
COILS
Lupas A Van Dyke M Stock J Predicting coiled coils from protein sequences Science 1991 252 1162 1164 2031185
Lupas A Prediction and analysis of coiled-coil structures Methods in Enzymology 1996 266 513 525 8743703
Eisenhaber F Bork P Wanted: Subcellular localization of proteins based on sequence Trends in Cell Biology 1998 8 169 170 9695832 10.1016/S0962-8924(98)01226-4
Meta_Annotator
Berman HM Battistuz T Bhat TN Bluhm WF Bourne PE Burkhardt K Feng Z Gilliland GL Iype L Jain S Fagan P Marvin J Padilla D Ravichandran V Schneider B Thanki N Weissig H Westbrook JD Zardecki C The protein data bank Acta Crystallographica Section D 2002 58 899 907 12037327
Altschul SF Madden TL Schaeffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: A new generation of protein database search programs Nucleic Acids Research 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389
Lennon G Auffray C Polymeropoulos M Soares MB The I.M.A.G.E. consortium: an integrated molecular analysis of genomes and their expression Genomics 1996 33 151 152 8617505 10.1006/geno.1996.0177
dbEST
Bannasch D Mehrle A Glatting KH Pepperkok R Poustka A Wiemann S LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system Nucleic Acids Research 2004 32 D505 D508 14681468 10.1093/nar/gkh022
Wiemann S Weil B Wellenreuther R Gassenhuber J Glassl S Ansorge W Bocher M Blocker H Bauersachs S Blum H Lauber J Dusterhoft A Beyer A Kohrer K Strack N Mewes HW Ottenwalder B Obermaier B Tampe J Heubner D Wambutt R Korn B Klein M Poustka A Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs Genome Research 2001 11 422 435 11230166 10.1101/gr.GR1547R
Büssow K Nordhoff E Lübbert C Lehrach H Walter G A human cDNA library for high-throughput protein expression screening Genomics 2000 65 1 8 10777659 10.1006/geno.2000.6141
Büssow K Cahill D Nietfeld W Bancroft D Scherzinger E Lehrach H Walter G A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library Nucleic Acids Research 1998 26 5007 5008 9776767 10.1093/nar/26.21.5007
Büssow K Quedenau C Sievert V Tischer J Scheich C Seitz H Hieke B Niesen FH Götz F Harttig U Lehrach H A catalogue of human cDNA expression clones and its application to structural genomics Genome Biology 2004 5 R71 15345055 10.1186/gb-2004-5-9-r71
The hEx1 Database
Hubbard T Barker D Birney E Cameron G Chen Y Clark L Cox T Cuff J Curwen V Down T Durbin R Eyras E Gilbert J Hammond M Huminiecki L Kasprzyk A Lehvaslaiho H Lijnzaad P Melsopp C Mongin E Pettett R Pocock M Potter S Rust A Schmidt E Searle S Slater G Smith J Spooner W Stabenau A Stalker J Stupka E Ureta-Vidal A Vastrik I Clamp M The Ensembl genome database project Nucleic Acids Research 2002 30 38 41 11752248 10.1093/nar/30.1.38
Aslanidis C de Jong PJ Ligation-independent cloning of PCR products (LIC-PCR) Nucleic Acids Research 1990 18 6069 6074 2235490
Scheich C Sievert V Büssow K An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography BMC Biotechnology 2003 3 12 12885298 10.1186/1472-6750-3-12
German Resource Centre (RZPD)
Scheich C Leitner D Sievert V Leidert M Schlegel B Simon B Letunic I Büssow K Diehl A Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis BMC Structural Biology 2004 4 4 15113422 10.1186/1472-6807-4-4
Brockmann C Leitner D Labudde D Diehl A Sievert V Büssow K Kühne R Oschkinat H The solution structure of the SODD BAG domain reveals additional electrostatic interactions in the HSP70 complexes of SODD subfamily BAG domains FEBS Letters 2004 558 101 106 14759524 10.1016/S0014-5793(03)01490-X
Soukenik M Diehl A Leidert M Sievert V Büssow K Leitner D Labudde D Ball LJ Lechner A Nagler DK Oschkinat H The SEP domain of p47 acts as a reversible competitive inhibitor of cathepsin L FEBS Letters 2004 576 358 362 15498563 10.1016/j.febslet.2004.09.037
McCall EJ Danielsson A Hardern IM Dartsch C Hicks R Wahlberg JM Abbott WM Improvements to the throughput of recombinant protein expression in the baculovirus/insect cell system Protein Expr Purif 2005 42 29 36 15939290 10.1016/j.pep.2005.03.021
Eshaghi S Hedren M Nasser MI Hammarberg T Thornell A Nordlund P An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins Protein Sci 2005 14 676 683 15689514 10.1110/ps.041127005
Li C Evans RM Ligation independent cloning irrespective of restriction site compatibility Nucleic Acids Research 1997 25 4165 4166 9321675 10.1093/nar/25.20.4165
ORFprimer
Inoue H Nojima H Okayama H High efficiency transformation of Escherichia coli with plasmids Gene 1990 96 23 28 2265755 10.1016/0378-1119(90)90336-P
Kyte J Doolittle RF A simple method for displaying the hydropathic character of a protein Journal of Molecular Biology 1982 157 105 132 7108955 10.1016/0022-2836(82)90515-0
Rice P Longden I Bleasby A EMBOSS: the European Molecular Biology Open Software Suite Trends in Genetics 2000 16 276 277 10827456 10.1016/S0168-9525(00)02024-2
Manjasetty BA Quedenau C Sievert V Büssow K Niesen F Delbrück H Heinemann U X-ray structure of human gankyrin, the product of a gene linked to hepatocellular carcinome Proteins: Structure, Function and Genetics 2004 55 214 217 10.1002/prot.20028
Manjasetty BA Niesen FH Delbruck H Gotz F Sievert V Bussow K Behlke J Heinemann U X-ray structure of fumarylacetoacetate hydrolase family member homo sapiens FLJ36880 Biological Chemistry 2004 385 935 942 15551868 10.1515/BC.2004.122
Manjasetty BA Delbrück H Pham DT Mueller U Fieber-Erdmann M Scheich C Sievert V Büssow K Niesen F Weihofen W Loll B Saenger W Heinemann U Crystal structure of Homo sapiens protein hp14.5 Proteins: Structure, Function and Bioinformatics 2004 54 797 800 10.1002/prot.10619
Turnbull AP Kümmel D Prinz B Holz C Schultchen J Lang C Niesen FH Hofmann KP Delbrück H Behlke J Müller EC Jarosch E Sommer T Heinemann U Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization EMBO Journal 2005 24 875 884 15692564 10.1038/sj.emboj.7600565
Kümmel D Müller JJ Roske Y Misselwitz R Büssow K Heinemann U Structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex EMBO Reports 2005 6 787 793 16025134 10.1038/sj.embor.7400463
Werner E Ziegler M Lerner F Schweiger M Heinemann U Crystal structure of human nicotinamide mononucleotide adenylyltransferase in complex with NMN FEBS Letters 2002 516 239 244 11959140 10.1016/S0014-5793(02)02556-5
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030340Book Reviews/Science in the MediaDevelopmentEvolutionNoneBringing Evo Devo to Life Book Review/Science in the MediaBrakefield Paul M 10 2005 11 10 2005 11 10 2005 3 10 e340Copyright: © 2005 Paul M. Brakefield.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.Sean Carroll's book shows how the central black box of development that maps phenotypes onto genotypes is revealing its secrets in many exquisite, and frequently unexpected, ways.
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Carroll SB (2005) Endless forms most beautiful: The new science of evo devo and the making of the animal kingdom. New York: W. W. Norton and Company. 350 p. ISBN (hardcover) 0-393-06016-0. US$25.95
The essential path of adaptive evolution is that genetic variation among individuals is translated by the process of development into phenotypic variability, which is then screened by natural selection for any design changes that enhance survival and reproductive success. In Endless Forms Most Beautiful, Sean Carroll shows how the central black box of development that maps phenotypes onto genotypes is revealing its secrets in many exquisite, and frequently unexpected, ways. This is at last enabling the whole evolutionary trail, from genes to adaptive phenotypes, to be traced—the promised synthesis begins to be realized. Changes in development generate the variation in morphology on which natural selection can act—no variation, no evolution [1]. To an evolutionary biologist, the exciting potential of evolutionary developmental biology, or “evo devo,” is the ability to open up this black box in an evolutionary context. Opening this black box will reveal not only how developmental processes themselves evolve, but also how these processes can in turn influence the paths and trajectories of morphological evolution.
Any new field in science needs its standard-bearers. In Sean Carroll evo devo has such a champion. His book, together with the new edition of an earlier book that he co-authored, called From DNA to Diversity [2], covers the whole research field. Evo devo illustrates as never before how Darwin's “endless forms most beautiful” have been and are still being made. Embryology has always been an integral component of the evidence for evolution and the principle of common descent. In Darwin's time, it was a challenge to demonstrate the common descent of all animals. Today, thanks to evo devo, it is a given fact underpinned by an increasingly broad comparative approach that makes use of phylogenetic trees, gene expression profiling, and other modern tools. Evo devo has produced many extraordinary insights. Perhaps the most unexpected insight is exemplified by the fact that not a single biologist ever anticipated that the same genes controlling the design of an insect's body and organs would also control the design of our own bodies. Indeed, Carroll cites Ernst Mayr as having predicted that “the search for homologous genes is quite futile except in very close relatives.” We now not only know much about how complexity is constructed from a single cell into a whole animal, but also fully appreciate how its foundation in an ancient genetic toolkit provides irrefutable evidence of the common descent of all animals. In addition, to me, it is exhilarating that genes crucial in specifying early embryonic and limb development in arthropods and other animals have been co-opted to tinker with the formation of butterfly wing patterns in the final throws of development [3].
In comparison to Carroll's earlier book [2], Endless Forms Most Beautiful is a much more personal account of how the field has become established, its major achievements to date, and where it is heading. For any biologist or scientist interested in learning more about the principles of development and the evolution of diversity in animal form, this is the book to read; for any evo devo researcher, this is a fascinating tale of how one of the leading figures in the field views its contributions to science and its future directions.
Carroll also reasons persuasively that evo devo provides wonderful opportunities for grabbing the interest of a new generation of biologists. His account illustrates how research in evo devo is fun as well as challenging. As he puts it, “Biology without evolution is like physics without gravity.” Evolution of form is change in development, and with the new insights, this process provides a route for inspirational teaching: there seems little doubt that, for many in the classroom, this will prove more attention-grabbing than a population-genetics approach based on the idea that “evolution is change in gene frequency.” Perhaps of no less importance than the up-to-date nature of the field is the aesthetic beauty of many images of development, such as those that fill the two sections of color plates in this book. While biologists have always been enthralled by diagrams of adaptive radiations starring those “endless forms most beautiful,” I defy anyone not to find Carroll's images of the patterns of gene expression in development just as captivating, especially when one appreciates how they reveal the underlying principles.
Carroll is clearly a master storyteller. The ease with which he weaves his story will come as no surprise to anyone who has experienced one of his performances at the lectern. His easygoing prose is matched by his infectious enthusiasm and open curiosity for all levels of diversity in biology. Although Carroll comes from a developmental biologist's background, he is well-read in evolutionary biology, including the history of the field. This book is characterized by phrases that provide evocative illustrations of how the evolution of form occurs. One doesn't usually come across words like “stunning” and “eureka” in the standard textbook accounts of a new field, but their usage adds to the effectiveness of this book in capturing the spirit of the new discoveries. Metaphors are used in a most effective way; for example, the evolution of forks or paper clips within recent human history illustrates how novelties are followed by elaboration and modification to yield a diversification of form and functional roles in different ecological environments. Scientific jargon is kept to an absolute minimum. These rhetorical devices all add to the excitement of the account, and make the book accessible to scientist or layman.
Endless Forms Most Beautiful can also fulfill an important role in the public outreach of our science. If I needed a single book to convince a skeptic or an advocate of intelligent design that evo devo is an exciting and influential field in modern biology, and that the principle of common descent is not theory but fact, I would have to look no further. As Carroll himself says, the discoveries of evo devo are illuminating the evolutionary process and particular evolutionary events in powerful new ways. There is fierce debate centered on evolution versus intelligent design in several countries, most prominently in the United States but also in the Netherlands, where I am based—this book would provide an invaluable primer for anyone uncertain about the rigor of the basis for evolution. Carroll makes it clear how complexity evolves, using examples that lend clarity to the account. The fine details may not yet be all there, but many of the underlying principles have been made much more assessable and robust by the advances of evo devo. One optimistic message of Carroll's book is that a complete understanding of the evolution of many large-scale and complex changes in animal design is within our grasp. Likewise, work in evo devo will also reveal precisely how changes evolve in the details of animal design across related species.
Whilst the scope of his book is very broad, Carroll uses a straightforward progression of topics to support his ideas. The first half of the book presents a primer on the principles of embryonic development. The remarkable discoveries of developmental biology, of how fruit fly genetics led to some of the profound insights of evo devo, come alive in Carroll's words. I can still recall the sheer frisson of excitement generated by waves of new discoveries at the first developmental biology meetings that I attended 15 years ago. Specific details and complexities, as well as epigenetic phenomena, may be glossed over in the text, but the reader interested in delving more deeply can use the thorough concluding section on scientific sources. Also, while on first reading, phrases such as “multiple genetic switches” may jar the reader, they end up being excellent choices in terms of explaining the basic principles to a very broad audience. There are also intriguing mysteries—why does it take as long to a make a 32-cell human embryo as it does to make a complete tadpole?
In the second half of his book, Carroll expands on four ideas about animal development: (1) the modularity of animal architecture, (2) the genetic toolkit for building animals, (3) the geography of the embryo in space and time, and (4) that genetic switches determine the coordinates of toolkit gene action in the embryo. All this is covered with broad references to the fossil record, the Cambrian explosion, and the opportunities arising from new ecological environments and innovations in animal form. Using numerous examples, often from evo devo work coming out of his own laboratory, he then shows how animal forms evolve through changes in the geography of the embryo, and that evolution of form is largely about “teaching very old genes new tricks.” For example, his team found that a primitive Australian onychophoran, or velvet worm, has no fewer Hox genes than flies and other arthropods [4]. Thus, the modularity and dramatic diversification of arthropod limbs and their parts has not evolved through increasing the toolkit of Hox genes but rather through evolution of the regulatory machinery.
Carroll argues that the arena of evolutionary action is, indeed, largely within the rich operating instructions for the toolkit genes, an area of special, current interest in his own laboratory [5]. Form evolves largely by elaborating on, and tinkering with, how these genes are regulated through batteries of multiple switches. The forms of animal parts are the products of large numbers of switches and proteins that organize patterns in time and space; the same genes and associated signaling pathways are used again and again at different times in development and in different tissues. Research is beginning to bring this to life, although it will still be a long time before all the exquisite details are revealed for particular toolkit genes and structures across groups of related animal species. Carroll makes some predictions for the future in terms of the complexity of the logic that underlies evolution of form. He shows how even the most complex of adaptations arise in this way, with multifunctionality and redundancy providing the ecological background to the evolution of specialization in specific environments. The remodeling of limbs to produce new forms and functions, whether in arthropods or in vertebrates, is a matter of changing the geography of limb development in terms of the patterns of expression of toolkit genes in time and space.
In a section near the end of the book, Carroll begins to apply the framework of evo devo to the evolution of humans, including the human mind. He discusses how the discoveries and perspectives of evo devo have already enriched and expanded on the foundations of evolutionary thinking—there is, however, clearly much more to come. He concludes with an eloquent plea that understanding the history of this planet is the key to our successful stewardship of it. I hope that he is right.
Endless Forms Most Beautiful reinforces the prediction that the foreseeable future will see such advances in evo devo that we will know in wonderful detail how both major and much more subtle changes in animal form are generated, and how the underlying developmental processes have evolved. One remaining challenge will be to match such a sophisticated understanding of how form and variation in form are generated with comparable detail from the perspectives of function and fitness in ecological environments. Another challenge will be to compare the properties of evolution in form and morphology with how life histories, behaviors, and other components of the whole functional phenotype of organisms evolve—it will be fascinating to see the results of an evo devo type of framework applied with equal force to these other facets of the phenotype. Finally, it remains to be seen how the processes of development themselves bias or channel evolution, such that even the most vivid examples of adaptive radiation are shaped not only by natural selection but also by how development works and is regulated.
Citation: Brakefield PM (2005) Bringing evo devo to life. PLoS Biol 3(10): e340.
Paul M. Brakefield is Professor of Evolutionary Biology in the Institute of Biology at the University of Leiden, Leiden, the Netherlands. E-mail: [email protected]
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References
Brakefield PM French V Zwaan BJ Development and the genetics of evolutionary change within insect species Annu Rev Ecol Evol Syst 2003 34 633 660
Carroll SB Grenier JK Weatherbee SD From DNA to diversity: Molecular genetics and the evolution of animal design, 2nd ed 2005 Oxford Blackwell Publishing 258
Beldade P Brakefield PM The genetics and evo-devo of butterfly wing patterns Nat Rev Genet 2002 3 442 452 12042771
Grenier J Garber T Warren R Whitington PM Carroll S Evolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade Curr Biol 1997 7 547 553 9259556
Gompel N Prud'homme B Wittkopp PJ Kassner VA Carroll SB Chance caught on the wing: cis -regulatory evolution and the origin of pigment patterns in Drosophila
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1620707610.1371/journal.pbio.0030341ObituaryInfectious DiseasesEpidemiology/Public HealthMedical HistoryEubacteriaA Path to Discovery: The Career of Maclyn McCarty ObituaryLederberg Joshua [email protected] Emil C 10 2005 11 10 2005 11 10 2005 3 10 e341Copyright: © 2005 Lederberg and Gotschlich.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.Maclyn McCarty (1911-2005) was best known for his part in the pioneering discovery that genes are made of DNA.
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Maclyn McCarty, who devoted his life as a physician-scientist to studying infectious disease organisms, was best known for his part in the monumental discovery that DNA, rather than protein, constituted the chemical nature of a gene. Uncovering the molecular secret of the gene in question—that for the capsular polysaccharide of pneumococcal bacteria—led the way to studying heredity not only through genetics but also through chemistry, and initiated the dawn of the age of molecular biology. McCarty was the youngest and longest surviving member of the research team responsible for this feat, which also included Oswald T. Avery and Colin MacLeod; he died on January 2, 2005, from congestive heart failure.
McCarty was born in 1911 in South Bend, Indiana, the second of four sons of a branch manager for the Studebaker Corporation while it was still a firm for horse-drawn carriages. In his teens, McCarty set himself the goal of becoming a physician-scientist, and he pursued a successful strategy to prepare for admission to, and early success in, Johns Hopkins University Medical School. As an undergraduate at Stanford University, he presciently began his studies in the nascent field of biochemistry, working with James Murray Luck on protein turnover in the liver. In 1937, he began his clinical training in pediatrics at the Harriet Lane Service at Johns Hopkins University. There McCarty developed a special interest in infectious diseases—in particular, antibacterial sulfonamide drug treatments that were just entering medicine—which he subsequently pursued by moving to New York University to work with William Tillett. A National Research Council Fellowship in the medical sciences and an opening in Oswald T. Avery's laboratory spurred his move to Rockefeller University in 1941.
At that time, research in the Avery laboratory was focused on the pneumococcal transformation, the heritable alteration of a pneumococcal strain from a nonvirulent rough form to a virulent smooth encapsulated form. McCarty's arrival at the Rockefeller Institute in September 1941 marked 13 years since this discovery, also known as the Griffith phenomenon. Prior to this discovery, the 1920s had been marked by a medley of disparate observations on Streptococcus pneumoniae that seemed to involve an exchange of receptors among diverse bacteria either grown together in liquid media or exposed to various kinds of extracts and supernatants. With rare exception, the early researchers in this area were utterly confused about the distinction between genotype and phenotype. No single experiment was carried forward to confirmation by other observers, so the entire field of “para agglutination” was in some disrepute.
Maclyn McCarty (June 9, 1911, to January 2, 2005) with Francis Crick and James D. Watson
(Photo: Marjorie McCarty)
However, in 1928, Fred Griffith, a leader in public-health research in Britain, demonstrated that the conversion of one strain to another could happen in vivo in mice. Shortly after the publication of his results, they were confirmed in several quarters, including Avery's lab. The analysis relied on serotyping: it was known that phenotypic differentiation of pneumoccocal groups could be diagnosed by their reactions with specific antisera, already recognized to reflect chemically distinct capsular polysaccharides. Griffith had neither the resources nor the inclination to purify and identify the responsible agent in pneumococcal extracts that induced the changes of serotype. But the phenomenon of transformation was at least vaguely understood to comprise an alteration of what we would now call genetic factors.
Though interrupted, sometimes for years at a time, these studies were from 1928 onwards the centerpiece of Avery's lab agenda. Around 1940, they were activated by Colin MacLeod's efforts to purify the chemical agent responsible for changes of serotype—whether protein, nucleic acid, or some other class of molecule—and demonstrate that it was necessary and sufficient to cause the Griffith phenomenon. Studies on pneumococcal transformation were grossly burdened by a wide variety of variables, which needed to be controlled to allow quantitative estimation of transforming activity in extracts undergoing various stages of purification. MacLeod, over a number of years of research, had resolved several thorny technical issues to render the experimental system somewhat more reliable as an assay for biological activity. By the time McCarty arrived at the Rockefeller University, Avery's team had just about decided that the active reagent was not a protein. But what was it then? Could it be a soluble saccharide, RNA, or, least likely, DNA? The progress of this research over the next three years is beautifully described in McCarty's memoir The Transforming Principle, written in the early 1980s [1].
As purification progressed, exposure of extracts to crystalline RNase and to proteinase preparations helped Avery's team determine that the biological activity of extracts was not dependent on RNA or protein. Crystalline DNase was not available until 1948, but biological activity was rapidly reduced by tissue extracts rich in DNase. McCarty's arrival at Rockefeller University was also marked by another milestone, namely, the development of a diphenylamine reagent assay to positively correlate DNA with biological activity. It gradually became evident that the active material in purified extracts had astonishingly high potency in micrograms of DNA that could consummate the pneumococcal transformation in vitro.
McCarty, MacLeod, and Avery wrestled with the standard of proof required to claim that they had accomplished pneumococcal transformation with highly purified DNA from extracts. After much self-inquiry, in 1944, they published in the Journal of Experimental Medicine that the active material was, indeed, DNA [2], bereft of protein or any other known polymer [3,4].
The vicissitudes of the acceptance of the concept that “genes are DNA” deserve the scholarly praise they have received [5,6]. The claim was, indeed, subject to a formidable, but predictable, round of organized skepticism. Some would say, even worse, that it was simply ignored, but that is manifestly untrue, at least in the case of the New York research institutions. The scientific community does not accept major scientific claims with ease, and in this case, there were challenges associated with research on S. pneumoniae, which made it especially difficult to attract other investigators to pursue this research. To begin with, few people had the necessary expertise with this pathogen from a biological perspective—it was dangerous to work with, and at the same time, it was finicky to grow. In order to assay its virulence, one needed to use mice as a selective filter. Most critically lacking as corroboration was the examination of other phenotypic markers, besides the capsular polysaccharide, to determine the extent that the findings on the gene for one pneumococcal antigen would apply to other metabolic markers of S. pneumoniae.
However, by 1953, influenced by the enormous impact of Watson and Crick's bihelical structure of DNA, the majority of researchers had fully accepted the 1944 paper. In fact, one might say, formal proof that DNA encoded genetic material was approximated only much later by the laboratory synthesis of oligonucleotides, and by the demonstration of genetic material's biological activity, for example, genes for tRNA or small DNA viruses. Long before this formal proof, most commentators had accepted the untrammeled heuristic value of the proposition that, indeed, genes were made of DNA.
Meanwhile, a physician-scientist through and through, McCarty turned his attention to diseases promoted by streptococci. So it happened that on the retirement of Homer Swift in 1946, McCarty was asked to head the laboratory established in 1922 to work on streptococci and rheumatic fever. This was the scientific home of Rebecca Lancefield, who developed the still powerful serological classification of streptococci. From innumerable clinical observations, combined with Lancefield's classification, it was clear that acute rheumatic fever, a severe sterile inflammatory condition affecting particularly the joints and the heart, was a complication of group A streptococcal pharyngitis, following the infection by several weeks. The causal chain of events still eludes us. McCarty attacked this problem by studying both the biology of group A streptococci and patients with acute rheumatic fever admitted to the Rockefeller Hospital.
Together with his students and collaborators, over the next 20 years, McCarty's work changed the understanding of the organism from a gram-positive streptococcus with a particular serological characteristic to one of the best characterized bacterial species. Work on bacterial cell-wall anatomy and chemistry was just beginning. His work led to the isolation of the streptococcal cell wall as a structural entity suitable for anatomic inspection by electronmicroscopy. Chemical dissection led to characterization of the group A–specific polysaccharide and the peptidoglycan, and the identification of its serological specificity in the terminal hexosamine. In order to prove this specificity, he first had to identify and purify a specific enzyme that cleaved hexosamine (a hexosaminidase) from a soil organism. Treating the polysaccharide with this enzyme abrogated its serological reactivity. McCarty further demonstrated the precise configuration of the hexosamine linkage by synthesizing both α- and β-N-acetyl-glucosamine ovalbumin and showing that only the second reacted with group A antisera. A similar analytical strategy indicated that the polysaccharide of group C streptococci differed by having a terminal β-N-acetyl galactosamine as the serological determinant.
In parallel, McCarty studied patients with rheumatic fever admitted to the Rockefeller Hospital as well as valuable specimen collections from military outbreaks of the disease during World War II. He and his collaborators found that antibody responses to several streptococcal antigens were significantly higher in the group of individuals that developed acute rheumatic fever than in individuals with uncomplicated infection. However, the response to unrelated antigens, for instance, diphtheria toxoid, was not enhanced. He found that group A streptococci secreted unusually high amounts of DNase, and established a test for the detection of antibodies produced in response to this antigen. This led to the discovery that streptococci were able to produce multiple isozymes of DNase. He purified human C-reactive protein through crystallization, produced a highly specific antiserum, and, using this much simpler and more sensitive test, found that C-reactive protein levels responded more rapidly and reliably than other inflammatory markers and could serve as the most accurate indicator of rheumatic inflammatory activity. Measuring C-reactive protein levels to detect inflammation is routine now in medical practice.
In his later years, McCarty increasingly served as a statesman of the biomedical sciences. He served for 14 years as the physician-in-chief of the Rockefeller University Hospital, and as a trusted adviser and the vice president of the Rockefeller University. Outside the university, his leadership was sought by the New York City Health Research Council, the Helen Hay Whitney Foundation, the Institute of Medicine (as a charter member), and numerous university visiting boards. For more than 40 years, as editor, he placed his stamp of excellence and integrity on the Journal of Experimental Medicine.
McCarty's scientific interests and energy had a counterpart in his rich personal life. Along with his wife, Marjorie, McCarty had a wide circle of very close friends, both in the United States and abroad, who cherished his personal warmth, his low key, spare, and pragmatic character, his wit, and his wide-ranging intellect. He loved English literature, theater, and symphonies. He loved to wander the streets and the museums of the great cities of the world, particularly, Paris, New York, and London, and frequently visited overseas following his retirement. Moreover, he remained close to his family; the four brothers, living in different parts of the country, never failed to meet for annual reunions. McCarty lived a full life, and he continues to inspire all who knew him.
Citation: Lederberg J, Gotschlich EC (2005) A path to discovery: The career of Maclyn McCarty. PLoS Biol 3(10): e341.
Joshua Lederberg and Emil C. Gotschlich are at the Rockefeller University, New York, New York, United States of America.
==== Refs
References
McCarty M The transforming principle: Discovering that genes are made of DNA 1985 New York W. W. Norton 252
Avery OT MacLeod CM McCarty M Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III J Exp Med 1944 79 137 158 19871359
McCarty M Avery OT Studies on the chemical nature of the substance inducing transformation of pneumococcal types. 2. Effect of desoxyribonuclease on the biological activity of the transforming substance J Exp Med 1946 83 89 96
McCarty M Avery OT Studies on the chemical nature of the substance inducing transformation of pneumococcal types. 3. An improved method for the isolation of the transforming substance and its application to Pneumococcus types II, III, and VI J Exp Med 1946 83 97 104
Amsterdamska O From pneumonia to DNA: The research career of Oswald T. Avery Hist Stud Phys Biol Sci 1993 24 1 40 11623400
Olby R The path to the double helix 1974 London Macmillan 510
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1620707710.1371/journal.pbio.0030349PrimerDevelopmentGenetics/Genomics/Gene TherapyArthropodsNematodesHow the Ecdysozoan Changed Its Coat PrimerEwer John 10 2005 11 10 2005 11 10 2005 3 10 e349Copyright: © 2005 John Ewer.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.External skeletons are found in a variety of animals, including arthropods and nematodes. Much remains to be learned about the process of replacing the exoskeleton (molting) during growth.
==== Body
Most people are aware that insects are crunchy on the outside. What they don't always realize is that the external crunchy parts form the animal's skeleton, and that this body design makes the apparently simple process of growing extremely complex.
In addition to providing a barrier against desiccation and protection from mechanical injury, an insect's external skeleton (exoskeleton) does what skeletons do for all animals: it gives the insect its shape and also provides the frame to which muscles are attached, and, therefore, is critical for its behaviors. External skeletons are found in a variety of animals, including arthropods (“jointed-foot invertebrates,” including insects, crustaceans, spiders, millipedes, and centipedes) and nematodes. Arthropods include some of the most successful organisms on earth. Ants alone are believed to represent up to one-third of the terrestrial animal biomass [1], and as many as half of earth's species may be insects.
The undeniable success of insects is most certainly due, in part, to the group's rugged exoskeleton. Nevertheless, living within an external skeleton immediately raises a logistical problem: how to grow. Although many insects, especially at immature stages, have elastic skeletons, continuous growth eventually requires that the skeleton be replaced with a larger one. The process that effects this replacement is called molting, and although the same term is used to describe the replacement of the outer skin layer of some vertebrates such as snakes, the process of replacing an exoskeleton is infinitely more difficult. The ability to replace an exoskeleton is currently believed to have evolved only once during animal evolution, giving rise to a clade of animals called Ecdysozoa, which includes arthropods and nematodes [2].
The Mechanics of Molting
The exoskeleton, or cuticle, is a well-defined inert structure that is secreted by, and strongly attached to, the underlying epidermal cells. Although its composition varies significantly among ecdysozoans (e.g., consider the skeleton of a beetle versus that of a crab), the process of molting itself is similar within the clade: the epidermis may undergo a round of cell division (thereby producing a larger surface) and separates from the exoskeleton. A new exoskeleton is then secreted by the epidermis, but is soft until the remains of the overlying old cuticle are shed at ecdysis. The new cuticle then expands and hardens.
In nature, this process appears simple only because ecdysozoans are experts at molting. But underlying this seamless process is a complex developmental feat, as the animal must replace its skeleton and survive the process in an unsheltered environment. For instance, despite the erosion of the cuticle's integrity that takes place during an insect molt, muscle attachments on the old cuticle remain functional until that cuticle is shed, at which time the corresponding attachments on the new cuticle are fully differentiated. Since the new cuticle is soft until it is sclerotized, rigidity is initially aided by an increase in internal pressure caused by swallowing of water or air at the time of ecdysis (shedding of the old cuticle). Like insects, nematodes are able to move during the lethargus that accompanies the molt, even though the old cuticle separates from the epidermis and muscle attachments are likely remodeled during this time. In insects, the sensory bristles attached to the cuticle remain functional until shortly before they are shed along with the rest of the exoskeleton. Then, over the course of just a few minutes, innervation by the same sensory neuron is transferred to the new bristle located on the underlying new cuticle. Finally, the lining of the main air ducts (trachea), which form a complex web of tubes that extends throughout the body, also molts. During this process, air exchange is likely compromised because of the presence of a new layer of developing cuticle and the molting fluid that is present between the old and new cuticles, which aids in the digestion of the old exoskeleton. While this complex process is taking place, the insect remains a potential prey, and is also prone to dehydration, since the old cuticle is weakened and the new cuticle has not yet been rendered impermeable by tanning. These considerations (and others) place tremendous constraints on the timing and choreography of the molting process.
Despite the complex internal upheaval, molting does not typically affect the insect's performance until the very end of the molt, when the remains of the old cuticle are shed at ecdysis and the new cuticle rapidly expands and hardens. It is mostly during this brief period that the animal is vulnerable: once the behavior of ecdysis starts, it cannot be interrupted, and even minor hitches can be fatal. For instance, failures to fully extricate a wing may cause a malformed wing to be hardened, forever compromising the adult's ability to fly.
This short list highlights some of the processes that must occur perfectly at every molt (some insects molt hundreds of times during their lifetime), and does not even include those processes for the much more complex process of insect metamorphosis. During a metamorphic molt the cuticle is also replaced; however, the whole animal is essentially redesigned at this time as it transforms from a larva to an adult. The different shape, appendages, sensory and motor capabilities, and diet of a caterpillar versus a butterfly give some idea of the magnitude of the changes that take place during metamorphosis. Nevertheless, a “simple” molt is complex enough, as the exoskeleton must be replaced as quickly as possible, and produce a functional animal when completed, with a cuticle that permits feeding and fleeing, and that provides a barrier to desiccation.
Molecular Control of Molting in Arthropods
Our understanding of the control of molting in insects stems from simple, clever experiments carried out in the early part of the 20th century by the founders of insect endocrinology, including Kopéc, Wigglesworth, and others [3]. Using parabiosis (the experimental joining of two organisms for the purpose of studying the effects of one on the other), ligatures, and transplants, they showed that molting and metamorphosis are controlled and coordinated by systemic hormonal signals. Thus, for example, Wigglesworth showed that molting could be induced in a nonmolting insect by joining it to an insect that was in the process of molting. Since the two insects only shared a circulatory system, this result indicated that molting was induced by a circulating hormone, now known to be the steroid 20-hydroxyecdysone (20E).
We also know that insect molting is initiated by the secretion of a brain neuropeptide called prothoracicotropic hormone in response to poorly understood signals that integrate the animal's size, weight, nutritional status, as well as photoperiodic information. This peptide acts on the peripheral prothoracic gland, causing it to synthesize and secrete the steroid hormone ecdysone (E). E is then converted by peripheral tissues to the active molting hormone, 20E.
20E is the key hormone that regulates the molt. The increase in 20E titers initiates the molt. For instance, it causes the epidermis to divide, separate from the old cuticle, and synthesize and secrete new cuticle components. The decrease in 20E titers at the end of the molt allows the molt to be completed. In particular, this fall is required to set in motion a complex hierarchy of interacting neuropeptides, which act on the nervous system to initiate ecdysis behaviors and on the peripheral tissues to the hardening of the new cuticle, among other peripheral actions [4].
20E acts mostly on members of the conserved nuclear receptor superfamily, which act as ligand-dependent transcription factors. Thus, 20E activates the complex transcriptional processes that underlie the many cellular and morphogenetic events that occur during molting. Being a circulating lipid-soluble (steroid) hormone gives 20E a built-in ability to affect all cells in the organism and cause widespread changes. Such coordination is critical for a successful molt, and is mostly effected by each tissue responding autonomously to the changes in 20E titers, with little tissue-to-tissue coordination. Although much more complex than “simple” molts, metamorphic molts are also directed by 20E. This global endocrine signal is then transduced into a very complex, tissue-specific pattern of gene expression by turning on (and off) 20E-regulated genes, which in turn activate or repress other genes in the pathway that eventually turns a caterpillar into a butterfly [5].
Comparatively less is known about how other ecdysozoans change their coat. Work in crustacea has established that a steroid hormone also controls molting within this class [6]. In fact, many crustacea produce E, which is then converted to the active hormone 20E, the exact same hormone that controls insect molting. Little is know about the mechanism that causes the onset of the molt and the downstream actions of 20E in crustacea, but there are bound to be interesting similarities as well as differences compared to the process in insects. For instance, we already know that 20E production in crustacea is regulated by a neuropeptide (molt-inhibiting peptide) that inhibits E production, rather than by a neuropeptide that stimulates E synthesis (as occurs in insects).
Molting in Worms
Relatively little is known about the process of molting in nematodes. However, this group of organisms is significant for several reasons. First, nematodes are quite different from arthropods; yet their membership in the ecdysozoan clade reflects a deep biological relatedness. Comparisons between molting in nematodes and molting in arthropods would reveal, for instance, which features are invariant (and presumably very constrained) aspects of molting, and which are different solutions to the same problem. Second, a number of serious human diseases are caused by parasitic nematodes. Agents that interfered with molting would be fatal to the parasite, but may not affect its host, to which the parasite is only very distantly related (“A worm, with very few exceptions, is not a human being” [7]). Finally, the nematode Caenorhabditis elegans is a model organism, with a sequenced genome and well-developed molecular genetic tools. These tools make it possible to carry out forward and reverse genetic screens, determine the time and place that candidate genes are expressed, investigate order of gene action, etc., and have been used to systematically analyze many aspects of the biology of this little worm.
Paradoxically, until now, molting has not been subjected to such systematic scrutiny. This may be because recognition of nematodes' membership in the ecdysozoan club is recent, so molting has not been viewed as a prominent feature of their biology. Nevertheless, a number of known features of molting in C. elegans make it especially intriguing. For instance, it has been difficult to demonstrate that molting in nematodes requires cholesterol, the precursor of steroid hormones, and C. elegans lacks key enzymes required for the synthesis of sterols [8]. In addition, despite encoding a bounty of 260 nuclear receptors, the C. elegans genome does not apparently encode a homolog of the 20E receptor or of ultraspiracle, its obligate partner.
The research article by Frand et al. [9] in this issue of PLoS Biology reports the results of the first systematic screen for genes involved in molting in nematodes. The authors used RNA interference to silence each of the 19,427 predicted C. elegans genes, and screened for the occurrence of worms that were unable to shed their old cuticle (Figure 1A). This screen produced a list of 159 genes whose interference produced molting defects in larvae. Significantly, interference with gene function could cause arrest at different larval molts, suggesting that many of these genes are required for molting, per se, not simply for the transition between two specific stages. Importantly, the 159 genes included nine genes that had previously been shown to be involved in C. elegans molting, as well as 28 that had previously been noted as causing an arrest during a molt when inactivated by RNA interference. The nine genes include nhr-25, the homolog of the orphan nuclear receptor ftz-f1 [10], which plays a key role in insect metamorphosis, as well as lpr-1 [11], a homolog of gp330-megalin, which is involved in vitamin D (a cholesterol derivative) uptake in mammals. Re-isolating these genes in a forward screen for molting defects lends further support to the (wishful) hypothesis that steroid hormones are involved in nematode molting, just as they are in molting in the arthropod members of this clade. Although the screen was based on a failure in the last step of the molt (shedding of the old cuticle), many of the candidates are likely to play a role in the earlier stages of the molting process. Indeed, the list of genes includes transcription factors, signaling proteins, and molecules involved in cuticle secretion and remodeling of basement membranes, as well as the expected list of proteins involved in the eventual release of the collagenous cuticle.
Figure 1 A C. elegans Molting Sampler
(A) A C. elegans larva unable to shed its old cuticle after RNA interference of the gene acn-1.
(B–D) Pairs of pictures show larvae expressing the GFP-tagged mlt-11 gene before (B), during (C), and after (D) the first molt.
(E) A collar of old cuticle found midway along the length of the animal mostly restricts expression of the GFP-tagged mlt-10 gene to the front half of the worm.
As expected for a first description of the results of a genome-wide screen, it is not yet possible to weave all the candidate genes into a solid story. Nevertheless, the authors do make a significant contribution to our understanding of how these genes might make the nematode change its coat by investigating in more detail eight genes representing the major functional categories. First, fusions to green fluorescent protein (GFP) revealed that all eight genes were expressed in epithelial cells, with a pulse of fluorescence prior to each molt (Figure 1B–1D). Thus, these genes are expressed in the right place at the right time. Second, the ability of RNA interference of molting genes to block GFP expression of the select tagged molting genes was used to order gene expression cascades. This analysis revealed, for instance, that interference of nhr-23 (encoding another orphan nuclear receptor previously implicated in molting [12]) affected the expression of the eight GFP-tagged molting genes, suggesting that nhr-23 may be generally required for the expression of molting genes. By contrast, blocking the molting gene mlt-9 caused the expected molting defect without affecting the expression of mlt-10 and mlt-8. As an additional bonus from this study, the authors noted that some genotypes tended to develop constrictions in their cuticle, and that in these animals, both GFP expression and molting were confined to the anterior part of the animal (Figure 1E). This result is reminiscent of the now classic experiments in insect endocrinology that demonstrated the role of circulating hormones in the control of molting [3]. Thus, Frand et al., in a single study, have carried out classic and molecular genetic experiments to provide a qualitative advance in our understanding of how nematodes molt.
The information gained from this screen is important in itself, since few genes involved in nematode molting had previously been identified. These genes can now be compared to the homologous genes in other ecdysozoans, and may also be useful for developing effective agents for controlling parasitic nematodes. Furthermore, modifications of the screen that was used (e.g., sensitized screens) may allow the nematode to answer some of the more intractable questions regarding the control of molting. For example, little is known about the mechanism that monitors the organism's size and signals that a molt must start. In insects, we know that it is dependent on size (weight), nutritional status, and time of day, but how this information is integrated and transduced is unknown for most species. Likewise, little is known about how ecdysozoans know how many immature stages they must complete before becoming an adult. For example, we do not know how a third-instar caterpillar determines that it still has two more caterpillar stages to go through before becoming a pupa (chrysalis), how a third-instar fruit fly larva knows that it has completed its larval life, and how a third-instar C. elegans knows that it still has a fourth instar to go through before becoming an adult. An interesting possibility is that the process of “instar counting” is regulated by so-called heterochronic genes [13]. Thus, is a failure to enter metamorphosis (for an insect) mechanistically related to adding an extra larval instar (for a worm)? The screen conducted by Frand et al. did not identify any known heterochronic genes, but this could simply reflect the criteria used for identifying candidate genes. Finally, we know little of both global checkpoints that must be cleared in order for molting to progress and any signals between molting tissues. Frand et al.'s research article paves the way for a new understanding of molting in Ecdysozoa.
I thank Alison Frand and Carl Thummel for comments. I am grateful to Alison Frand for providing Figure 1.
Citation: Ewer J (2005) How the ecdysozoan changed its coat. PLoS Biol 3(10): e349.
John Ewer is in the Entomology Department, Cornell University, Ithaca, New York, United States of America. E-mail: [email protected]
Abbreviations
20E20-hydroxyecdysone
Eecdysone
GFPgreen fluorescent protein
==== Refs
References
Hölldobler B Wilson EO The ants 1990 Cambridge (Massachusetts) Harvard University Press 746
Aguinaldo AM Turbeville JM Linford LS Rivera MC Garey JR Evidence for a clade of nematodes, arthropods and other moulting animals Nature 1997 387 489 493 9168109
Nijhout HF Insect hormones 1994 Hoboken (New Jersey) Princeton University Press 280
Ewer J Reynolds S Pfaff DW Arnold AP Fahrbach SE Etgen AM Rubin RT Neuropeptide control of molting in insects 2002 San Diego Academic Press 1 92 Hormones, brain and behavior
Thummel CS Files on steroids—Drosophila metamorphosis and the mechanisms of steroid hormone action Trends Genet 1996 12 306 310 8783940
Chang ES Comparative endocrinology of molting and reproduction: Insects and crustaceans Annu Rev Entomol 1993 38 161 180 8424625
Brooks M Young Frankenstein [film] 1974 Los Angeles 20th Century Fox/Gruskoff/Venture Film/Crossbow Productions/Jouer Limited
Kurzchalia TV Ward S Why do worms need cholesterol? Nat Cell Biol 2003 5 684 688 12894170
Frand AR Russel S Ruvkun G Functional genomic analysis of C. elegans molting PLoS Biol 2005 3 e312 10.1371/journal.pbio.0030312 16122351
Gissendanner CR Sluder AE nhr-25, the Caenorhabditis elegans ortholog of ftz-f1 , is required for epidermal and somatic gonad development Dev Biol 2000 221 259 272 10772806
Yochem J Tuck S Greenwald I Han M A gp330/megalin-related protein is required in the major epidermis of Caenorhabditis elegans for completion of molting Development 1999 126 597 606 9876188
Kostrouchova M Krause M Kostrouch Z Rall JE Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans
Proc Natl Acad Sci U S A 2001 98 7360 7365 11416209
Thummel CS Molecular mechanisms of developmental timing in C. elegans and Drosophila
Dev Cell 2001 1 453 465 11703937
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1620707710.1371/journal.pbio.0030349PrimerDevelopmentGenetics/Genomics/Gene TherapyArthropodsNematodesHow the Ecdysozoan Changed Its Coat PrimerEwer John 10 2005 11 10 2005 11 10 2005 3 10 e349Copyright: © 2005 John Ewer.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.External skeletons are found in a variety of animals, including arthropods and nematodes. Much remains to be learned about the process of replacing the exoskeleton (molting) during growth.
==== Body
Most people are aware that insects are crunchy on the outside. What they don't always realize is that the external crunchy parts form the animal's skeleton, and that this body design makes the apparently simple process of growing extremely complex.
In addition to providing a barrier against desiccation and protection from mechanical injury, an insect's external skeleton (exoskeleton) does what skeletons do for all animals: it gives the insect its shape and also provides the frame to which muscles are attached, and, therefore, is critical for its behaviors. External skeletons are found in a variety of animals, including arthropods (“jointed-foot invertebrates,” including insects, crustaceans, spiders, millipedes, and centipedes) and nematodes. Arthropods include some of the most successful organisms on earth. Ants alone are believed to represent up to one-third of the terrestrial animal biomass [1], and as many as half of earth's species may be insects.
The undeniable success of insects is most certainly due, in part, to the group's rugged exoskeleton. Nevertheless, living within an external skeleton immediately raises a logistical problem: how to grow. Although many insects, especially at immature stages, have elastic skeletons, continuous growth eventually requires that the skeleton be replaced with a larger one. The process that effects this replacement is called molting, and although the same term is used to describe the replacement of the outer skin layer of some vertebrates such as snakes, the process of replacing an exoskeleton is infinitely more difficult. The ability to replace an exoskeleton is currently believed to have evolved only once during animal evolution, giving rise to a clade of animals called Ecdysozoa, which includes arthropods and nematodes [2].
The Mechanics of Molting
The exoskeleton, or cuticle, is a well-defined inert structure that is secreted by, and strongly attached to, the underlying epidermal cells. Although its composition varies significantly among ecdysozoans (e.g., consider the skeleton of a beetle versus that of a crab), the process of molting itself is similar within the clade: the epidermis may undergo a round of cell division (thereby producing a larger surface) and separates from the exoskeleton. A new exoskeleton is then secreted by the epidermis, but is soft until the remains of the overlying old cuticle are shed at ecdysis. The new cuticle then expands and hardens.
In nature, this process appears simple only because ecdysozoans are experts at molting. But underlying this seamless process is a complex developmental feat, as the animal must replace its skeleton and survive the process in an unsheltered environment. For instance, despite the erosion of the cuticle's integrity that takes place during an insect molt, muscle attachments on the old cuticle remain functional until that cuticle is shed, at which time the corresponding attachments on the new cuticle are fully differentiated. Since the new cuticle is soft until it is sclerotized, rigidity is initially aided by an increase in internal pressure caused by swallowing of water or air at the time of ecdysis (shedding of the old cuticle). Like insects, nematodes are able to move during the lethargus that accompanies the molt, even though the old cuticle separates from the epidermis and muscle attachments are likely remodeled during this time. In insects, the sensory bristles attached to the cuticle remain functional until shortly before they are shed along with the rest of the exoskeleton. Then, over the course of just a few minutes, innervation by the same sensory neuron is transferred to the new bristle located on the underlying new cuticle. Finally, the lining of the main air ducts (trachea), which form a complex web of tubes that extends throughout the body, also molts. During this process, air exchange is likely compromised because of the presence of a new layer of developing cuticle and the molting fluid that is present between the old and new cuticles, which aids in the digestion of the old exoskeleton. While this complex process is taking place, the insect remains a potential prey, and is also prone to dehydration, since the old cuticle is weakened and the new cuticle has not yet been rendered impermeable by tanning. These considerations (and others) place tremendous constraints on the timing and choreography of the molting process.
Despite the complex internal upheaval, molting does not typically affect the insect's performance until the very end of the molt, when the remains of the old cuticle are shed at ecdysis and the new cuticle rapidly expands and hardens. It is mostly during this brief period that the animal is vulnerable: once the behavior of ecdysis starts, it cannot be interrupted, and even minor hitches can be fatal. For instance, failures to fully extricate a wing may cause a malformed wing to be hardened, forever compromising the adult's ability to fly.
This short list highlights some of the processes that must occur perfectly at every molt (some insects molt hundreds of times during their lifetime), and does not even include those processes for the much more complex process of insect metamorphosis. During a metamorphic molt the cuticle is also replaced; however, the whole animal is essentially redesigned at this time as it transforms from a larva to an adult. The different shape, appendages, sensory and motor capabilities, and diet of a caterpillar versus a butterfly give some idea of the magnitude of the changes that take place during metamorphosis. Nevertheless, a “simple” molt is complex enough, as the exoskeleton must be replaced as quickly as possible, and produce a functional animal when completed, with a cuticle that permits feeding and fleeing, and that provides a barrier to desiccation.
Molecular Control of Molting in Arthropods
Our understanding of the control of molting in insects stems from simple, clever experiments carried out in the early part of the 20th century by the founders of insect endocrinology, including Kopéc, Wigglesworth, and others [3]. Using parabiosis (the experimental joining of two organisms for the purpose of studying the effects of one on the other), ligatures, and transplants, they showed that molting and metamorphosis are controlled and coordinated by systemic hormonal signals. Thus, for example, Wigglesworth showed that molting could be induced in a nonmolting insect by joining it to an insect that was in the process of molting. Since the two insects only shared a circulatory system, this result indicated that molting was induced by a circulating hormone, now known to be the steroid 20-hydroxyecdysone (20E).
We also know that insect molting is initiated by the secretion of a brain neuropeptide called prothoracicotropic hormone in response to poorly understood signals that integrate the animal's size, weight, nutritional status, as well as photoperiodic information. This peptide acts on the peripheral prothoracic gland, causing it to synthesize and secrete the steroid hormone ecdysone (E). E is then converted by peripheral tissues to the active molting hormone, 20E.
20E is the key hormone that regulates the molt. The increase in 20E titers initiates the molt. For instance, it causes the epidermis to divide, separate from the old cuticle, and synthesize and secrete new cuticle components. The decrease in 20E titers at the end of the molt allows the molt to be completed. In particular, this fall is required to set in motion a complex hierarchy of interacting neuropeptides, which act on the nervous system to initiate ecdysis behaviors and on the peripheral tissues to the hardening of the new cuticle, among other peripheral actions [4].
20E acts mostly on members of the conserved nuclear receptor superfamily, which act as ligand-dependent transcription factors. Thus, 20E activates the complex transcriptional processes that underlie the many cellular and morphogenetic events that occur during molting. Being a circulating lipid-soluble (steroid) hormone gives 20E a built-in ability to affect all cells in the organism and cause widespread changes. Such coordination is critical for a successful molt, and is mostly effected by each tissue responding autonomously to the changes in 20E titers, with little tissue-to-tissue coordination. Although much more complex than “simple” molts, metamorphic molts are also directed by 20E. This global endocrine signal is then transduced into a very complex, tissue-specific pattern of gene expression by turning on (and off) 20E-regulated genes, which in turn activate or repress other genes in the pathway that eventually turns a caterpillar into a butterfly [5].
Comparatively less is known about how other ecdysozoans change their coat. Work in crustacea has established that a steroid hormone also controls molting within this class [6]. In fact, many crustacea produce E, which is then converted to the active hormone 20E, the exact same hormone that controls insect molting. Little is know about the mechanism that causes the onset of the molt and the downstream actions of 20E in crustacea, but there are bound to be interesting similarities as well as differences compared to the process in insects. For instance, we already know that 20E production in crustacea is regulated by a neuropeptide (molt-inhibiting peptide) that inhibits E production, rather than by a neuropeptide that stimulates E synthesis (as occurs in insects).
Molting in Worms
Relatively little is known about the process of molting in nematodes. However, this group of organisms is significant for several reasons. First, nematodes are quite different from arthropods; yet their membership in the ecdysozoan clade reflects a deep biological relatedness. Comparisons between molting in nematodes and molting in arthropods would reveal, for instance, which features are invariant (and presumably very constrained) aspects of molting, and which are different solutions to the same problem. Second, a number of serious human diseases are caused by parasitic nematodes. Agents that interfered with molting would be fatal to the parasite, but may not affect its host, to which the parasite is only very distantly related (“A worm, with very few exceptions, is not a human being” [7]). Finally, the nematode Caenorhabditis elegans is a model organism, with a sequenced genome and well-developed molecular genetic tools. These tools make it possible to carry out forward and reverse genetic screens, determine the time and place that candidate genes are expressed, investigate order of gene action, etc., and have been used to systematically analyze many aspects of the biology of this little worm.
Paradoxically, until now, molting has not been subjected to such systematic scrutiny. This may be because recognition of nematodes' membership in the ecdysozoan club is recent, so molting has not been viewed as a prominent feature of their biology. Nevertheless, a number of known features of molting in C. elegans make it especially intriguing. For instance, it has been difficult to demonstrate that molting in nematodes requires cholesterol, the precursor of steroid hormones, and C. elegans lacks key enzymes required for the synthesis of sterols [8]. In addition, despite encoding a bounty of 260 nuclear receptors, the C. elegans genome does not apparently encode a homolog of the 20E receptor or of ultraspiracle, its obligate partner.
The research article by Frand et al. [9] in this issue of PLoS Biology reports the results of the first systematic screen for genes involved in molting in nematodes. The authors used RNA interference to silence each of the 19,427 predicted C. elegans genes, and screened for the occurrence of worms that were unable to shed their old cuticle (Figure 1A). This screen produced a list of 159 genes whose interference produced molting defects in larvae. Significantly, interference with gene function could cause arrest at different larval molts, suggesting that many of these genes are required for molting, per se, not simply for the transition between two specific stages. Importantly, the 159 genes included nine genes that had previously been shown to be involved in C. elegans molting, as well as 28 that had previously been noted as causing an arrest during a molt when inactivated by RNA interference. The nine genes include nhr-25, the homolog of the orphan nuclear receptor ftz-f1 [10], which plays a key role in insect metamorphosis, as well as lpr-1 [11], a homolog of gp330-megalin, which is involved in vitamin D (a cholesterol derivative) uptake in mammals. Re-isolating these genes in a forward screen for molting defects lends further support to the (wishful) hypothesis that steroid hormones are involved in nematode molting, just as they are in molting in the arthropod members of this clade. Although the screen was based on a failure in the last step of the molt (shedding of the old cuticle), many of the candidates are likely to play a role in the earlier stages of the molting process. Indeed, the list of genes includes transcription factors, signaling proteins, and molecules involved in cuticle secretion and remodeling of basement membranes, as well as the expected list of proteins involved in the eventual release of the collagenous cuticle.
Figure 1 A C. elegans Molting Sampler
(A) A C. elegans larva unable to shed its old cuticle after RNA interference of the gene acn-1.
(B–D) Pairs of pictures show larvae expressing the GFP-tagged mlt-11 gene before (B), during (C), and after (D) the first molt.
(E) A collar of old cuticle found midway along the length of the animal mostly restricts expression of the GFP-tagged mlt-10 gene to the front half of the worm.
As expected for a first description of the results of a genome-wide screen, it is not yet possible to weave all the candidate genes into a solid story. Nevertheless, the authors do make a significant contribution to our understanding of how these genes might make the nematode change its coat by investigating in more detail eight genes representing the major functional categories. First, fusions to green fluorescent protein (GFP) revealed that all eight genes were expressed in epithelial cells, with a pulse of fluorescence prior to each molt (Figure 1B–1D). Thus, these genes are expressed in the right place at the right time. Second, the ability of RNA interference of molting genes to block GFP expression of the select tagged molting genes was used to order gene expression cascades. This analysis revealed, for instance, that interference of nhr-23 (encoding another orphan nuclear receptor previously implicated in molting [12]) affected the expression of the eight GFP-tagged molting genes, suggesting that nhr-23 may be generally required for the expression of molting genes. By contrast, blocking the molting gene mlt-9 caused the expected molting defect without affecting the expression of mlt-10 and mlt-8. As an additional bonus from this study, the authors noted that some genotypes tended to develop constrictions in their cuticle, and that in these animals, both GFP expression and molting were confined to the anterior part of the animal (Figure 1E). This result is reminiscent of the now classic experiments in insect endocrinology that demonstrated the role of circulating hormones in the control of molting [3]. Thus, Frand et al., in a single study, have carried out classic and molecular genetic experiments to provide a qualitative advance in our understanding of how nematodes molt.
The information gained from this screen is important in itself, since few genes involved in nematode molting had previously been identified. These genes can now be compared to the homologous genes in other ecdysozoans, and may also be useful for developing effective agents for controlling parasitic nematodes. Furthermore, modifications of the screen that was used (e.g., sensitized screens) may allow the nematode to answer some of the more intractable questions regarding the control of molting. For example, little is known about the mechanism that monitors the organism's size and signals that a molt must start. In insects, we know that it is dependent on size (weight), nutritional status, and time of day, but how this information is integrated and transduced is unknown for most species. Likewise, little is known about how ecdysozoans know how many immature stages they must complete before becoming an adult. For example, we do not know how a third-instar caterpillar determines that it still has two more caterpillar stages to go through before becoming a pupa (chrysalis), how a third-instar fruit fly larva knows that it has completed its larval life, and how a third-instar C. elegans knows that it still has a fourth instar to go through before becoming an adult. An interesting possibility is that the process of “instar counting” is regulated by so-called heterochronic genes [13]. Thus, is a failure to enter metamorphosis (for an insect) mechanistically related to adding an extra larval instar (for a worm)? The screen conducted by Frand et al. did not identify any known heterochronic genes, but this could simply reflect the criteria used for identifying candidate genes. Finally, we know little of both global checkpoints that must be cleared in order for molting to progress and any signals between molting tissues. Frand et al.'s research article paves the way for a new understanding of molting in Ecdysozoa.
I thank Alison Frand and Carl Thummel for comments. I am grateful to Alison Frand for providing Figure 1.
Citation: Ewer J (2005) How the ecdysozoan changed its coat. PLoS Biol 3(10): e349.
John Ewer is in the Entomology Department, Cornell University, Ithaca, New York, United States of America. E-mail: [email protected]
Abbreviations
20E20-hydroxyecdysone
Eecdysone
GFPgreen fluorescent protein
==== Refs
References
Hölldobler B Wilson EO The ants 1990 Cambridge (Massachusetts) Harvard University Press 746
Aguinaldo AM Turbeville JM Linford LS Rivera MC Garey JR Evidence for a clade of nematodes, arthropods and other moulting animals Nature 1997 387 489 493 9168109
Nijhout HF Insect hormones 1994 Hoboken (New Jersey) Princeton University Press 280
Ewer J Reynolds S Pfaff DW Arnold AP Fahrbach SE Etgen AM Rubin RT Neuropeptide control of molting in insects 2002 San Diego Academic Press 1 92 Hormones, brain and behavior
Thummel CS Files on steroids—Drosophila metamorphosis and the mechanisms of steroid hormone action Trends Genet 1996 12 306 310 8783940
Chang ES Comparative endocrinology of molting and reproduction: Insects and crustaceans Annu Rev Entomol 1993 38 161 180 8424625
Brooks M Young Frankenstein [film] 1974 Los Angeles 20th Century Fox/Gruskoff/Venture Film/Crossbow Productions/Jouer Limited
Kurzchalia TV Ward S Why do worms need cholesterol? Nat Cell Biol 2003 5 684 688 12894170
Frand AR Russel S Ruvkun G Functional genomic analysis of C. elegans molting PLoS Biol 2005 3 e312 10.1371/journal.pbio.0030312 16122351
Gissendanner CR Sluder AE nhr-25, the Caenorhabditis elegans ortholog of ftz-f1 , is required for epidermal and somatic gonad development Dev Biol 2000 221 259 272 10772806
Yochem J Tuck S Greenwald I Han M A gp330/megalin-related protein is required in the major epidermis of Caenorhabditis elegans for completion of molting Development 1999 126 597 606 9876188
Kostrouchova M Krause M Kostrouch Z Rall JE Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans
Proc Natl Acad Sci U S A 2001 98 7360 7365 11416209
Thummel CS Molecular mechanisms of developmental timing in C. elegans and Drosophila
Dev Cell 2001 1 453 465 11703937
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1620707810.1371/journal.pbio.0030355EssayBiophysicsBiotechnologyNeurosciencePhysiologyMammalsFrom Art to Engineering? The Rise of In Vivo Mammalian Electrophysiology via Genetically Targeted Labeling and Nonlinear Imaging EssayKleinfeld David [email protected] Oliver 10 2005 11 10 2005 11 10 2005 3 10 e355Copyright: © 2005 Kleinfeld and Griesbeck.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 convergence of technical advancements in neuroscience has begun to transform mammalian electrophysiology from an art into a precise practice.
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For close to half a century, neurophysiologists have been able to record electrical signals from the millions of individual neurons that compose even the smallest mammalian brain. Despite this long history, which has led to significant strides toward understanding how neuronal activity translates into brain function, much of the way electrophysiological data is gathered is more of an art form than a science. Reproducibility, a cornerstone of scientific progress, hasn't always been forthcoming when recording from individual neurons in the brain, largely because of the improbability or uncertainty that different investigators make their measurements from the same neurons or even the same select subpopulations of neurons. How does one put together the information about activity in single neurons that are recorded by different investigators in different ways? Further still, how does one combine this information with knowledge of the underlying circuitry to make sense of the firing patterns that underlie normal brain function? Progress will come largely from the ability to reproducibly record voltages, as well as other variables that define physiological function, from identified neuronal cell types. The ability to record from the same subpopulation of cells on a routine basis is the singular means to validate measurements across different laboratories and move electrophysiology beyond its current, largely anecdotal status.
By definition, measurements from identified neuronal cell types depend on a means to visualize the cells in question. For simple neuronal circuits in invertebrates, in which the function of a cell is often well correlated with its physical location within a ganglion, simple light microscopy imaging is adequate to uniquely identify a neuron. Similarly, in a mammalian brain slice, gross architectonic features can be discerned from the visual texture of the tissue, while individual neuronal boundaries may be identified with optical techniques that minimize the interfering effects of scattered light. For the case of recording from brains in living mammals, the technical challenges that must be surmounted to record from identified cells are far greater. Antidromic activation of projection neurons, a heroic approach, provides selectivity in some instances [1]. Yet, at present, much of in vivo recording is performed blind, in the sense that cell morphology and phenotype are confirmed only from post hoc histology.
What advances lie ahead to advance the qualitative nature of mammalian in vivo recording? In particular, can electrophysiology approach the level of precision and reproducibility that one associates, for example, with biochemistry or molecular biology? The confluence of three avenues of technical advance—one in imaging, one in labeling, and one in behavioral training—suggest that in vivo electrical and optical recording from identified neuronal phenotypes in the central nervous system of awake behaving mice should soon be a common reality. That's the good news. But before we get too enthusiastic, it is important to realize that the major stumbling block in electrophysiology has yet to be solved. Electrophysiology remains a labor-intensive art form. Data gathering involves many manually controlled processes that require an extended and constant level of vigilance. This is to be contrasted with molecular biology, where standard tools and high levels of automation make acquisition relatively cheap in terms of time and expense, and thus shift the focus to conceptual synthesis. Time will tell if the technical advances described below advance not just the reliability of electrophysiology but further serve as a tipping point for its transition from an art to an engineering process.
Labeling of Specific Neuronal Phenotypes—Mus musculus as a “Simple” Nervous System
The age of transgenic animals and fluorescence labeling drives forward with ever greater abandon. Neurobiology is one of the great beneficiaries of the development of a rainbow spectrum of fluorescent proteins (XFPs) [2–4], in the sense that transgenic expression of these proteins reveals the three-dimensional outlines of individual living neurons with minimal cytosolic perturbation. For the electrophysiologist, this portends the engineering of mice in which defined subclasses of neurons express a fluorescence label. While this technique in mammals is not quite at the level of precision it has in invertebrates, where one can often identify individual neurons in vivo, such mice offer the possibility of allowing researchers to return to the same phenotypically defined neurons within a given brain region. A demonstration of the power of this approach is a well appreciated series of transgenic mice labeled via nonhomologous incorporation of an expression cassette (a short sequence of DNA) that codes for the pan-neuronally expressed Thy-1 promoter, a selected XFP, and ribosome binding [5]. The type of expression varies significantly from line to line as a result of strong positional and context sensitivity of the Thy-1 expression cassette when integrated into the genome. The resultant mosaic labeling is valuable for certain studies, but more importantly, it serves to illustrate that considerable “artistic” elements are currently at work in labeling the brain.
What are the essential difficulties in reproducibility and predictability in the generation of mice with labeled neurons? The use of expression cassettes in mammals suffers from the difficulty of identifying key regulatory elements, such as enhancers or silencers, that are necessary for the correct expression of a transgene [6]. A related source of variability is that expression of the label is influenced by the DNA sequences that flank the inserted DNA, yet the site of integration into the genome differs between transgenic animals. These difficulties are diminished through the use of bacterial artificial chromosomes (BACs) [7,8], which incorporate the entire transcription unit and large pieces of sequence 5′ and 3′ of it (Figure 1A). Although this approach is not perfect and can still miss out on important regulatory elements in some cases (Figure 1B), in the ideal case the BAC includes all necessary elements to express a reporter gene in the correct manner.
Figure 1 Schematic for the Production of a Modified BAC for the Targeted Expression of an XFP or an XFP-Based Reporter in Mice
(A) A library of suitable BAC clones is scanned using bioinformatics and an appropriate clone, encoding a suitable cell-type-specific transcriptional unit with ample flanking regions, is selected. Note that only a few of the many possible enhancer and silencer regions are drawn. An exon that lies downstream of the ATC start sequence is selected to be replaced by the XFP/reporter sequence by homologous recombination (exon 2 in this example), and a shuttle vector that codes for the label together with flanking regions around the exon (“a” and “b”) is constructed. The enzyme RecA is used to interchange the sequence for the exon and the label to form a modified BAC clone that codes for the label. The modified clone is injected into a mouse oocyte, where the dominant incorporation into the host DNA occurs through nonhomologous recombination.
(B) Many factors influence the phenotype of a given transgenic mouse, and thus the same clone may result in a number of lines with slightly different properties. The insert shows the XFP expression pattern for a line based on a BAC clone that contains the transcriptional unit of a glycine transporter.
(Image: Jean-Marc Fritschy and Hanns-Ulrich Zeilhofer)
Methods to incorporate reporter genes into BAC constructs are relatively straightforward (Figure 1A) and have led to an almost industrial-scale effort to generate and characterize a collection of mice with defined labeled neurons for further anatomical and physiological analysis [9]. Recent examples of transgenic mouse technology based on BAC clones demonstrate the accurate labeling of neurons containing the neurotransmitter glycine in the spinal cord, brainstem, and cerebellum (Figure 1B) [10], and the labeling of neurons expressing both parvalbumin and GABA throughout neocortex [11]. Other examples used clones with the gene for glutamic acid dehydrogenase (GAD-27) to select for all GABAergic neurons, but observed expression in only the parvalbumin-positive subpopulation [12,13]. It is to be expected that the precision of molecular biology will further evolve to produce mice with ever increasing specificity of subtype labeling.
In Vivo Visually Guided Recording of Labeled Cortical Neurons—Laser Jocks Turned Neuroscientists
Making mice with fluorescent neurons is only the first step; the second requires the means to visualize the axons and dendrites of these neurons, which can be less than a micrometer in thickness. In vivo two-photon laser scanning microscopy (TPLSM) [14,15] provides a unique means to image fluorescently labeled neurons that lie below the surface of the brain [16]. When used in conjunction with transgenic mice that are labeled by the expression of a fluorescent protein, TPLSM provides the necessary visualization to target a fine glass electrode to the membrane surface of one's neuron of choice [17] (Figure 2). TPLSM can image deep into scattering brain tissue, in excess of 500 μm under normal conditions [18] and down to 1,000 μm under special circumstances [19]. Although technical challenges—such as increasing the rate at which images are scanned and compensating for optical aberrations—one can, in principle, image and thus target neurons throughout almost the entire depth of mouse cortex.
Figure 2 Targeted Electrical Recording of Transgenically Labeled Inhibitory Interneurons in Mouse Cortex
(A) The two-photon laser scanning microscope is shown schematically. The critical features are the use of separate fluorophores, one for the label (GFP in this example) and another to mark the intracellular fluid of electrode (Alexa in this example) that have overlapping excitation spectra and different emission spectra (see [B]). The intracellular voltage shows a trace obtained under whole-cell patch of the response to vibrissa stimulation. Alexa, Alexa 594 dye; fs laser, titanium:sapphire mode-locked laser with 100 fs output pulse width; GFP, green fluorescent protein; PMT, photomultiplier tube.
(B and C) Emission spectra and fluorescent images from the GFP and Alexa channels. Confirmation of whole-cell patch is achieved by injecting Alexa into the GFP-filled cell, as illustrated in the overlay.
(Images: Troy Margrie)
Biomolecular Reporters and Drivers of State Variables—Proteins as Spies and Membrane Provocateurs
Apart from their role as phenomenally good, noninvasive labels of neuronal structure, XFPs have become the basis for a series of sensors of physiological variables and events, such as membrane-potential fluctuations and intracellular messenger dynamics [20]. Genetically encoded, these sensors are generated inside cells, do not require cofactors, and do not leak out of cells even during prolonged studies. These sensors will benefit considerably from the increasing accuracy of neuronal labeling via modified BAC clones (see Figure 1). Indicators of synaptic release [21–23] or intracellular [Ca2+] dynamics [24–28] might initially be the most appealing. While issues, such as signal strength and response kinetics, have still to be sorted out, recent work on transgenic mice that express these and other probes proved the feasibility of the approach (Table 1). An exquisite example is the expression of the pH indicator synaptopHluorin in olfactory sensory neurons of the mouse, which allowed for the in vivo imaging of patterns of activation in the olfactory bulb after odorant stimulation [21]. To the extent that optical microscopy is able to resolve their pattern of expression, XFP-based molecular probes offer a means to read out activity not only from a few but ideally from whole populations of identified neurons.
Table 1 XFP-Based Indicators Expressed and Tested in Transgenic Mice
a A subjective measure in which + signifies a population response or a multi-spike single-cell response in brain slice and ++ signifies a population response in vivo.
b Collaboration of O. Griesbeck and T. Örtner laboratories.
c Collaboration of E. M. Callaway, E. Y. Isacoff. and R. M. Siegel laboratories.
The complement to optical-based probes of neuronal state variables is optical-based perturbation mediated by intrinsic chromophores. The ability to perturb the state of neuronal activation plays two essential roles in systems identification. The first is to determine the effect of a depolarizing perturbation in neighboring as well as downstream cells. The challenge has been met, in non-mammalian systems, through the use of cloned photoreceptor complexes [29] and photolabile organic cages that release agonists of excitatory neurotransmission onto cloned channels that are expressed in defined phenotypes [30]. The second role is the inactivation of neuronal pathways as a means to open feedback loops and determine the direction of signal flow. For example, a modified K+ channel in which photoisomerization drives the reversible transition between closed and conducting states has been demonstrated in vitro [31]. One clear challenge is the functional incorporation of these and related photo-activated agents in defined mammalian cells.
Targeted Recording from the Awake Rodent—Molecular Biology Meets Consciousness
Immobilization is generally necessary for most forms of recording. Needless to say, immobilization achieved by anesthesia blatantly disrupts neural function, and the whole notion of attentive-based activation as well as motor output per se is lost. This problem is avoided with primates through the use of “head-fixed” animals that are trained to sit quietly while they perceive the world through arrays of projectors and tactile pads. The same form of constraint can be brought to studies with rodents through the use of head-fixed preparations [32], which has proved to be of critical importance for the study of behavioral [33] and electrophysiological [34] aspects of whisking. This strategy has also provided a means to record both optically and electrically from individual neurons that are labeled with organic [Ca2+] indicators (Figure 3A; J. Waters and F. Helmchen, unpublished data), and it is anticipated that recording and perturbation from cells labeled via viral transfection will soon be forthcoming [35]. The near-term challenge is to record from awake head-fixed mice.
Figure 3 Prospects for Recording from Awake but Head-Restrained Animals
(A) Photograph of a trained rat that is awake and head-restrained, ready for imaging of organic [Ca2+]-sensitive dyes. All aspects of the recording procedure demonstrated in primates are expected hold for mice as well. (Image: Jack Waters)
(B) Photograph and set-up of visual virtual reality for rodents. In this example, the rat is body-fixed, and can rotate on an axis, but is not head-fixed. The visual world of the animal is controlled by projected images, and reward is administered through a food tube. (Images: Hansjuergen Dahmen)
A final issue concerns the extent of behavior that may be expected with head-fixed animals, especially as a large block of research concerns spatial tasks and hippocampal function. Both primate electrophysiological studies [36] and human psychophysical studies [37] have advanced with the use of virtual reality. Recently, the same level of sophistication has been brought to bear on rodent studies [38] (Figure 3B), where body-fixed rats are constrained to walk on a near frictionless ball while they observe a virtual visual world. This advance already provides a means to record from rats when the tether, such as that for a head-mounted scanner [39], is too short for use with animals in mazes. In the best of worlds, this advance is a stepping stone to recording from head-fixed mice as they respond to novel environments.
Putting It All Together
The tools are there to perform targeted electrical and optical-based ion recording, and stimulation, of identified neuronal phenotypes in mice. Nonlinear microscopy, while still a tool of the aficionado, is approaching maturity [40]. The design of endogenous molecular sensors of cell function, while in early days, has attained a set of heuristics and material successes (Table 1). This suggests that signaling and circuitry in the mammalian nervous system may be addressed in a reliable and logical, if painstaking, way. Other recent work, involving methods to automate histology at the synaptic [41] and cellular [42] levels, will help place physiological measurements in the framework of detailed architectonics. The greatest challenges for in vivo electrophysiology appear to lie primarily in the areas of molecular biology and behavior. Gene expression through the use of BACs has been successfully targeted to only a few neuronal subtypes so far, yet must be pushed to all cell types. This highlights a need for better phenotyping of neurons, both by conventional histochemistry and by microarray analysis of gene expression, and a better understanding of the transcription factor logic that defines expression. Automated means for shaping animal behavior need to be advanced [43]. Critically, while the bias that “mice cannot be trained” is pervasive, there has been little concerted effort to breed and train calm mice that could be the background for transgenesis. Behavioral issues aside, it is a good bet that a mixture of genetics and optics will play a dominant role in delimiting the algorithms of brain function.
The ideas in this essay originated from presentations and discussions at the Imaging Neurons and Neural Activity: New Methods, New Results biannual conference and the Imaging Structure and Function in the Nervous System annual summer school, both held at Cold Spring Harbor Laboratory in 2005. We thank Eve Marder and Ofer Tchernichovski for additional discussions and Ed Callaway and Beth Friedman for critical reading of the essay.
Citation: Kleinfeld D, Griesbeck O (2005) From art to engineering? The rise of in vivo mammalian electrophysiology via genetically targeted labeling and nonlinear imaging. PLoS Biol 3(10): e355.
David Kleinfeld is with the Department of Physics and a member of the Graduate Program in Neurosciences, University of California at San Diego, La Jolla, California, United States of America. Oliver Griesbeck is with the Department of Cellular Dynamics, Max-Planck Institute for Neurobiology, Martinsried, Germany.
Note Added in Proof
A recent report demonstrates viral incorporation of a photo-activated cation channel into mammalian neurons and the use of this channel to gate spiking [54].
Abbreviations
BACbacterial artificial chromosome
TPLSMtwo-photon laser scanning microscopy
XFPfluorescent protein
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References
Swadlow HA Waxman SG Rosene DL Latency variability and the identification of antodromically activated neurons in mammalian brain Exp Brain Res 1978 32 439 443 98342
Prasher DC Eckenrode VK Ward WW Prendergast FG Cormier MJ Primary structure of the Aequorea victoria green-fluorescent protein Gene 1992 111 229 233 1347277
Tsien RY The green fluorescent protein Annu Rev Biochem 1998 67 509 544 9759496
Shaner NC Campbell RE Steinbach PA Giepmans BN Palmer AE Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein Nat Biotechnol 2004 22 1567 1572 15558047
Feng G Mellor RH Bernstein M Keller-Peck C Nguyen QT Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP Neuron 2000 28 41 51 11086982
Rulicke T Hubscher U Germ line transformation of mammals by pronuclear microinjection Exp Physiol 2000 85 589 601 11187955
Yang XW Model P Heintz N Homologous recombination based modification in Escherichia coli and germline transmission in artificial mice of a bacterial artificial chromosome Nat Biotechnol 1997 15 859 865 9306400
Gong S Zheng1 C Doughty ML Losos K Didkovsky N A gene expression atlas of the central nervous system based on bacterial artificial chromosomes Nature 2003 425 917 925 14586460
Hatten ME Heintz N Large-scale genomic approaches to brain development and circuitry Annu Rev Neurosci 2005 28 89 108 16022591
Zeilhofer HU Studler B Arabadzisz D Schweizer C Ahmadi S Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice J Comp Neurol 2005 482 123 141 15611994
Meyer AH Kartona I Blatow M Rozov A Monyer H In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons J Neurosci 2002 22 7055 7064 12177202
Ango F di Cristo G Higashiyama H Bennett V Wu P Ankyrin-based subcellular gradient of neurofascin, an immunoglobulin family protein, directs GABAergic innervation at Purkinje axon initial segment Cell 2004 119 257 272 15479642
Chattopadhyaya B Cristo GD Higashiyama H Knott GW Kuhlman SJ Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period J Neurosci 2004 24 9498 9611
Denk W Delaney KR Kleinfeld D Strowbridge B Tank DW Anatomical and functional imaging of neurons and circuits using two photon laser scanning microscopy J Neurosci Methods 1994 54 151 162 7869748
Svoboda K Denk W Kleinfeld D Tank DW In vivo dendritic calcium dynamics in neocortical pyramidal neurons Nature 1997 385 161 165 8990119
Denk W Svoboda K Photon upmanship: Why multiphoton imaging is more than a gimmick Neuron 1997 18 351 357 9115730
Margrie TW Meyer AH Caputi A Monyer H Hasan MT Targeted whole-cell recordings in the mammalian brain in vivo Neuron 2003 39 911 918 12971892
Oheim M Beaurepaire E Chaigneau E Mertz J Charpak S Two-photon microscopy in brain tissue: Parameters influencing the imaging depth J Neurosci Methods 2001 111 29 37 11574117
Theer P Hasan MT Denk W Two-photon imaging to a depth of 1000 micron in living brains by use of a Ti: Al2O3 regenerative amplifier Opt Lett 2003 28 1022 1024 12836766
Tsien RY Imagining imaging's future 2003 Nat Rev Mol Cell Biol Suppl SS16 SS21
Bozza T McGann JP Mombaerts P Wachowiak M In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse Neuron 2004 42 9 21 15066261
Li Z Burrone J Tyler WJ Hartman KN Albeanu DF Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin Proc Natl Acad Sci U S A 2005 102 6131 6136 15837917
Okumoto S Looger LL Micheva KD Reimer RJ Smith SJ Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors Proc Natl Acad Sci U S A 2005 102 8740 8745 15939876
Miyawaki A Griesbeck O Heim R Tsien RY Dynamic and quantitative Ca2+ measurements using improved cameleons Proc Natl Acad Sci U S A 1999 96 2135 2140 10051607
Nakai J Ohkura M Imoto K A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein Nat Biotechnol 2001 19 137 141 11175727
Heim N Griesbeck O Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein J Biol Chem 2004 279 14280 14286 14742421
Nagai T Yamada S Tominaga T Ichikawa M Miyawaki A Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins Proc Natl Acad Sci U S A 2004 101 10554 10559 15247428
Dýez-Garcýa J Matsushita S Mutoh H Nakai J Ohkura O Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein Eur J Neurosci 2005 22 627 635 16101744
Lima SQ Miesenböck G Remote control of behavior through genetically targeted photostimulation of neurons Cell 2005 121 141 152 15820685
Zemelman BV Lee GA Ng M Miesenbock G Selective photostimulation of genetically ChARGed neurons Neuron 2002 33 15 22 11779476
Baghart M Borges K Isacoff EY Kramer RH Light-activated ion channels for remote control of neuronal firing Nat Neurosci 2004 7 1381 1386 15558062
Ono T Nakamura K Nishijo H Fukuda M Hypothalamic neuron involvement in integration of reward, aversion and cue signals J Neurophysiol 1986 56 63 79 3746401
Harvey MA Bermejo R Zeigler HP Discriminative whisking in the head-fixed rat: Optoelectronic monitoring during tactile detection and discrimination tasks Somatosens Mot Res 2001 18 211 222 11562084
Kleinfeld D Sachdev RN Merchant LM Jarvis MR Ebner FF Adaptive filtering of vibrissa input in motor cortex of rat Neuron 2002 34 1021 1034 12086648
Callaway EM A molecular and genetic arsenal for systems neuroscience Trends Neurosci 2005 28 196 201 15808354
Schwartz AB Direct cortical representation of drawing Science 1994 265 540 542 8036499
Kahana MJ Sekuler R Caplan JB Kirschen M Madsen JR Human theta oscillations exhibit task dependence during virtual maze navigation Nature 1999 339 781 784
Holscher C Schnee A Dahmen H Setia L Mallot HA Rats are able to navigate in virtual environments J Exp Biol 2005 208 561 569 15671344
Helmchen F Fee MS Tank DW Denk W A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals Neuron 2001 31 903 912 11580892
Zipfel WR Williams RM Webb WW Nonlinear magic: Multiphoton microscopy in the biosciences Nat Biotechnol 2003 21 1369 1377 14595365
Denk W Horstmann H Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure PLoS Biol 2004 2 e319 10.1371/journal.pbio.0020329
Tsai PS Friedman B Ifarraguerri AI Thompson BD Lev-Ram V All-optical histology using ultrashort laser pulses Neuron 2003 39 27 41 12848930
Kafkafi N Benjamini Y Sakov A Elmer GI Golani I Genotype–environment interactions in mouse behavior: A way out of the problem Proc Natl Acad Sci U S A 2005 102 4619 4624 15764701
Miyawaki A Llopis J Heim R McCaffery JM Adams JA Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin Nature 1997 388 882 885 9278050
Hasan MT Friedrich RW Euler T Larkum ME Giese GG Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control PLoS Biol 2004 2 763 775 10.1371/journal.pbio.0020163
Baird GS Zacharias DA Tsien RY Circular permutation and receptor insertion within green fluorescent proteins Proc Natl Acad Sci U S A 1999 96 11241 11246 10500161
Griesbeck O Baird GS Campbell RE Zacharias DA Tsien RY Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications J Biol Chem 2001 276 29188 29194 11387331
Pologruto TA Yasuda R Svoboda K Monitoring neural activity and [Ca2+ ] with genetically encoded Ca2+ indicators J Neurosci 2004 24 9572 9579 15509744
Nagai T Sawano A Park ES Miyawaki A Circularly permuted green fluorescent proteins engineered to sense Ca2+
Proc Natl Acad Sci U S A 2001 98 3197 3202 11248055
Siegel MS Isacoff EY A genetically encoded optical probe of membrane voltage Neuron 1997 19 735 751 9354320
Guerrero G Siegel MS Roska B Loots E Isacoff EY Tuning FlaSh: Redesign of the dynamics, voltage range, color of the genetically encoded optical sensor of membrane potential Biophys J 2002 83 3607 3618 12496128
Metzger F Repunte-Canonigo V Matsushita S Akemann W Diez-Garcia J Transgenic mice expressing a pH and Cl− sensing yellow-fluorescent protein under the control of a potassium channel promoter Eur J Neurosci 2002 15 40 50 11860505
Miesenböck G De Angelis DA Rothman JE Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins Nature 1998 394 192 195 9671304
Boyden ES Zhang F Bamberg E Nagel G Deisseroth K Millisecond-timescale, genetically targeted optical control of neural activity Nat Neurosci 2005 8 1263 1268 16116447
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1752326410.1371/journal.pbio.0030367EditorialScience PolicyNonePublished and Not Perished EditorialParthasarathy Hemai 10 2005 11 10 2005 11 10 2005 3 10 e367Copyright: © 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.
PLoS Biology launches an electronic letters service.
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Once upon a time, you formulated a hypothesis and designed the experiments to test it. You applied for a grant. You were awarded the money to pursue your line of inquiry. You did the work. You wrote the paper. Your colleagues reviewed your work and found it to be true. You published your paper. The conclusions of the paper joined the cannon of scientific knowledge. The End.
This platonic ideal of the scientific method is, sadly, at best science fiction and at worst history of science. The reality, as everyone knows, is much less linear—simultaneously more frustrating and more exciting. Publishing a paper is somewhere within an iterative loop that involves proving your point before you write the grant, working backwards to the rationale from a completely unexpected finding, and ultimately receiving a set of mixed peer reviews, which an editor interprets into a binary decision to publish your paper or not, in one peer-reviewed journal or another. And quite often, it is only with publication of the paper, that its worth is judged in earnest. Ultimately, worth is assessed by whether the scientific community decides to build upon a particular finding. More immediately, results are evaluated in journal clubs and scientific meetings, and a whole discussion surrounding a particular paper can be transmitted by word-of-mouth within a particular community, without ever being documented.
Last month, PLoS Biology launched its e-letters service, an electronic forum for responses to our published articles. Each article in PLoS Biology (including all content in the magazine section) could potentially have appended to it a series of comments that praise, criticize, clarify, or speculate about ideas presented in the original article.
Although open online discussion of content is quite common in some fields (e.g., medicine), and is all the rage in entirely different contexts (e.g., http://www.amazon.com), biology journals have been slow to embrace the discussion forum. Enthusiasm for the concept of attaching moderated blogs or discussion boards (e.g., http://www.slashdot.org) to research articles seems to emanate from the more junior members of the scientific community. Post-docs and graduate students seem especially sensitive to the wealth of discussion that surrounds published research articles that is never formally documented. Faced with an exponentially growing scientific literature, being “in the know” can provide a substantial advantage in pursuing promising leads and ignoring spurious tracks.
We hope that e-letters serve as the first step in implementing a broader program that provides a more interactive forum for publication of research. Although we have had requests to allow anonymous postings, we feel that there is enough anonymity in the publication process, and that transparent discussion—both positive and negative—should be encouraged. Ultimately, we would like to institute a system in which questions such as “How useful did you find this article?” and other sorts of immediate feedback mechanisms could serve as a tool to aid readers in allocating their limited time and attention.
Comments will be posted on PLoS Biology at the discretion of the editors. We will apply liberal policies to our screening process, but we will censor anything that is abusive, redundant, scientifically bogus, and/or extremely tangential to the issues addressed in the related article. As the BMJ noted on the anniversary of its 50,000th posting, there are downsides to a policy in which “just about anything that isn't libellous or doesn't breach confidentiality” is posted (BMJ 330: 1284). Those downsides included the following: “Some respondents feel the urge to opine on any given topic, and pile in early and often, despite having little of interest to say. Others have pet topics, which they return to obsessively, finding almost any peg to hang their views on. Some respondents don't seem to feel they're really alive until they've sparked off an angry response from someone else” (BMJ 330: 1284).
PLoS Medicine e-letters
Thus, we will endeavor to provide some level of oversight to foster a constructive dialogue about issues relevant to a particular article. Obviously, the degree of editorial oversight we can provide will, in part, depend on the volume of comments we receive. But we will cross that bridge when we get to it. Our sister journal, PLoS Medicine, launched e-letters last year, and at the present time, has posted 122 letters and rejected 45, a healthy but not unmanageable response.
We welcome your feedback on this new service, and we hope that you will use it to make open-access publishing a still more valuable resource within your scientific community. Write to us!
Citation: Parthasarathy H (2005) Published and not perished. PLoS Biol 3(10): e367.
Hemai Parthasarathy is Managing Editor for PLoS Biology. E-mail: [email protected]
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030373CorrectionImmunologyMus (Mouse)Correction: Directed Migration of Positively Selected Thymocytes Visualized in Real Time CorrectionWitt Colleen M Raychaudhuri Subhadip Schaefer Brian Chakraborty Arup K Robey Ellen A 10 2005 11 10 2005 11 10 2005 3 10 e373Copyright: © 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.
Directed Migration of Positively Selected Thymocytes Visualized in Real Time
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In PLoS Biology, volume 3, issue 6: DOI: 10.1371/journal.pbio.0030160
The following statement in the last paragraph of the Results contains a factual error: “A total of 34% of cortical thymocytes expressing the P14 TCR had motility rates greater than 13 μm/min compared to approximately 2% for wild-type thymocytes expressing diverse TCRs.” The sentence should read as follows: “A total of 20% of cortical thymocytes expressing the P14 TCR had motility rates greater than 13 μm/min compared to approximately 2% for wild-type thymocytes expressing diverse TCRs.”
The authors apologize for any confusion this error may have caused.
This correction note may be found online at DOI: 10.1371/journal.pbio.0030373. Published October 11, 2005
Citation: (2005) Correction: Directed migration of positively selected thymocytes visualized in real time. PLoS Biol 3(10): e373.
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030376CorrectionEcologyEvolutionGenetics/Genomics/Gene TherapyOtherHomo (Human)Correction: Traces of Archaic Mitochondrial Lineages Persist in Austronesian-Speaking Formosan Populations CorrectionTrejaut Jean A Kivisild Toomas Loo Jun Hun Liang Lee Chien He Chun Lin Hsu Chia Jung Lee Zheng Yuan Lin Marie 10 2005 11 10 2005 11 10 2005 3 10 e376Copyright: © 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.
Traces of Archaic Mitochondrial Lineages Persist in Austronesian-Speaking Formosan Populations
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In PLoS Biology, volume 3, issue 8: DOI: 10.1371/journal.pbio.0030247
The seventh author's name was originally misspelled as Zheng Yuan Li; it should be Zheng Yuan Lee.
The first sentence of the Materials and Methods should read as follows: “In this study the sequence variation of the mtDNA D-loop HVS-I and HVS-II regions, nps 16006 to 16397 and nps 53 to 404, respectively, was analyzed in 640 samples drawn from nine Taiwan indigenous mountain tribes representative of most languages, cultures, and geographical settlements seen on the island before the last four centuries.”
The first two sentences in the section “Data Sequence Analysis” in the Materials and Methods should read as follows: “Using primer pairs L15997–H16401 and L048–H408 [59], segments of 404 bps and 360 bps from the D-loop HVS-I and HVS-II, respectively, were obtained. Primer pairs 5, 7, 8, 11–14, 19, and 24 F&R described in Rieder et al. [60]….”
This correction note may be found online at DOI: 10.1371/journal.pbio.0030376. Published October 11, 2005
Citation: (2005) Correction: Traces of archaic mitochondrial lineages persist in Austronesian-speaking Formosan populations. PLoS Biol 3(10): e376.
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PLoS PathogPLoS PathogppatplpaplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1622014610.1371/journal.ppat.001001105-PLPA-RA-0073R2plpa-01-02-01Research ArticleEvolutionMicrobiologyVirologyGenetics/Population GeneticsVirusesMus (Mouse)Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice Attenuation of High-Fidelity PoliovirusPfeiffer Julie K. Kirkegaard Karla *Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of AmericaManchester Marianne EditorScripps Research Institute, United States of America* To whom correspondence should be addressed. E-mail: [email protected] 2005 7 10 2005 1 2 e1117 6 2005 25 8 2005 Copyright: © 2005 Pfeiffer and Kirkegaard.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.RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase.
Synopsis
RNA viruses have the highest error rates in nature, resulting in the likelihood that each virus differs from other viruses in the population by one or more mutations. The consequence of this “infidelity” is that the viral population as a whole, under selective pressure from the immune system or antiviral drugs, may benefit from adaptive changes in a subset of its members. Therefore, it has been theorized that RNA viruses need high error rates to survive in complex environments. We tested this hypothesis using a drug-resistant poliovirus that contains a mutation in its polymerase that reduces errors during replication. We found that this high-fidelity mutant virus has reduced growth in mice, a complex environment where mutations may be required for growth and spread within the infected animal. At least part of this attenuation is likely due to the high fidelity of this mutant virus, since it was unable to compete with the low-fidelity version of the virus in the context of a defined selective pressure. Therefore, it is likely that mutations do benefit viral populations, especially in complex environments such as an infected animal or human.
Citation:Pfeiffer JK, Kirkegaard K (2005) Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog 1(2): e11.
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Introduction
RNA viruses have the highest replicative error rates in nature, with approximately one mistake made per 1,000–100,000 nucleotides copied [1–3]. As a result, each viral genome is likely to differ from every other virus by one or more point mutations. The complex populations thus generated, called quasispecies, have been hypothesized to be important for survival of the population as a whole in the presence of selective pressure, under which a few viruses that contain beneficial mutations would survive and act as founders for the next generation [1,4]. For example, viruses with mutations that confer resistance to neutralizing antibodies would benefit the virus population as a whole under selective pressure from the host immune response. However, the vast majority of errors made during replication are deleterious, resulting in debilitation of a high percentage of the population. Therefore, RNA viruses live on the edge of “error catastrophe,” where the cost of deleterious mutations is in dynamic balance with the benefit of adaptive mutations [1,5,6].
A promising new class of antiviral drugs, RNA virus mutagens, takes advantage of the cost of error catastrophe. Ribavirin is a nucleoside analog mutagen, which is incorporated into the viral genome during replication and increases error frequency by promiscuous base-pairing [3]. Ribavirin debilitates the viral population over several rounds of replication by causing accumulation of deleterious mutations and error catastrophe [5]. Previously, we isolated a poliovirus variant able to survive serial passage in ribavirin, and mapped this phenotype to a single mutation in the viral polymerase, 3D-G64S [7]. The mechanism of ribavirin resistance in 3D-G64S mutant poliovirus was shown to be due to increased fidelity of RNA replication by showing that it has lower reversion frequency at the resistance mutation for the anti-viral compound guanidine [7], confers resistance to other mutagens as well [7], by direct sequencing of viral genomes [8], and by demonstration that the purified 3D-G64S poliovirus displays increased fidelity in solution that correlates in a delayed conformational change before phosphoryl transfer compared to the wild-type enzyme [8].
If a viral quasispecies is important to the survival either of individual genomes or of the population during growth under standard laboratory conditions, the higher fidelity of 3D-G64S virus should result in an attenuated phenotype. Therefore, we wondered if the 3D-G64S virus would have growth defects in tissue culture, a relatively simple environment, or in mice, a complex environment with many selective pressures. Laboratory strains of mice are not susceptible to poliovirus infection; however, mice transgenic for the human poliovirus receptor, CD155, are susceptible to injected poliovirus [9–11]. When poliovirus receptor (PVR) mice are given poliovirus by intramuscular (IM) injection in the leg, animals develop paralysis and eventually die of paralytic disease. Poliovirus inoculated by IM injection is thought to traffic up the sciatic nerve, to the spinal cord, and then to the brain [12,13], during which many different environments are likely to play a role in its propagation.
In this study, we found that 3D-G64S poliovirus is less pathogenic than wild-type virus in mice, but displays little growth defect in tissue culture. This attenuation in mice was at least partially due to the increased fidelity of the 3D-G64S virus in the context of a defined selected pressure, since it was unable to compete with 3D wild-type virus in the context of a revertible attenuating point mutation. Therefore, we argue that, in the face of the myriad selective pressures encountered by poliovirus during murine infection, the decreased variability in the population generated by the relatively high-fidelity 3D-G64S polymerase may be a distinct disadvantage for the growth and spread of the mutant virus.
Results
3D-G64S Virus Is Less Pathogenic than Wild-Type Virus in Mice
To test whether the pathogenicity of high-fidelity 3D-G64S virus was comparable to that of wild-type virus in infected animals, mice that express the human poliovirus receptor [9] were inoculated by IM injection with 5 × 106 PFU (plaque-forming units), an amount slightly over the LD50 for these animals [9]. Animals infected in this way develop symptoms similar to poliomyelitis in humans [10]. First, animals display lethargy, then weakness and paralysis in the inoculated leg, then paralysis of both legs, followed quickly by death. For this study, we monitored the time required for the first symptoms, weakness and paralysis of the inoculated limb, to develop. Figure 1 shows time courses for 48 mice inoculated with either wild-type or 3D-G64S virus. The 3D-G64S-inoculated animals developed disease significantly more slowly than wild-type infected animals, and fewer animals succumbed to disease (Figure 1). Therefore, the 3D-G64S virus is less pathogenic than wild-type virus in mice.
Figure 1 Pathogenesis of Wild-Type and 3D-G64S Viruses in PVR Mice
PVR mice were given IM injections with 5 × 106 PFU of either the wild-type or 3D-G64S viruses and were monitored for symptoms of disease. Mice were scored as having symptoms when weakness was first observed in the inoculated limb. Asterisks denote time points where statistically significant differences (p < 0.05, determined by two-sample Student's t-test [d 3, 0.0025; d 4, 0.0053; d 5, 0.043]) were observed between 3D-G64S and wild-type viruses.
DOI: 10.1371/journal.ppat.0010011.g001
Competition between 3D-G64S and Wild-Type Virus in Mice
To test whether the decreased pathogenicity of the 3D-G64S virus could be observed in mixed infections, we infected PVR mice with mixtures of wild-type and 3D-G64S virus. A 1:2 ratio of wild-type:3D-G64S virus was chosen because, as will be shown in subsequent figures, this ratio was required to achieve equal RNA yields in mixed infections of HeLa cells. A total of 2 × 107 total PFU of mixed virus were given as IM injections and mice were sacrificed at the first sign of disease, usually 3 or 4 d post-infection. To monitor the relative accumulation of the mutant and wild-type RNAs of mixed infections, we took advantage of the presence of an additional BstBI restriction enzyme site engineered at the position of the 3D-G64S mutation. As shown in Figure 2A, BstBI digestion of end-labeled RT-PCR reaction products from mixtures of wild-type and 3D-G64S RNAs is expected to produce products of different sizes. For the sets of primers used here, a 558-basepair (bp) fragment is produced from RT-PCR analysis of wild-type RNA, and a 396-bp product from 3D-G64S RNA. Although this assay was not designed to quantify the total amounts of these viral RNAs, it should accurately determine their relative proportions within a population. Inoculated leg muscles and brains were harvested from mice infected with a mixture of wild-type and 3D-G64S viruses, RNA was extracted, and the products of reverse transcription, PCR amplification, end-labeling and BstBI digestion were quantified. As shown in Figure 2B and quantified in Figure 2E, both wild-type and 3D-G64S viruses were able to replicate within the inoculated muscle tissue. However, when products from brains were analyzed (Figures 2C and 2F), a distribution of outcomes was observed: in many mice (69%), only the wild-type band was present (e.g., mice 1, 2, 4, 6); in some mice (22%), the 3D-G64S band was predominately or exclusively present (e.g., mouse 5), and only rarely (9%) were both bands present (e.g., mouse 7). These results support two conclusions; first, 3D-G64S virus has a defect in spreading to the brain following IM infection, and second, because usually only one of the two viruses was found in the brain, there is a bottleneck between the injection site and the brain. This bottleneck to poliovirus spread has been observed previously in both humans [14,15] and in mice [15] (J. K. Pfeiffer, unpublished data).
Figure 2 Viral Competition Assay in Poliovirus Receptor-Expressing Mice
(A) Diagram of poliovirus genome showing BstBI restriction enzyme sites within the coding region for 3D polymerase. Digestion of 1,024-bp 32P end-labeled (*) RT-PCR products yields a 558-bp labeled band for wild-type and a 396-bp labeled band for 3D-G64S. 6-wk-old mice were given IM injections with a 1/2 mixture of wild-type to 3D-G64S poliovirus (2 × 107 PFU total virus per mouse). Tissues were harvested when animals first developed paralysis, between days 3 and 5. PCR products were end-labeled with 32P during synthesis by inclusion of a radiolabeled downstream primer and products were digested with BstBI before electrophoresis on a denaturing polyacrylamide gel. Products from muscle samples from the IM infections are shown in (B) while products from brain samples of the IM infections are shown in (C). In (D), 2-wk-old mice were given IC injections with a 1/1 mixture of wild-type to 3D-G64S virus, and brain tissue was harvested when mice became lethargic or paralyzed. For (B), (C), and (D), representative gels are shown. In (E), (F), and (G) all mouse data obtained from the types of experiments shown in (B), (C), and (D), respectively, are quantified, and show the total number of mice whose tissues contained predominately wild-type virus, 3D-G64S virus, or both. The total number of mice per condition are as follows: IM-muscle samples, 31 mice; IM-brain samples, 54 mice; IC-brain samples, 10 mice.
DOI: 10.1371/journal.ppat.0010011.g002
To ensure that both viruses were able to replicate and be detected in the brain at the same time, intracerebral (IC) inoculations were performed using a 1:1 input ratio of wild-type:3D-G64S virus. Samples from the brains of these mice (Figures 2D and 2G) showed that both viruses were able to replicate in the brain when inoculated into this tissue. Therefore, the preferential spread of wild-type virus from the infected muscle to the brain did not result from an inability of 3D-G64S virus to replicate in murine brain tissue.
3D-G64S Displays a Slight Growth Defect in Cultured Cells
The decreased virulence of the high-fidelity 3D-G64S mutant virus (Figure 1) could result from: (1) an advantage conferred to the wild-type virus by the increased complexity of its quasispecies population, or (2) a growth disadvantage conferred to the 3D-G64S mutant virus by some other property of the mutant polymerase than its increased fidelity. To monitor the replication of high-fidelity 3D-G64S poliovirus in tissue culture, several kinds of viral growth assays were performed. After infection of HeLa cell monolayers with wild-type and 3D-G64S virus at a multiplicity of infection (MOI) of 10 PFU/cell, the growth curves of single-cycle infections were found to be indistinguishable (Figure 3A). Furthermore, when wild-type and 3D-G64S virus growth was compared in plaque assays (Figure 3B), the plaques were of similar size at all temperatures tested, indicating equivalent growth of the viruses for several rounds of replication in HeLa cells.
Figure 3 Growth of Wild-Type and 3D-G64S Poliovirus in HeLa Cells
(A) Single-cycle growth curve. 2 × 106 HeLa cells were infected at an MOI of 10 PFU/cell with either wild-type or 3D-G64S viruses at 37 °C. Cell-associated virus was harvested at indicated times and titered by plaque assay.
(B) Plaque assays. Wild-type or 3D-G64S poliovirus was plated on HeLa cell monolayers and incubated under an agar overlay for 48 h at indicated temperatures before staining the cells with crystal violet.
(C) Competition assay titration with plasmid DNA. Plasmid DNAs that encode wild-type and 3D-G64S poliovirus were diluted and mixed at the ratios indicated: for example, 1/100 denotes a ratio of 0.1 ng of wild-type viral cDNA to 10 ng of 3D-G64S cDNA. End-labeled PCR products were digested with BstBI and were electrophoresed on denaturing polyacrylamide gels.
(D) Competition assay in HeLa cells infected with wild-type and 3D-G64S virus. HeLa cells (2 × 106) were infected at an MOI of 0.1 or 10 PFU/cell, as indicated, with wild-type virus, 3D-G64S virus, or both, and cells were harvested 5 h after infection. RNA was extracted, subjected to RT-PCR as in (C), and digested with BstBI. Control bands “uncut” and “mix” were amplified from cDNAs. Products from MOI 0.1 single infections with wild-type, 3D-G64S- and mock-infected cells are shown, as are mixed infections with a 1/1 ratio of wild-type and 3D-G64S virus (MOI of 0.1 PFU/cell or 10 PFU/cell for each virus) and a 1/2 ratio (MOI of 5 and 10 PFU/cell, respectively). Products were quantified by Phosphorimager and the percentage 3D-G64S is shown.
DOI: 10.1371/journal.ppat.0010011.g003
To test for a defect in RNA accumulation in the 3D-G64S mutant virus, we performed competition assays in HeLa cells with wild-type and G64S mutant virus and monitored the relative proportions of RNA after single-cycle infections. To determine the sensitivity of the competition assay, plasmid DNAs that contained wild-type and 3D-G64S cDNAs were mixed at various ratios and the 3D polymerase coding regions were amplified and end-labeled by PCR. The BstBI-digested products from the wild-type and 3D-G64S genomes (Figure 2A) displayed equivalent intensities when obtained from a 1:1 mixture of wild-type and 3D-G64S mutant cDNAs (Figure 3C). At 1:10 input ratios, however, the bands derived from the minority species were barely visible, and disappeared altogether at 1:100 input ratios. Therefore, this assay is capable of detecting both the wild-type and 3D-G64S-derived products, as long as the input amounts are within 10-fold of each other (Figure 3C). When the relative amounts of RNA accumulation of wild-type and 3D-G64S virus from single and mixed infections of HeLa cell monolayers were monitored, a slight RNA accumulation defect for the 3D-G64S virus was observed. As shown in Figure 3D, a 1:1 input ratio of the two viruses displayed a decreased accumulation of 3D-G64S RNA relative to that of wild-type. Using twice as much 3D-G64S virus in the mixed infection equalized the bands (Figure 3D). Therefore, although no defect was observed in viral growth assays in HeLa cells, 3D-G64S virus displayed a slight RNA accumulation defect during a single cycle of replication.
Viral growth in HeLa cells, however, may not mirror replication capacity in murine cells, and subtle growth defects may be more apparent in primary cells. Therefore, we compared the growth of wild-type and 3D-G64S viruses in mouse embryo fibroblasts (MEF) derived from PVR mice [9]. In single-cycle growth curves in these primary cells, 3D-G64S virus showed slightly reduced accumulation at early time points (Figure 4A), displaying 2- to 4-fold titer reduction compared to wild-type virus at 3, 4, and 5 h post-infection. Despite this slight growth defect in a single-cycle growth curve, wild-type and 3D-G64S viruses displayed similar plaque sizes in the PVR-MEF (Figure 4B). To test whether this slight growth defect would prevent the 3D-G64S virus from propagating during multiple passage in the PVR-MEF, RNA from serially passaged mixtures of wild-type and G64S virus was analyzed. PVR-MEF were inoculated with a 1:1 input ratio of mutant:wild-type virus at high MOI (5–30 PFU/cell). As shown in Figure 4C, the 3D-G64S and wild-type product amounts were comparable over three rounds of serial passage, indicating no significant growth defect exists for the mutant virus in PVR-MEF even during multiple rounds of infection. It should be noted that poliovirus replication is 10- to 100-fold less efficient in MEF compared with HeLa cells (Figures 3A, 4A and 4B); therefore, MEF represent a stringent environment, where growth defects may be more apparent. The fact that 3D-G64S virus matches the replication of wild-type virus in MEF indicates the mutant virus has only a slight growth defect in cultured cells.
Figure 4 Growth of Wild-Type and 3D-G64S Poliovirus in Primary PVR-MEF
(A) Single-cycle growth curve. 2 × 105 PVR-MEF were infected at an MOI of 10 PFU/cell with either wild-type or 3D-G64S viruses at 37 °C. Cell-associated virus was harvested at indicated times and titered by plaque assay.
(B) Plaque assays. Wild-type or 3D-G64S poliovirus was plated on HeLa or PVR-MEF monolayers and incubated under an agar overlay for 72 h at 37 °C before staining the cells with crystal violet. The amount of virus plated, according to HeLa titration, is shown underneath as “input.”
(C) Serial passage competition assay in PVR-MEF. 3D-G64S and wild-type virus were mixed in a 1:1 ratio and used to infect PVR-MEF at an MOI of 10 PFU/cell at 37 °C. At 6 h post-infection, cell-associated RNA and virus were harvested, and the virus was used to infect fresh cells at high MOI (5–30 PFU/cell). This cycle was repeated a total of three times; BstBI digestion assay products are shown for passages 1, 2, and 3 (P1, P2, P3), and the Phosphorimager-quantified percentage of the G64S product is shown.
DOI: 10.1371/journal.ppat.0010011.g004
Effect of a Defined Selective Pressure on the Fitness of 3D-G64S Mutant Virus: Characterization of the Selective Pressure
The reduced pathogenesis of the 3D-G64S mutant virus could result, as stated previously, from a growth defect or the quasispecies restriction caused by increased polymerase fidelity. In the latter case, the increased mutation rate in the wild-type virus population would presumably aid in viral spread in the diverse environments encountered during spread through the animal. We do not know what these diverse selection pressures might be; therefore, we chose to exert a defined selective pressure on the viral mixtures of wild-type and G64S mutant virus and inquire whether the high-fidelity phenotype of the 3D-G64S virus became a liability. Specifically, a single amino acid change (F28S) in the coding region for poliovirus protein 2C is known to confer temperature sensitivity to the virus in cell culture [16] (J. K. Pfeiffer, unpublished data). We engineered two versions of the F28S amino acid change: one version that contained a single nucleotide substitution [2C-F28S(1m)], which should be able to revert readily during propagation, and a second version that contained three nucleotide substitutions [2C-F28S(3m)], to reduce the probability of reversion to the wild-type codon (Figure 5). These two versions of the F28S mutation were cloned into wild-type and 3D-G64S backgrounds and the resulting viruses were plaque-purified and propagated at 32 °C, the permissive temperature. No differences in the rates of growth of viruses that contained the 2C-F28S(1m) and 2C-F28S(3m) mutations were observed during single-cycle infections in cell culture (J. K. Pfeiffer, unpublished data). As shown in Figure 5, the presence of either the single-nucleotide (1m) or the three-nucleotide (3m) 2C-F28S mutation did not change the ability of the 3D-G64S virus to compete with virus that was wild-type at the 3D locus during serial passage in HeLa cells.
Figure 5 Serial Passage Competition Assay in HeLa Cells for Mixtures of Wild-Type and 3D-G64S Viruses Containing the 2C-F28S Temperature-Sensitive Attenuating Mutation
One-to-one mixtures of 3D-G64S to WT virus containing the indicated 2C-F28S mutations were used to infect HeLa cells at an MOI of 0.1 PFU/cell at 32 °C. At 12 h post-infection, cell-associated RNA and virus were harvested, and the virus was titered to determine input for the next passage. This cycle was repeated three to five times, and representative gels of digestion products, with maps indicating the mutations at the 2C locus in the mixtures of viruses that were wild-type and 3D-G64S at the 3D locus, are shown: (A) wild-type, (B) 2C-F28S(1m), (C) 2C-F28S(3m). X denote the numbers of nucleotide substitutions at the 2C-F28S site, while O denotes the presence of the 3D-G64S mutation.
DOI: 10.1371/journal.ppat.0010011.g005
To determine the effect of the 2C-F28S(1m) and 2C-F28S(3m) mutations on pathogenesis and growth in mice, and the role of the probability of genetic reversion in these processes, we inoculated PVR mice intramuscularly with the three different mixtures of viruses, designated by their 2C allele, depicted in Figure 5. The three mixtures were generated as follows: wild-type, containing a 1:10 mixture of wild-type and 3D-G64S virus; 2C-F28S(1m), containing a 1:10 mixture of 2C-F28S(1m) and 2C-F28S(1m)/3D-G64S virus; and 2C-F28S(3m), containing a 1:10 mixture of 2C-F28S(3m) and 2C-F28S(3m)/3D-G64S virus (Figure 6A). The ratio of 1:10 (wild-type: 3D-G64S) at the 3D locus was chosen to enhance the detection of 3D-G64S-containing RNAs, should they compete poorly with virus that was wild-type at the 3D polymerase locus. 2-wk-old mice were inoculated by IM injection with the three mixtures of viruses and neuropathological symptoms were monitored as a function of time. Upon injection of 5 × 106 PFU, mice inoculated with virus mixtures that were wild-type at the 2C locus showed symptoms by the third day post-infection, whereas mice inoculated with the 2C-F28S(1m) virus mixture showed a small, but significant, delay in disease onset (Figure 6A). Even more strikingly, the 2C-F28S(3m) virus mixture was found to cause no disease at the input dose of 5 × 106 PFU (Figure 6A). At higher doses, however, the 2C-F28S(3m) virus mixture caused disease (Figure 6B).
Figure 6 Pathogenesis of Mixtures of Wild-Type and 3D-G64S Viruses Containing the 2C-F28S Temperature-Sensitive Attenuating Mutation
PVR mice were injected intramuscularly with 5 × 106 (A) or 2 × 108 (B) PFU of total virus. The viral inocula were 1/10 mixtures of the wild-type/3D-G64S derivatives to allow 3D-G64S viruses a chance to compete. For example, 2C-F28S(1m) was a mixture of 5 × 105 PFU of 2C-F28S(1m) and 4.5 × 106 PFU of 2C-F28S(1m)/3D-G64S, giving a total of 5 × 106 PFU. The numbers of mice in each group, as designated by the identity of the 2C allele, were: wild-type, 26 mice; 2C-F28S(1m), 32 mice; 2C-F28S(3m), 4 and 27 mice in (A) and (B), respectively. Asterisks denote time points where statistically significant differences (p < 0.05, determined by two-sample Student's t-test, [d 2, 0.00125; d 3, 0.00125; d 4, 0.0112]) were observed between 2C-F28S(1m) and wild-type viruses. (C) Sequence analysis of RT-PCR products from viral RNA pools (“input”) used to inoculate the mice shown in (A) and from muscle tissue after development of disease (“output”). For output samples, samples from at least three mice were sequenced, and a representative example for each is shown.
DOI: 10.1371/journal.ppat.0010011.g006
We hypothesized that the 2C-F28S mutation is highly attenuating, as shown by the profoundly reduced pathogenesis of the 2C-F28S(3m) virus mixture. However, because the 2C-F28S(1m) mutations could readily revert to a wild-type sequence, the 2C-F28S(1m) virus mixture caused much more disease than the 2C-F28S(3m) mixture. To test this hypothesis, the viral RNAs present in muscle tissue from diseased mice [wild-type and 2C-F28S(1m) from Figure 6A, and 2C-F28S(3m) from Figure 6B] were amplified and sequenced in the region of the 2C mutations. Portions of the resulting chromatograms, as well as sequences obtained from RT-PCR products of the viral RNAs in the input pools, are shown in Figure 6C. As predicted, the sequences near the site of the mutations at the 2C locus from the wild-type pool, and the 2C-F28S(3m) pool did not change during passage in the mouse. However, virus isolated from mice infected with the 2C-F28S(1m) pool showed a high frequency of reversion to wild-type sequence at the F28S(1m) codon (Figure 6C). Therefore, the increased pathogenesis of the F28S(1m) virus pool, relative to that of the F28S(3m) virus pool, was accompanied by direct reversion of the F28S(1m) mutation, implying that there was strong selective pressure for wild-type sequence at this codon.
Effect of a Defined Selective Pressure on the Fitness of 3D-G64S Mutant Virus: Growth in Mouse Tissue
To ask whether the presence of the strong selective pressure to revert the 2C-F28C mutation would decrease the competitiveness of 3D-G64S virus, we analyzed the relative ratios of 3D-G64S and wild-type RNA sequence at the 3D locus by analysis of muscle samples from many of the mice shown in Figure 6. For the mixture of viruses that were wild-type at the 2C locus (Figure 7A), the 3D-G64S genomes were more highly represented than genomes that were wild-type at the 3D locus, which was an expected result considering the 10-fold excess of the 3D-G64S virus in the inoculum. However, in the context of the “revertible” attenuating mutation, 2C-F28S(1m), the 3D-G64S products were poorly represented in the resulting population (Figure 7B). However, in the presence of the “non-revertible” 2C-F28S(3m) mutations, the 3D-G64S products were once again well-represented in the population (Figure 7C). These results, quantified in Figure 7D, argue that the high fidelity of the 3D-G64S virus decreases its fitness in the presence of a defined selective pressure, if errors introduced by the wild-type polymerase can bring about escape from that pressure.
Figure 7 Viral Competition Assay in the Context of the 2C-F28S Temperature-Sensitive Attenuating Mutations
Muscle tissue from the experiment shown in Figure 6 was processed and the BstBI digestion assay was performed. Representative gels of digestion products, with maps indicating the mutations at the 2C locus are shown: (A) wild-type, (B) 2C-F28S(1m), (C) 2C-F28S(3m). (D) Quantitation of the gels in A–C. The percent 3D-G64S is shown, and represents an average from 5–10 mice for each condition.
DOI: 10.1371/journal.ppat.0010011.g007
Discussion
It has been theorized that RNA viruses need high error rates to survive in complex environments [1]. With the isolation of a high-fidelity poliovirus, we were in a unique position to address this hypothesis.
In tissue culture, we found a slight RNA accumulation defect of 3D-G64S poliovirus in HeLa cells (Figure 3), which may result from the decreased rate of nucleotide incorporation observed in reactions with purified 3D-G64S polymerase [8]. In murine embryo fibroblasts derived from PVR-expressing mice, a slight growth defect was also observed in single-cycle infections (Figure 4A). Nevertheless, in both HeLa cells and PVR-MEF, the 3D-G64S could compete with wild-type virus over several rounds of serial passage (Figures 4 and 5). Our results differ from those of Arnold et al, where population sequencing suggested the 3D-G64S virus could not compete with wild-type virus even during one round of competition passage [8]. We do not know the basis for this discrepancy, but it could be due to differences in the virus clones used, the method of detection, or the cell lines.
In mouse infections, the 3D-G64S virus was less pathogenic than wild-type virus, as measured by the time of symptom onset and the number of animals acquiring disease (Figure 1). When mice were inoculated with a mixture of the wild-type and 3D-G64S viruses, both were able to replicate in muscle and both were capable of spreading to the brain, although 3D-G64S virus spread to the brain less often than wild-type virus. Interestingly, we found that generally, only one of the two input viruses was present in the brain of infected animals. This so-called poliovirus “bottleneck effect” has been observed before, both in mice and in humans [14,15]. Other pathogens also encounter bottlenecks during spread within a host. The plant RNA virus Cucumber mosaic virus undergoes striking bottlenecks during spread within an infected plant [17], and Salmonella strains and Pneumocystis carnii experience bottlenecks during spread in rodents [18–20]. We will describe the nature of the poliovirus bottleneck effect in more detail in the future.
The virulence and growth defects of 3D-G64S poliovirus in mice could be due to its enhanced fidelity or to the slight growth defect documented here. To determine whether enhanced fidelity could play a role in the fitness of poliovirus, we competed 3D-G64S virus against virus that was wild-type at the 3D locus in the context of a defined selective pressure, a revertible attenuating point mutation in the viral 2C coding region. Indeed, the 3D-G64S virus was unable to compete with virus that was wild-type at the 3D locus in the presence of a revertible version of the 2C mutation. This did not result from a growth defect of the 3D-G64S virus, because, in the absence of the 2C mutation, or in the presence of a non-revertible version of the 2C mutation, the 3D-G64S-containing virus could compete well with virus that was wild-type at the 3D polymerase locus (Figure 7).
The results presented here argue that a high-fidelity poliovirus variant that contains the 3D-G64S mutation in the coding region for its RNA-dependent RNA polymerase is less able to revert attenuating point mutations. Therefore, in the context of known, harmful, mutations, a virus that is wild-type at the 3D polymerase locus is more fit than the higher-fidelity 3D-G64S virus. Most of the mutations made during viral growth are likely to be deleterious, and it is possible that reversion of attenuating mutations is important for survival of RNA viruses. However, why would it not be advantageous for a virus to encode a higher-fidelity polymerase and simply not to make so many errors in the first place?
Genetic change can be very rapid in the presence of a low-fidelity polymerase (Figure 6C). In a complex environment such as within a mouse, the ability to generate a quasispecies may allow virus populations to respond to the different environments they encounter during spread between tissues and in reaction to changing pressure of the host immune response. In monkeys infected with a vaccine strain of poliovirus, distinct mutations were observed to occur within 2 d post-infection, only to revert to the inoculated sequence later in the course of disease [21]. Furthermore, HIV is known to change its cell tropism from macrophage-tropic to T cell-tropic, during spread in many infected individuals [22–23]. It would be interesting to determine whether the pathogenesis of HIV is reduced by increasing the fidelity of its replicative machinery. We have shown that, in the presence of a defined selective pressure, the increased fidelity of the 3D-G64S mutant virus decreases its fitness. Given the slight growth defect of the 3D-G64S mutant virus, we further suggest that, the decreased virulence of high-fidelity 3D-G64S mutant poliovirus in the mouse is due, at least in part, to its relative inability to generate variants suitable for the variety of selection pressures faced during the normal course of infection of a complex organism.
Materials and Methods
Cells, mice, and viruses.
HeLa cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% calf serum [24]. PVR mice expressing the human poliovirus receptor (CD155) from a β-actin promoter were obtained from R. Andino (University of California at San Francisco) [9]. Primary MEF were harvested from PVR mice. Day 14–18 embryos were harvested, washed in phosphate-buffered saline, the heads and viscera were removed, and the body was minced with a sterile razor blade. The slurry was trypsinized and homogenized with a pipet for 10 min and then plated in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum. Experiments with MEF were performed between passages two and five (see below). Mahoney serotype 1 poliovirus was grown from a single plaque generated by transfection of the viral cDNA clone [25]. Individual plaques were grown into high-titer virus stocks by infecting monolayers of HeLa cells at 37 °C. Plasmids that encoded cDNAs for temperature-sensitive mutant viruses were transfected at 32 °C, and small plaques were picked 4 d later. In all cases, care was taken to ensure that the specific infectivities were comparable to wild-type plasmid cDNAs transfected at the same time. Multiple individual plaques were chosen from the transfections and propagated for three cycles of infection at 32 °C to generate high-titer stocks. The phenotypes of these stocks were then compared to those of the original plaque isolates to ensure that no additional mutations were acquired during propagation.
Plasmid construction.
The 3D-G64S mutation in the viral 3D-coding region was introduced by site-specific mutagenesis, which added a BstBI restriction enzyme site (sense primer: 5′ATTTTCTCCAAGTACGTTTCGAACAAAATTACTGAAGTG 3′). This BstBI-marked 3D-G64S derivative was engineered to contain three nucleotide substitutions (GGT to TCG) to minimize reversion at this site. A mutation in the 2C coding region, F28S, which confers temperature sensitivity [16], was introduced into both the 3D-G64S and wild-type poliovirus clones in two forms: one with nucleotide substitution [TTC to TCC, termed 2C-F28S(1m)] and the other with three nucleotide substitutions [TTC to AGT, termed 2C-F28S(3m)]. In each case, the entire PCR-generated region was confirmed by sequencing.
Tissue culture infections.
For single-cycle viral growth curves, 2 × 106 HeLa cells or 2 × 105 PVR-MEF were infected at 37 °C at an MOI of 10 PFU/cell, harvested at various times post-infection, and lysed by repeated freeze-thawing. Cell-associated virus was quantified by plaque assay on HeLa cell monolayers [7]. Each growth curve was performed at least twice and results were similar in all replicates. For competition experiments, as shown in Figure 3D, 2 × 106 HeLa cells were infected at an MOI of 0.1 or 10 PFU/cell with wild-type virus, 3D-G64S virus, or both viruses, at varying ratios for 5 h at 37 °C. Cells were harvested and RNA was extracted using TRIZOL (Invitrogen, Carlsbad, California, United States) according to the manufacturer's instructions. For serial passage competition experiments shown in Figure 5, 2 × 106 HeLa cells were infected at 32 °C with a 1:1 mixture of wild-type and 3D-G64S viruses at low MOI (0.1 PFU/cell). At 12 h post-infection, cell-associated virus and RNA were harvested (as above), and the cycle was repeated three to five times, titering the virus each time to determine input for the next cycle. Serial passage experiments in PVR-MEF (Figure 4C) were performed as above, except that 2 × 106 MEF were infected at a high MOI (5–30 PFU/cell) at 37 °C, and cell-associated virus and RNA were harvested at 6 h post-infection.
Mouse infections and tissue processing.
All animal work was performed according to protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care. For IM inoculations, 50 μl of virus was injected into the thigh muscle of the right hind leg of mice that were 2–10 wk old, as indicated, using a 28–gauge needle. Before IC inoculations, 2-wk old animals were anesthetized by intraperitoneal injection with 11 μl of a solution containing 2.5% avertin/g animal weight. Once animals were unconscious, 20 μl of virus stock was injected into the cerebrum through the right temple. Mice were observed for symptoms twice per day, and were euthanized at the first sign of disease, weakness in the inoculated limb, or, for IC-inoculated mice, ruffled fur and general lethargy. Tissues, including the inoculated leg muscle, brain, or both, were harvested and frozen at −80 °C until studied. Tissues were ground into a fine powder under liquid nitrogen using a mortar and pestle. RNA was extracted from approximately 300 mg of total tissue using TRIZOL (Invitrogen) according to the supplied protocol.
Reverse transcription-PCR digestion assay.
RNA was reverse transcribed using 5–10 μg of RNA per cDNA synthesis reaction using Superscript II reverse transcriptase (Invitrogen) and reverse primer 5′ATAGGTTCCCAGAA GCCATTCTC3′ for the 3D polymerase-coding region or 5′CCATGTCGAACGCAGAGCGCCTGG3′ for the 2C-coding region. Twenty percent of the reverse transcription reaction was PCR amplified using 5′CCGCATTTGGAAGTGGATCTACTCAGCAG3′ and 5′CCATGTCGAACGCAGAGCGCCTGG3′ primers for the 2C-coding region and primers 5′GCTTCACCTGGTGAAAGCATTGTGATCG3′ and 5′ATAGGTTCCCAGAA GCCATTCTC3′ primers for the 3D-coding region. The 3D reverse primer was end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, Massachusetts, United States), and 125 μCi [γ-32P] ATP (Perkin-Elmer, Boston, Massachusetts, United States), and was used at a 1:5 ratio with the unlabeled 3D reverse primer in PCR reactions. The PCR reactions contained 1 unit Taq (Qiagen, Valencia, California, United States) and were done according to the supplier protocol, using a buffer that contained 50 μM potassium glutamate, 20 μM potassium HEPES (pH 8.4), 3 μM MgCl2 and 0.1 mg/ml BSA. The PCR products were digested with BstBI (New England Biolabs) at 65 °C for 3–16 h, and products were ethanol-precipitated in the presence of glycogen (Invitrogen). Pellets were resuspended in denaturing gel loading dye (80% formamide, 10 mM EDTA, 0.025% bromophenol blue and xylene cyanol, 0.1% SDS), heated to 80 °C for 2 min, and loaded on gels that contained 7% polyacrylamide and 8M urea. Gels were dried under vacuum on DE81 paper (Whatman, Florham Park, New Jersey, United States) and exposed to PhosphorImager screens.
Sequence analysis.
The cDNAs products from viral RNAs purified using the QiaAmp kit (Qiagen) or from cDNA-encoding plasmids were PCR-amplified for sequence analysis at the F28S locus. The PCR reactions were performed as above, except that no radiolabel was added. Products were run on 1% agarose gels and products were excised, purified using the Qiaex II kit (Qiagen), and sequenced commercially (Sequetech, Mountain View, California, United States).
Accession Number
The Genbank (http://www.ncbi.nlm.nih.gov/entrez) accession number for serotype 1 poliovirus is NC002058.
We thank Jomaquai Jenkins and Elizabeth Stillman for training on animal work. We are grateful to Shane Crotty and Raul Andino for the provision of poliovirus receptor-expressing mice. This work was supported by a National Institutes of Health grant (#AI-48756), and JKP is a Rebecca Ridley Kry Fellow of the Damon Runyon Cancer Research Foundation.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. JKP and KK conceived and designed the experiments. JKP performed the experiments. JKP and KK analyzed the data, and wrote the paper.
Abbreviations
bpbasepair
ICintracerebral
IMintramuscular
MEFmouse embryo fibroblast
MOImultiplicity of infection
PFUplaque-forming unit
PVRpoliovirus receptor
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References
Domingo E Holland JJ 1997 A virus mutations and fitness for survival Annu Rev Microbiol 51 151 178 9343347
Drake JW Charlesworth B Charlesworth D Crow JF 1998 Rates of spontaneous mutation Genetics 148 1667 1686 9560386
Crotty S Maag D Arnold JJ Zhong W Lau JY 2000 The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen Nat Med 6 1375 1379 11100123
Domingo E Menendez-Arias L Holland JJ 1997 RNA virus fitness Rev Med Virol 7 87 96 10398474
Crotty S Cameron CE Andino R 2001 RNA virus error catastrophe: Direct molecular test by using ribavirin Proc Natl Acad Sci U S A 98 6895 6900 11371613
Biebricher CK Eigen M 2005 The error threshold Virus Res 107 117 127 15649558
Pfeiffer JK Kirkegaard K 2003 A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity Proc Natl Acad Sci U S A 100 7289 7294 12754380
Arnold JJ Vignuzzi M Stone JK Andino R Cameron CE 2005 Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase J Biol Chem 280 25706 25716 15878882
Crotty S Hix L Sigal LJ Andino R 2002 Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: Age-dependent paralysis and a mucosal route of infection J Gen Virol 83 1707 1720 12075090
Ren RB Costantini F Gorgacz EJ Lee JJ Racaniello VR 1990 Transgenic mice expressing a human poliovirus receptor: A new model for poliomyelitis Cell 63 353 362 2170026
Koike S Taya C Aoki J Matsuda Y Ise I 1994 Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene–Influence of the transgene expression on pathogenesis Arch Virol 139 351 363 7832641
Ohka S Yang WX Terada E Iwasaki K Nomoto A 1998 Retrograde transport of intact poliovirus through the axon via the fast transport system Virology 250 67 75 9770421
Ohka S Nomoto A 2001 Recent insights into poliovirus pathogenesis Trends Microbiol 9 501 506 11597452
Hovi T Lindholm N Savolainen C Stenvik M Burns C 2004 Evolution of wild-type 1 poliovirus in two healthy siblings excreting the virus over a period of 6 months J Gen Virol 85 369 377 14769894
Georgescu MM Balanant J Ozden S Crainic R 1997 Random selection: A model for poliovirus infection of the central nervous system J Gen Virol 78 1819 1828 9266975
Crowder S Kirkegaard K 2005 Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses Nat Genet 37 701 709 15965477
Li H Roossinck MJ 2004 Genetic bottlenecks reduce population variation in an experimental RNA virus population J Virol 78 10582 10587 15367625
Nevola JJ Stocker BA Laux DC Cohen PS 1985 Colonization of the mouse intestine by an avirulent Salmonella typhimurium strain and its lipopolysaccharide-defective mutants Infect Immun 50 152 159 3899931
Uzzau S Gulig PA Paglietti B Leori G Stocker BA Rubino S 2000 Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice FEMS Microbiol Lett 188 15 18 10867227
Keely SP Cushion MT Stringer JR 2003 Diversity at the locus associated with transcription of a variable surface antigen of Pneumocystis carinii as an index of population structure and dynamics in infected rats Infect Immun 71 47 60 12496148
Lu Z Asher DM Levenbook IS Chumakov KM 1996 Succession of mutations in the Sabin strain of type 3 poliovirus replicating in the central nervous system of monkeys Virology 220 285 289 8661379
Connor RI Sheridan KE Ceradini D Choe S Landau NR 1997 Change in coreceptor use coreceptor use correlates with disease progression in HIV-1–infected individuals J Exp Med 185 621 628 9034141
Glushakova S Grivel JC Fitzgerald W Sylwester A Zimmerberg J 1998 Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency Nat Med 4 346 349 9500611
Kirkegaard K Baltimore D 1986 The mechanism of RNA recombination in poliovirus Cell 47 433 443 3021340
Racaniello VR Baltimore D 1981 Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome Proc Natl Acad Sci U S A 78 4887 4891 6272282
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1620183610.1371/journal.pbio.0030357Research ArticleBioinformatics/Computational BiologyEvolutionGenetics/Genomics/Gene TherapyHomo (Human)PrimatesEukaryotesEmergence of Young Human Genes after a Burst of Retroposition in Primates Emergence of Young Human RetrogenesMarques Ana Claudia
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Kaessmann Henrik [email protected]
1
1Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland,2Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandWolfe Ken Academic EditorUniversity of DublinIreland11 2005 11 10 2005 11 10 2005 3 11 e35711 7 2005 19 8 2005 Copyright: © 2005 Marques 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.
Retrocopied Genes May Enhance Male Fitness
The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past ∼63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome−derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.
In humans, retroposition--integration into the genome of DNA reverse transcribed from mRNA--has contributed to the formation of recent functional genes selected to enhance male germline function.
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Introduction
Together with more subtle genetic modifications such as gene expression changes and point substitutions, new genes with novel functions may have significantly contributed to the evolution of new phenotypes specific to humans and their closest evolutionary relatives. New duplicate genes may originate through (segmental) gene duplication by intra- or interchromosomal transposition of gene-containing segments [1,2]. Another mechanism, retroposition, generates new intronless gene copies (retrocopies) by reverse transcription of mRNAs derived from source genes (“parental” genes), followed by reintegration of the resulting cDNA in the genome [2,3]. Retroposition was commonly thought to generate nonfunctional gene copies (retropseudogenes) that accumulate disablements such as premature stop codons and frameshift mutations [4], because the copied mRNA is generally lacking regulatory elements. However, we and others have recently shown that retroposition has generated a significant number of new functional genes (retrogenes) in mammalian and invertebrate animal genomes [3,5,6].
Multiple studies have suggested a high rate of retroposition on the primate and rodent lineages [7−9], probably driven by the activity of L1 retrotransposable elements [10]. Thus, retroposition may also have provided abundant raw material for the formation of new genes on the primate lineage leading to humans, potentially generating many more retrogenes than the four primate-specific retrogenes (present in the human genome) with functional roles and/or expression in testis, brain, and lymphocytes previously described [11−14].
To assess the importance of retroposition for the creation of new genes on the primate lineage leading to humans, we systematically screened the human genome for retrogenes that emerged during the primate burst of retroposition. Our results suggest an important role of retroposition in the formation of new genes and phenotypes in the recent evolution of the human genome.
Results
Age Distribution of Human Retrocopies
We identified 3,951 retrocopies (and their corresponding parental genes) in the human genome using a refinement of a previously published procedure [5] (see Materials and Methods). Among these, 705 retrocopies (∼18%) are found to be “intact,” i.e., they show no disablements such as premature stop codons or frameshift mutations when compared to the open reading frame (ORF) of their parental genes. To assess the age distribution of retrocopies, we calculated nucleotide divergence at silent sites (K
S) between retrocopies and their parental genes (Figure 1). Assuming neutral mutation rates of 1−1.3 × 10−9 substitutions per site per year [15], the high number of retrocopies with K
S ≍ 0.1 suggests that the burst of retroposition reached its peak approximately 38−50 million years ago (MYA) on the primate lineage, in agreement with previous estimates [7,8]. The vast majority of retrocopies (91%) also show a divergence at silent sites much lower than that observed between human and mouse genes (Figures 1 and S1), indicating that they arose after the human−mouse split. Therefore, our data are consistent with a high retroposition activity on the primate lineage.
Figure 1 KS Distribution for 3,951 Retrocopies
The peak at K
S ≍ 0.1 suggests a burst of retroposition on the primate lineage (see also text and Figure S1). Retrocopies with K
S > 1 were pooled in a single bin.
Rate of New Retrogene Formation
To estimate the number of recent human retrogenes, we compared signatures of selective constraint between intact, potentially functional retrocopies, and retropseudogenes (assumed to be nonfunctional, i.e., evolving neutrally). To this end, we calculated the ratio of nonsynonymous to synonymous substitutions per site (K
A/K
S) for retrocopy/parental gene pairs with a synonymous divergence of less than 0.15. This value approximately reflects the deepest neutral divergence in the primate tree between humans and the most divergent extant primate lineage [16,17], the lemurs, and corresponds to around 63 million years of primate evolution [18].
This analysis reveals a difference in the K
A/K
S distributions between intact copies and retropseudogenes (which may show low K
A/K
S ratios by chance), with a highly significant excess of intact copies for K
A/K
S < 0.5 (Figure 2; p < 10−6, Fisher's exact test). K
A/K
S significantly less than one is indicative of purifying selection [19]. However, in a pairwise analysis, where K
A/K
S reflects the average selective constraint on the retrocopy and parental gene, K
A/K
S < 0.5 is indicative of purifying selection (i.e., K
A/K
S < 1) on both copies [5]. The 16% excess of intact retrocopies relative to retropseudogenes at K
A/K
S < 0.5 corresponds to approximately 76 retrogenes that were fixed on the primate lineage leading to humans through natural selection in the past 63 million years.
Figure 2 KA
/K
S Distributions for 475 Intact Retrocopies and 1,554 Retropseudogenes with K
S < 0.15
Note that we tested that the differences observed for K
A
/K
S < 0.5 are not explained by differences in GC content (see Materials and Methods for details). The bin with K
A
/K
S > 1.5 includes estimates where K
S = 0 (K
A
/K
S = ∞).
Based on a subset of the data for which a mouse ortholog of the human parental gene is available as an outgroup, we performed a similar analysis to calculate the K
A/K
S ratio on the retrocopy lineage itself (Figure S2). Again, we observed a significant excess (p < 2 × 10−3) of intact retrocopies with low K
A/K
S values. When we extrapolate this excess to the whole dataset (475 intact retrocopies with K
S < 0.15), this indicates that approximately 57 retrogenes in the human genome emerged in primates. This result is similar to the estimate based on the whole dataset using the pairwise K
A/K
S approach.
Together, these analyses suggest that approximately one retrogene per million years has emerged on the primate lineage leading to humans. It should be noted that the estimates based on this approach are restricted to cases with low K
A/K
S values averaged over the entire sequence, despite the fact that retrogenes may be found with higher K
A/K
S values due to the action of positive selection, a neutral phase of evolution upon emergence, or both (see Discussion).
Functional Retrogenes
To identify and characterize individual functional retrogenes in the human genome that emerged recently in primate evolution, we selected 38 intact retrocopies with low divergence at silent sites from their parental genes (K
S < 0.15) for further study (Table S1). To obtain an unbiased view of new retrogene formation, we chose these retrocopies independent of their average pairwise K
A/K
S values, as new genes may show high, intermediate, or low K
A/K
S values, depending on the type and extent of selection acting after the duplication event [3]. We determined the age of the 38 retrocopies by screening for their presence or absence in eight primate genomes.
This phylogenetic dating approach revealed retrocopies that emerged throughout primate evolutionary history (Table S1). For instance, we identified five retrocopies present in all Old World primates, five hominoid-specific retrocopies, and six copies unique to humans. Our dating revealed that the PGAM3 retrogene, previously shown to have been shaped by positive selection [11], originated recently in the ancestor of humans and the African apes, less than 14 MYA [18]. We also found that the PABP3 retrogene, for which a function in testis was recently demonstrated [20], emerged in primates.
In order to identify functional retrogenes among these dated retrocopies, we used an approach that combines comparative genomic sequencing and evolutionary simulations. First, we selected only retrocopies with a minimum sequence length (>600 bp) and age (>8 million years; i.e., presence in humans and African apes), characteristics estimated to provide sufficient statistical power for the simulation approach (see Materials and Methods). We sequenced these copies in all species carrying them. Sequence alignments show that eight of these 23 retrocopies are intact in all species, whereas the remaining copies carry one or more stop codons and/or frameshift mutations in one or more lineages (Table 1).
Table 1 Retrocopies Tested for Identifying Retrogenes
Next, we used a simulation approach (see Materials and Methods), which is based on the basic assumption that under neutrality, an intact retrocopy will accumulate deleterious mutations (stop codons or frameshifts) over time that will disrupt its ORF and may eventually preclude gene function, whereas under functional constraint, natural selection will prevent the accumulation of deleterious mutations in the retrocopy sequence.
Our simulation approach estimates the probability that a gene copy would have retained its ORF since the duplication event, in all or most species in which it is present, if it had evolved neutrally along all lineages of the species tree. In parallel, this approach tests whether the number of nonsynonymous substitutions that accumulated since the retroposition event along the different branches of the species phylogeny is consistent with neutral evolution.
The simulations revealed that seven retrocopies are unlikely to have remained intact in all (or most) species if they had evolved neutrally throughout their evolutionary history, even after correcting for multiple tests (p < 0.05; Table 1; Figures S3−S8). For example, a retrocopy on Chromosome 1 (RBMXL1), which we find to be intact in all six Old World primates carrying it, showed at least one disablement in each of 105 simulations during which the retrocopy was evolving neutrally after the duplication event (Figure 3A). This strongly suggests that the ORFs of all seven retrocopies were selectively preserved after duplication. Therefore, these copies very likely represent functional genes. Among these seven genes is also PABP3, for which a functional protein has been previously described [20], confirming that our simulation approach correctly predicts the functionality of recent genes.
Figure 3 Illustration of the Simulation Results Used to Support Functionality of Retrogenes for One Case (RBMXL1)
(A) Distribution of the number of disablements observed in 105 simulations of the RBMXL1 retrogene evolution under neutrality. The frequency distribution of stop codons is shown in white, and that of deleterious indels in black. All of the simulations showed at least one mutation disrupting the ORF (see text); simulations without stop codons all showed several frame-disrupting indels (the minimum number of such indels in each simulation is four).
(B) Nonsynonymous (N
A) and synonymous (N
S) substitutions observed in 105 simulations of neutral RBMXL1 retrogene evolution (diamonds). The black square indicates the observed nonsynonymous and synonymous substitutions in the RBMXL1 primate phylogeny.
(C) Ratio of nonsynonymous to synonymous substitutions in 105 simulations of RBMXL1 retrogene evolution in the primate lineages. The arrow indicates the observed ratio of nonsynonymous to synonymous substitutions in the RBMXL1 primate phylogeny.
Five of the seven copies accumulated fewer nonsynonymous substitutions than expected under neutrality, lending further support to the notion that these copies were preserved through natural selection (Figures 3B, 3C, and S3−S8). The remaining genes (NACA2 and GMCL2) may have been affected by positive selection at a subset of sites or may have experienced a period of relaxed selective constraint after duplication, rendering the average number of nonsynonymous substitutions not significantly different from that expected under a neutral evolutionary process.
The seven retrogenes identified here (Table 1) originated between 18 and 63 MYA [18] in the ancestor of hominoids (CDC14B2, eIF-2-gamma2, and GMCL2), Old World primates (RBMXL1 and KIF4b), and anthropoid primates (NACA2 and PABP3). On the basis of the functions of their parental genes (Table S1) or gene family members [20−28], these retrogenes can be predicted to play diverse functional roles in RNA processing and transport (RBMXL1), initiation of translation (eIF-2-gamma2 and PABP3), mRNA stability (PABP3), transcriptional regulation and protein biosynthesis (CDC14B2, GMCL2, and NACA2), and chromosome condensation and segregation (KIF4b).
Evolutionary Fate of New Retrogenes
Newly emerged retrogenes may evolve new functional roles through adaptive evolution of encoded proteins and/or by developing new spatial or temporal expression patterns. To trace the functional adaptation of the seven novel retrogenes identified here, we reconstructed phylogenetic trees based on the primate retrocopy and parental gene sequences and then scrutinized substitutional patterns on the retrogene branches in a maximum likelihood selection framework (Table 2). We also analyzed spatial gene expression patterns in 20 human tissues using RT-PCR.
Table 2 PAML Analyses for the Seven Retrogenes Identified by the Simulations Approach
Strikingly, we found that all seven retrogenes are exclusively or predominantly transcribed in testis, whereas transcripts of their parental genes were detected in all tissues tested (Figure 4). Three of these retrogenes (eIF-2-gamma2, RBMXL1, and KIF4b) derive from parental genes located on the X chromosome (see Table 1). Our selection analyses show that substitutional models allowing for sites under purifying selection and neutrally evolving sites on the retrogene lineages after the duplication event provide the best fit for these genes. In agreement with our simulations (Table 1), purifying selection has shaped most of their codons (54%−77%; see Table 2), which suggests that ancestral/parental protein functions are likely preserved in these genes.
Figure 4 Expression Pattern of Retrogenes and Parents Determined by RT-PCR
Black boxes indicate retrogenes; hatched boxes indicate parental genes. Note that in all cases testis expression of the retrogene was the strongest, as indicated by the semiquantitative PCR procedure (data not shown).
We have previously shown that X chromosomal genes in mammals generated a statistically significant excess of (autosomal) retrogenes relative to genes on other chromosomes [5]. One possible explanation for this pattern was that X chromosomal genes produced functional counterparts on autosomes that can be recruited during male meiosis when X chromosomal genes are silenced or during haploid stages of spermatogenesis [29,30]. Our findings that the coding sequences of the three recent X-derived genes identified here appear to be preserved by purifying selection at early stages of their evolution and that all genes are expressed (exclusively or most strongly) in testis (Figure 4) lend further support to this hypothesis. These retrogenes (eIF-2-gamma2, RBMXL1, and KIF4b) also support our previous notion that the generation of functional autosomal substitutes for genes on the X chromosome is an ongoing process [5]. In fact, this gene “movement” appears to have progressively enhanced male germline functions in primate evolution.
The four remaining genes stem from autosomes (see Table 1). Interestingly, the Drosophila ortholog (germ cell-less) of GMCL1—the parental gene of the hominoid-specific retrogene GMCL2 identified here—was shown to be essential for germ cell formation [26,31,32]. Furthermore, the mouse ortholog of GMCL1 [33] shows its highest expression in testis and has been shown to function as a transcriptional repressor [27]. Together, these results suggest that GMCL2 might have been preserved through male selection to enhance testis function in hominoids.
The other three retrogenes (CDC14B2, NACA2, and PABP3) show a statistically significant excess of nonsynonymous to synonymous substitutions (K
A/K
S > 1, p < 0.01) for a subset of sites (∼4.7%, ∼27.6%, and ∼28.4% of sites, respectively), indicative of accelerated protein evolution driven by positive Darwinian selection (see Table 2). This may suggest new or more adapted functional roles of these retrogenes in transcriptional regulation and protein biosynthesis in testis.
For PABP3, the maximum likelihood procedure identifies many codons as being positively selected (Table 2; Figure 5). Positively selected sites are present in all major domains of the PABP3-encoded protein such as the poly(A)-binding domain (Figure 5). Interestingly, a recent study not only supports the presence and functionality of the PABP3-encoded protein but also provides evidence for altered poly(A)-binding affinity [20]. However, positively selected sites particularly cluster in a region that was shown in PABP proteins to be involved in interactions with not only other proteins such as translation initiation factors but also viruses that target this region to shut off protein synthesis in the host cell (Figure 5) [20]. This may indicate that PABP3 has evolved new or enhanced protein interaction properties and/or an altered viral susceptibility compared to its parent, PABP1. Testis expression of PABP3 appears to be restricted to a later phase of spermatogenesis, during which the activity of PABP1 is repressed [20]. This suggests that PABP3 functionally replaces its parent to enhance translation and/or RNA stability during male meiosis.
Figure 5 Evidence for Positive Selection on PABP3 Codons
The rectangles correspond to the different domains of the PABP1 protein: RNA poly(A)-tail-binding domains (vertical lines), RNA-binding domains (hatched), protein–protein interaction domain (white), and PABP homo-oligomerization domain (black). Positions of positively selected codons with high posterior probabilities (>0.95) are indicated by arrows.
PABP3 provides an intriguing example of a retrogene that has adapted functionally by evolving a new spatial and temporal expression pattern as well as new protein properties relative to its parent. We have shown that this adaptation was driven by positive selection and occurred within the past ∼35−63 million years since the duplication event that gave rise to this gene in the common ancestor of anthropoid primates [18]. The high K
A/K
S ratio (2.8) on the human lineage after the separation from that of the chimpanzee (Figure S9) might suggest that adaptation shaped human PABP3 properties until recently in human evolution.
Discussion
Functional Retrogenes
Although gene duplications of different types have been prevalent in primate evolution, a more detailed picture with respect to the functionality of individual gene copies and their potential to contribute to human- and/or primate-specific phenotypes is only beginning to emerge [12,13,34−38]. Demonstrating the functionality of recently duplicated genes is hampered by their close similarity to original copies, which complicates both statistical and experimental inferences. Here, we have used a combination of comparative genomic sequencing, evolutionary analysis, and gene expression experiments to estimate the number of recent human genes that arose by retroposition and to characterize their functions.
Our study almost triples the number of described primate-specific retrogenes from four to 11 [11−14]. However, on the basis of a systematic analysis of selective signatures in retrocopy sequences, we estimate that approximately 57−76 retrogenes emerged during and after the primate burst of retroposition. This tentative estimate represents a lower bound for several reasons. First, our in silico approach (comparing K
A/K
S values between intact and retropseudogene copies) only detects copies with low K
A/K
S values, whereas newly emerged genes often show higher K
A/K
S values owing to the action of positive selection at a subset of sites (K
A/K
S > 1) and/or a neutral phase of evolution after duplication [3,12]. Second, retrocopies with disablements in their ORFs (as defined by their parents) are treated as pseudogenes in this analysis, although new retrogenes may emerge from truncated coding regions [3,13]. It is also known that new splicing signals in a coding region that contains frameshifts or premature stop codons may evolve to define a new intron or to generate chimeric transcripts with nearby or “host” genes [3]. Finally, duplicate “pseudogene” copies may play functional roles by virtue of their RNAs regulating closely related paralogous genes [39,40]. At any rate, our results suggest that in addition to other types of duplications [1], retroposition significantly contributed to new gene formation in primates.
Retrogenes and Male Functions
It is remarkable that all seven retrogenes identified in this report are expressed predominantly or exclusively in testis, whereas their parents are all expressed ubiquitously. A preliminary survey of retrocopy transcription using expressed sequence tag databases suggests that this observation may reflect a general pattern (data not shown). Several factors may contribute to this effect. For example, chromatin remodeling [41] and abundance of RNA polymerase II complexes during late phases of male meiosis [42] lead to a state of “hypertranscription” [43], which may allow retrocopies to become initially transcribed in testis. This may also have facilitated transcription of new genes arising from pericentromeric segmental duplications [44,45]. Thus, there is a mechanistic bias that may favor testis expression of new genes.
However, our results suggest that testis expression is often not merely a by-product of new retrogene formation but that natural selection may have favored the recruitment of testis-specific regulatory elements to enhance the beneficial effects of the initial mechanistically driven testis transcription. Consistently, we can infer a testis function for five of the seven primate retrogenes identified here and for two of the four previously identified retrogenes (TAF1L and UTP14C; [13,14]). Five retrogenes (eIF-2-gamma2, RBMXL1, KIF4b, TAF1L, and UTP14C) stem from the X chromosome and probably either substitute for their parental genes during male meiosis [30] or otherwise enhance male germline function [46]. For one retrogene (GMCL2), a function in sperm formation can be postulated based on studies of parental orthologs. Finally, PABP3 functionally adapted to late spermatogenesis both on the protein sequence level and by developing a highly specific expression pattern [20].
Sex- and reproduction-related genes are generally recognized as a class of rapidly evolving genes, particularly genes involved in male reproduction [47]. Possible causes include sperm competition, sexual conflict, and selection for reproductive isolation [48]. A comparison of the human and mouse genomes revealed an excess of lineage-specific expansions of genes related to reproduction as well as an accelerated protein evolution of such genes [49]. Together, these observations suggest that duplicate gene copies may have provided important raw material for rapid testis evolution in primates. Specifically, gene duplication may allow one copy of the duplicate pair to specialize in testis function, while the other is selectively preserved to sustain a role in somatic tissues [50−52]. Our data suggest that retroduplication may have provided a means to allow for such decoupling of functions in primates. Indeed, we show that selection to attain enhanced male germline function has progressively fixed and adapted retroposed gene copies on the primate lineage leading to humans.
Materials and Methods
Retrocopy screen
We retrieved all peptide sequences (categories: known and novel) from the Ensembl ([53]; http://www.ensembl.org/index.html) database (version 29). To screen for retrocopies, these peptide sequences were used as queries in translated similarity searches against the complete human genome (NCBI genome release 35) sequence using tBLASTn [54]. Adjacent homology matches were merged in a series of parsing steps using Perl scripts, combining only nearby matches (distance < 40 bp) that were likely not separated by introns. We also required that query and merged target sequences had significant similarity on the amino acid level (amino acid identity > 50%) and aligned to one another over more than 70% of the length of their sequence (minimum length: 50 amino acids). Next, we performed similarity searches of the merged sequences against all Ensembl genes (intron-containing and intronless) using FASTA. We kept only copies where the closest hit was an Ensembl peptide with multiple coding exons (putative parental gene). Merged sequences for which the closest match was an intronless gene were excluded from the data (e.g., to avoid intronless genes of other types such as olfactory receptor genes). We also confirmed the absence of introns in these retrocopies by mapping parental intron locations onto the alignments. We required that parental introns map within the alignments between parents and retrocopies and be larger than 80 bp. This threshold was chosen to ensure that real introns are missing in the retrocopies; 80 bp is larger than the gap size (40 bp) allowed in the merging step, it avoids mapping of small gaps in parental exons erroneously annotated as introns, and it takes into account that the majority of human introns are ∼80 bp or larger [55].
Samples
Primate DNA samples were mainly obtained from the E
CACC repository (Wiltshire, United Kingdom): chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), gibbon (Hylobates lar), Old World monkey (African green monkey, Cercopithecus aethiops sabaeus), and New World monkey (owl monkey, Aotus trivirgatus). Lemur (Lemur catta) and tupaia (Tupaia glis) DNA samples were obtained from Institut des Sciences de l'Evolution, Montpellier University 2.
PCR and sequencing reactions
PCR amplifications were performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany) using either Taq DNA Polymerase or ProofStart DNA Polymerase from Qiagen (Valencia, California, United States). PCRs were performed according to the instructions of the manufacturer. For sequencing, amplified PCR products were reamplified using a pair of nested primers. The resulting PCR products were purified using the MinElute PCR Purification Kit or QIAquick Gel Extraction Kit from Qiagen. From these PCR products, both strands of the retrogene coding sequence were determined using the BigDye 3.1 cycle sequencing kit (PerkinElmer, Wellesley, California, United States). The sequencing reactions were run on an ABI 3730 automated sequencer (Applied Biosystems, Foster City, California, United States). Parental and retrogene expression patterns were analyzed using PCR and a cDNA panel of 20 different human tissues. Experiments were repeated twice to confirm the expression pattern. Unique primer pairs were designed for both parental gene and retrogene, based on ClustalX alignments of parental and retrogene cDNA sequences. The cDNA panel was synthesized using the FirstChoice Human Total RNA Survey panel from Ambion (Austin, Texas, United States) and a SuperScript II First-Strand Synthesis System RT-PCR (Invitrogen, Carlsbad, California, United States). Reactions without reverse transcriptase were done in parallel as negative controls for all 20 tissues. RT-PCR amplifications were performed in a Mastercycler gradient (Eppendorf) using JumpsStart DNA Polymerase (Sigma-Aldrich, St. Louis, Missouri, United States) using standard conditions as recommended by the supplier. Products were purified using the MinElute PCR Purification Kit from Qiagen and sequenced using the same pair of primers. Obtained sequences for each retrogene were then aligned with both retrogene and parental gene sequences using ClustalX. To ensure that RT-PCR products were derived from the retrogene, nucleotides at diagnostic sites that discriminate between retrogene and parental gene were manually confirmed. All oligonucleotide sequences used for PCR and sequencing are available upon request.
Age of retrocopies
We estimated the age of retroposition events by calculating coding sequence divergence at synonymous sites (K
S) between each retrocopy and the corresponding parental gene. The same analysis was performed for parental genes and their mouse orthologs. Codon sequences were aligned on the basis of the translated sequence alignment using the EMBOSS package [56]. In all alignments, the coding sequence of the parental gene was used as a reference. Pairwise K
S statistics were estimated using the YN00 program of PAML [57] version 3.14. We note that the ages of retrocopies may be slightly underestimated by this approach, because silent sites are not always completely neutral ([58] and references therein).
Using a phylogenetic dating approach, we determined the age of individual retrocopies by screening for their presence or absence in primate genomes using PCR with primers flanking the insertion site. We confirmed that the insertion site in species not carrying the copy reflects the expected size of the ancestral state (before retrocopy insertion [12]). For five of the retrogenes analyzed in detail, the ancestral state of the insertion site was further confirmed by sequencing. For the two retrogenes (NACA2 and PABP3) present in all anthropoid primates (hominoids, Old World monkey, and New World monkey), we confirmed their absence in lemur and tupaia using several different primer pairs located in their coding regions, as the insertion site could not be amplified using primers in the flanking region.
Rate of retrogene formation
Pairwise K
A and K
S statistics for all retrocopies were estimated using the YN00 program of PAML [57] version 3.14. To estimate K
A/K
S on the retrocopy lineage itself, we performed the same analysis but compared the retrocopy and the ancestral sequence of the retrocopy at the time point of retroposition (estimated by a maximum likelihood procedure; using the codeml program of PAML [57] and the mouse ortholog of the parent as outgroup). K
A/K
S is influenced by the GC content at synonymous sites of the parent as well as by the GC content of the genomic region surrounding the retrogene [59]. In particular, retrocopies derived from parental genes with high GC that insert into regions of low GC may show low K
A/K
S driven by local adaptation to local GC. To test whether GC differences between intact and retropseudogene copies with low K
A/K
S (<0.5) explain differences in K
A/K
S between these two types of sequences, we first estimated the GC content at 4-fold degenerate sites and in regions (20 kb) upstream and downstream of the retrocopies, according to the previous analysis [59]. Intact retrocopies and retropseudogenes showed no significant difference when analyzing copies stemming from high-GC (>60% at 4-fold degenerate sites) parents that inserted into low-GC (lower than median value of GC) regions (52 of 130 intact retrocopies versus 60 of 172 retropseudogene copies, p = 0.4). Thus, the difference in the distributions for K
A/K
S < 0.5 between the two types of retrocopies is not accounted for by differences in GC but is likely explained by purifying selection on a number of intact retrocopies.
Functionality of retrocopies
Codon sequences were aligned on the basis of the translated sequence alignments using the EMBOSS package [56]. Phylogenetic trees were based on the established evolutionary relationships of primates [18]. In the simulation approach used to support functionality of retrocopies, we reconstructed the ancestral state of the retrocopy at the time point of duplication based on this phylogeny using the codeml program of PAML [57] and the parent as an outgroup. Then, we repeatedly simulated the evolution of this ancestral sequence throughout the phylogeny assuming neutral evolution (i.e., point mutations and indels accumulate according to a neutral model of sequence evolution). We used the Kimura-2 parameter model [60] for sequence evolution (assuming a transition/transversion ratio of two), a point mutation rate of 1.0 × 10−9 per site per year as suggested previously for hominoids and Old World monkeys [15], and an indel rate of 1.0 × 10−10 per site per year [61]. Indels with a multiple of three nucleotides (17%) were assumed to be nondeleterious as they do not disrupt the ORF. The simulations provided a probability (P
dis) for each gene, which corresponds to the number of simulated datasets with a number of deleterious mutations on the different lineages that is smaller or equal to our observation. In parallel, the accumulation of nonsynonymous and synonymous substitutions in the simulated phylogenies was monitored. Thus, we could compare the observed ratio of nonsynonymous to synonymous substitutions to its null distribution estimated by the simulations. The parental genes of the seven retrogenes for which functionality was supported showed low to medium GC content (22%−52%) at 4-fold degenerate, similar to the GC content of the regions flanking their insertions sites (33%−47%). Thus, GC effects (see above; [59]) are unlikely to explain nonsynonymous/synonymous distribution patterns, which are therefore indicative of purifying selection for several cases.
Selection analysis using PAML
To test for the presence of sites under diversifying selection (K
A/K
S > 1) on the retrogene lineages, we compared model M1 and model A as implemented in codeml from the PAML package [57] using likelihood ratio tests [62]. Model M1 assumes two classes of sites for the sequences in the whole phylogeny: sites under purifying selection (K
A/K
S < 1) and neutral sites (K
A/K
S = 1). Model A adds a third class of sites in the retrogene lineages, with K
A/K
S as a free parameter, allowing for sites with K
A/K
S > 1. We also compared this model A to a modified model where K
A/K
S is fixed at one. Sites under positive selection in the retrogene lineages were identified using the Bayesian approach as implemented in codeml [63]. Note that with respect to CDC14B2, the human and chimpanzee sequences have lost the original translation initiation codon (methionine) used by the parental gene (which may have led to the annotation of this gene as a VEGA pseudogene, OTTHUMG00000033880) and gained a putatively new methionine start codon at position 31. The selection tests show similar (statistically significant) results when either the original full-length sequence alignment or a shorter alignment starting from position 31 is used (data not shown).
Supporting Information
Figure S1 Distribution of K
S between Parental Genes and Their Orthologs in Mouse
(644 KB EPS).
Click here for additional data file.
Figure S2
K
A
/K
S on the Retrocopy Lineage: Comparison of the K
A
/K
S Distributions for Intact Retrocopies and Retropseudogenes
The mode of the K
A
/K
S distributions is smaller than one (usually expected under neutrality), owing to the effect previously described [59]. White bars correspond to intact retrocopies, and dark bars to retropseudogene copies.
(636 KB EPS).
Click here for additional data file.
Figure S3 Simulation Results for CDC14B2
See legend of Figure 3.
(7.9 MB PDF).
Click here for additional data file.
Figure S4 Simulation Results for eIF-2-gamma2
See legend of Figure 3.
(6.6 MB PDF).
Click here for additional data file.
Figure S5 Simulation Results for GMCL2
See legend of Figure 3.
(6.6 MB PDF).
Click here for additional data file.
Figure S6 Simulation Results for KIF4B
See legend of Figure 3.
(6.6 MB PDF).
Click here for additional data file.
Figure S7 Simulation Results for NACA2
See legend of Figure 3.
(6.7 MB PDF).
Click here for additional data file.
Figure S8 Simulation Results for PABP3
See legend of Figure 3.
(6.6 MB PDF).
Click here for additional data file.
Figure S9 Phylogenetic Trees for the Seven Retrogenes Identified in This Study
Maximum likelihood K
A
/K
S values and the estimated number of nonsynonymous versus synonymous substitutions (in parentheses) for each branch are indicated.
(791 KB EPS).
Click here for additional data file.
Table S1 Dated Retrocopies
(107 KB DOC).
Click here for additional data file.
Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the primate sequences generated for this paper are DQ120612−DQ120720. They are detailed in Table S1. The Ensembl (http://www.ensembl.org/) accession numbers for other genes discussed in this paper are GMCL1 (ENSG00000087338) and PABP1 (ENSG00000152520).
We thank Corinne Peter, Lukasz Potrzebowski, and Lia Rosso for technical help; Victor Jongeneel and the Vital-IT unit for computational support; Max Ingman for comments on the manuscript; and Christian Roos and F. M. Catzeflis for primate and treeshrew DNA samples. This research was supported by funds available to HK from the Center for Integrative Genomics (University of Lausanne), the Swiss National Science Foundation (grant 3100A0–104181), and the European Union (grant PKB140404).
Competing interests. The authors have declared that no competing interests exist.
Author contributions. ACM, ID, NV, and HK conceived and designed the experiments. ACM and NV performed the experiments. ACM, ID, NV, and HK analyzed the data. ID and AR contributed reagents/materials/analysis tools. ACM, ID, NV, and HK wrote the paper.
Citation: Marques AC, Dupanloup I, Vinckenbosch N, Reymond A, Kaessmann H (2005) Emergence of young human genes after a burst of retroposition in primates. PLoS Biol 3(11): e357.
Abbreviations
MYAmillion years ago
ORFopen reading frame
==== Refs
References
Samonte RV Eichler EE Segmental duplications and the evolution of the primate genome Nat Rev Genet 2002 3 65 72 11823792
Brosius J Retroposons—Seeds of evolution Science 1991 251 753 1990437
Long M Betran E Thornton K Wang W The origin of new genes: Glimpses from the young and old Nat Rev Genet 2003 4 865 875 14634634
Mighell AJ Smith NR Robinson PA Markham AF Vertebrate pseudogenes FEBS Lett 2000 468 109 114 10692568
Emerson JJ Kaessmann H Betran E Long M Extensive gene traffic on the mammalian X chromosome Science 2004 303 537 540 14739461
Betran E Long M Expansion of genome coding regions by acquisition of new genes Genetica 2002 115 65 80 12188049
Zhang Z Harrison PM Liu Y Gerstein M Millions of years of evolution preserved: A comprehensive catalog of the processed pseudogenes in the human genome Genome Res 2003 13 2541 2558 14656962
Ohshima K Hattori M Yada T Gojobori T Sakaki Y Whole-genome screening indicates a possible burst of formation of processed pseudogenes and Alu repeats by particular L1 subfamilies in ancestral primates Genome Biol 2003 4 R74 14611660
Zhang Z Carriero N Gerstein M Comparative analysis of processed pseudogenes in the mouse and human genomes Trends Genet 2004 20 62 67 14746985
Esnault C Maestre J Heidmann T Human LINE retrotransposons generate processed pseudogenes Nat Genet 2000 24 363 367 10742098
Betran E Wang W Jin L Long M Evolution of the phosphoglycerate mutase processed gene in human and chimpanzee revealing the origin of a new primate gene Mol Biol Evol 2002 19 654 663 11961099
Burki F Kaessmann H Birth and adaptive evolution of a hominoid gene that supports high neurotransmitter flux Nat Genet 2004 36 1061 1063 15378063
Wang PJ Page DC Functional substitution for TAF(II)250 by a retroposed homolog that is expressed in human spermatogenesis Hum Mol Genet 2002 11 2341 2346 12217962
Bradley J Baltus A Skaletsky H Royce-Tolland M Dewar K An X-to-autosome retrogene is required for spermatogenesis in mice Nat Genet 2004 36 872 876 15258580
Yi S Ellsworth DL Li WH Slow molecular clocks in Old World monkeys, apes, and humans Mol Biol Evol 2002 19 2191 2198 12446810
Liu G Zhao S Bailey JA Sahinalp SC Alkan C Analysis of primate genomic variation reveals a repeat-driven expansion of the human genome Genome Res 2003 13 358 368 12618366
Locke DP Jiang Z Pertz LM Misceo D Archidiacono N Molecular evolution of the human chromosome 15 pericentromeric region Cytogenet Genome Res 2005 108 73 82 15545718
Goodman M The genomic record of humankind's evolutionary roots Am J Hum Genet 1999 64 31 39 9915940
Li WH Molecular evolution 1997 Sunderland MA Sinauer Associates 487
Feral C Guellaen G Pawlak A Human testis expresses a specific poly(A)-binding protein Nucleic Acids Res 2001 29 1872 1883 11328870
Wiedmann B Sakai H Davis TA Wiedmann M A protein complex required for signal-sequence-specific sorting and translocation Nature 1994 370 434 440 8047162
Soulard M Della Valle V Siomi MC Pinol-Roma S Codogno P hnRNP G: Sequence and characterization of a glycosylated RNA-binding protein Nucleic Acids Res 1993 21 4210 4217 7692398
Mazumdar M Sundareshan S Misteli T Human chromokinesin KIF4A functions in chromosome condensation and segregation J Cell Biol 2004 166 613 620 15326200
Zhu C Jiang W Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis Proc Natl Acad Sci U S A 2005 102 343 348 15625105
Zhu C Zhao J Bibikova M Leverson JD Bossy-Wetzel E Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference (RNAi) Mol Biol Cell 2005 16 3187 3199 15843429
Leatherman JL Levin L Boero J Jongens TA
germ cell-less acts to repress transcription during the establishment of the Drosophila germ cell lineage Curr Biol 2002 12 1681 1685 12361572
Masuhara M Nagao K Nishikawa M Kimura T Nakano T Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less Biochem Biophys Res Commun 2003 308 927 932 12927808
Ehrmann IE Ellis PS Mazeyrat S Duthie S Brockdorff N Characterization of genes encoding translation initiation factor eIF-2gamma in mouse and human: Sex chromosome localization, escape from X-inactivation and evolution Hum Mol Genet 1998 7 1725 1737 9736774
Richler C Soreq H Wahrman J X inactivation in mammalian testis is correlated with inactive X-specific transcription Nat Genet 1992 2 192 195 1345167
McCarrey JR Thomas K Human testis-specific PGK gene lacks introns and possesses characteristics of a processed gene Nature 1987 326 501 505 3453121
Jongens TA Hay B Jan LY Jan YN The germ cell-less gene product: A posteriorly localized component necessary for germ cell development in Drosophila
Cell 1992 70 569 584 1380406
Jongens TA Ackerman LD Swedlow JR Jan LY Jan YN Germ cell-less encodes a cell type-specific nuclear pore-associated protein and functions early in the germ-cell specification pathway of Drosophila
Genes Dev 1994 8 2123 2136 7958883
Leatherman JL Kaestner KH Jongens TA Identification of a mouse germ cell-less homologue with conserved activity in Drosophila
Mech Dev 2000 92 145 153 10727854
Paulding CA Ruvolo M Haber DA The Tre2 (USP6) oncogene is a hominoid-specific gene Proc Natl Acad Sci U S A 2003 100 2507 2511 12604796
Johnson ME Viggiano L Bailey JA Abdul-Rauf M Goodwin G Positive selection of a gene family during the emergence of humans and African apes Nature 2001 413 514 519 11586358
Ciccarelli FD von Mering C Suyama M Harrington ED Izaurralde E Complex genomic rearrangements lead to novel primate gene function Genome Res 2005 15 343 351 15710750
Zhang J Rosenberg HF Nei M Positive Darwinian selection after gene duplication in primate ribonuclease genes Proc Natl Acad Sci U S A 1998 95 3708 3713 9520431
Cooper DN Human gene evolution 1999 Oxford BIOS Scientific Publishers 490
Podlaha O Zhang J Nonneutral evolution of the transcribed pseudogene Makorin1-p1 in mice Mol Biol Evol 2004 21 2202 2209 15306660
Hirotsune S Yoshida N Chen A Garrett L Sugiyama F An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene Nature 2003 423 91 96 12721631
Kleene KC A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells Mech Dev 2001 106 3 23 11472831
Schmidt EE Schibler U High accumulation of components of the RNA polymerase II transcription machinery in rodent spermatids Development 1995 121 2373 2383 7671803
Schmidt EE Transcriptional promiscuity in testes Curr Biol 1996 6 768 769 8805310
She X Horvath JE Jiang Z Liu G Furey TS The structure and evolution of centromeric transition regions within the human genome Nature 2004 430 857 864 15318213
Mudge JM Jackson MS Evolutionary implications of pericentromeric gene expression in humans Cytogenet Genome Res 2005 108 47 57 15545715
Wu CI Xu EY Sexual antagonism and X inactivation—The SAXI hypothesis Trends Genet 2003 19 243 247 12711214
Swanson WJ Vacquier VD The rapid evolution of reproductive proteins Nat Rev Genet 2002 3 137 144 11836507
Nielsen R Bustamante C Clark AG Glanowski S Sackton TB A scan for positively selected genes in the genomes of humans and chimpanzees PLoS Biol 2005 3 e170 10.1371/journal.pbio.0030170 15869325
Waterston RH Lindblad-Toh K Birney E Rogers J Abril JF Initial sequencing and comparative analysis of the mouse genome Nature 2002 420 520 562 12466850
Torgerson DG Singh RS Rapid evolution through gene duplication and subfunctionalization of the testes-specific alpha4 proteasome subunits in Drosophila
Genetics 2004 168 1421 1432 15579695
Lynch M Force A The probability of duplicate gene preservation by subfunctionalization Genetics 2000 154 459 473 10629003
Ohno S Evolution by gene duplication 1970 Berlin Springer-Verlag 160
Hubbard T Barker D Birney E Cameron G Chen Y The Ensembl genome database project Nucleic Acids Res 2002 30 38 41 11752248
Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Gapped BLAST and PSI-BLAST: A new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694
Lander ES Linton LM Birren B Nusbaum C Zody MC Initial sequencing and analysis of the human genome Nature 2001 409 860 921 11237011
Rice P Longden I Bleasby A EMBOSS: The European Molecular Biology Open Software Suite Trends Genet 2000 16 276 277 10827456
Yang Z PAML: A program package for phylogenetic analysis by maximum likelihood Comput Appl Biosci 1997 13 555 556 9367129
Chamary JV Hurst LD Biased codon usage near intron-exon junctions: Selection on splicing enhancers, splice-site recognition or something else? Trends Genet 2005 21 256 259 15851058
Bustamante CD Nielsen R Hartl DL A maximum likelihood method for analyzing pseudogene evolution: Implications for silent site evolution in humans and rodents Mol Biol Evol 2002 19 110 117 11752196
Kimura M A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J Mol Evol 1980 16 111 120 7463489
Zhang J Webb DM Evolutionary deterioration of the vomeronasal pheromone transduction pathway in catarrhine primates Proc Natl Acad Sci U S A 2003 100 8337 8341 12826614
Yang Z Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution Mol Biol Evol 1998 15 568 573 9580986
Yang Z Wong WS Nielsen R Bayes empirical Bayes inference of amino acid sites under positive selection Mol Biol Evol 2005 22 1107 1118 15689528
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030399SynopsisBioinformatics/Computational BiologyEvolutionGenetics/Genomics/Gene TherapyHomo (Human)PrimatesEukaryotesRetrocopied Genes May Enhance Male Fitness Synopsis11 2005 11 10 2005 11 10 2005 3 11 e399Copyright: © 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.
Emergence of Young Human Genes after a Burst of Retroposition in Primates
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“Retrocopied” genes were long viewed as evolutionary dead ends, with little functional relevance. Such a gene copy is generated by a circuitous route. First, a normal gene is copied to make a messenger RNA, which in the usual scheme of things is sent out of the cell nucleus, used to code for protein, and eventually destroyed. Once in a great while, though, the messenger RNA is “reverse transcribed,” coded back into a DNA sequence by the enzyme reverse transcriptase. It can then be inserted back into a chromosome, quite possibly a different chromosome than the one on which its parent gene resides. This process, which is driven by the legions of transposable elements that litter the genome, usually creates a functionless “retropseudogene,” stranded without a promoter and unable to be expressed.
But occasionally, the new gene copy recruits a promoter by, as yet, largely unknown mechanisms, and may thus potentially become functional, able to code for a protein. In the primate lineage, four such genes have been identified to date. In this issue, Henrik Kaessman and colleagues announce the discovery of seven more, and estimate that on average, one such new gene arises every million years. Many of these genes are expressed predominantly in the testes, where at least some of them probably substitute for X-chromosome genes that are inactivated during sperm development.
The authors first used bioinformatics methods to identify almost 4,000 retrocopies in the human genome. Retrocopied genes can be distinguished from their parents because the introns, or noncoding sequences, of the parent are edited out of the original messenger before retrocopying; thus, the DNA coding sequence is initially the same, but lacks the intervening introns of the parent. Of these 4,000, about 700 had not been disabled by mutations that interrupt the coding sequence. The number of other harmless mutations each had accumulated was then used to estimate the time each retrocopy was formed, based on molecular evolution theory that such neutral mutations occur at a predictable rate. While retrocopies have been created continuously over many millions of years of mammalian evolution, the authors' analysis showed a peak around 40 million years ago, after the emergence of primates, but before establishment of the human line. They estimate that 57 functional retrogenes arose in primates, about one per million years of primate evolution.
Approximately one new retrogene (a re-inserted copy of a gene) per million years emerged on the primate lineage leading to humans. (Image: © 2005 Hybrid Medical Animation)
To pinpoint individual retrogenes, the authors first conducted an evolutionary simulation to estimate, based on sequence changes, which retrocopies were likely to still be functional. They found seven, which originated between 18 and 63 million years ago. These genes play a variety of roles in transcription and translation, as well as chromosome condensation and segregation, which occur just prior to cell division.
They next looked at expression patterns for these genes in 20 human tissues, and discovered that for all seven, expression was restricted mostly or entirely to the testes. Three of the seven genes were copied from genes on the X chromosome to Chromosome 1, 5, and 12, respectively. The authors suggest that such retrogenes functionally replace their silenced parental genes on the X chromosome during spermatogenesis, a resourceful maneuver that may enhance the reproductive fitness of the organism expressing them. This increase in fitness, in turn, preserves the functional retrocopy through natural selection. For two other genes, the authors also infer a function in spermatogenesis based on the parental gene function. One of these genes as well as the two remaining genes appear to have been selectively driven to evolve new or more adapted functional properties compared to their parents. Together, the results suggest that retrogenes were often recruited during primate evolution to enhance male germline functions. —Richard Robinson
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1620183510.1371/journal.pmed.0020312Research ArticleHematologyHematology (including Blood Transfusion)Drugs and adverse drug reactionsA C1173T Dimorphism in the VKORC1 Gene Determines Coumarin Sensitivity and Bleeding Risk Coumarin Sensitivity and BleedingReitsma Pieter H
1
*van der Heijden Jeroen F.
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Groot Angelique P
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Rosendaal Frits R
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Büller Harry R
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1Laboratory for Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands,2Departments of Clinical Epidemiology and Hematology, Leiden University Medical Center, Leiden, The Netherlands,3Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The NetherlandsGreaves Michael Academic EditorUniversity of AberdeenUnited Kingdom*To whom correspondence should be addressed. E-mail: [email protected]
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: PHR and HRB designed the study. PHR drafted the report and incorporated all suggestions. JFV was responsible for collecting all patient data and specimens. APG drafted the genetic assay and performed the lab analyses. FRR oversaw the statistical analyses and other methodological issues of the report.
10 2005 11 10 2005 2 10 e31228 4 2005 29 7 2005 Copyright: © 2005 Reitsma 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.
Pharmacogenetics in the Management of Coumarin Anticoagulant Therapy: The Way Forward or an Expensive Diversion?
Understanding Bleeding Risk after Anticoagulation
Background
A C1173T polymorphism in intron 1 of the VKORC1 gene has been claimed to determine the interindividual variability in the response to vitamin K antagonist therapy (VKA), but it is unknown whether it also influences bleeding risk. We aimed to confirm the relationship between C1173T status and phenprocoumon or acenocoumarol use, and to examine the risk of severe bleeding for the various genotypes.
Methods and Findings
We studied this in a case-control study of 110 patients who bled during VKA therapy and 220 control patients free of bleeding under the same therapy. To achieve the same target INR, CT genotype and TT genotype control patients required less phenprocoumon (CC genotype 2.9 mg/d [95% confidence interval (CI): 2.6–3.2], CT genotype 2.6 mg/d [95% CI: 2.1–3.1], TT genotype 1.4 mg/d [95 % CI: 1.1–1.7]) or acenocoumarol (CC genotype 3.2 mg/d [95% CI: 2.9–3.5], CT genotype 2.3 mg/d [95% CI: 2.1–2.5], TT genotype 1.7 mg/d [95% CI: 1.3–2.1]) than CC genotype control patients. Compared with CC genotype individuals, carriers of at least one T allele had an increased risk of bleeding in the phenprocoumon users (crude odds ratio = 2.6, 95% CI: 1.2–5.7), but not in acenocoumarol users (crude odds ratio = 1.2, 95% CI: 0.6–2.3).
Conclusion
These findings encourage taking further steps towards the evaluation of the use of VKORC1 genetic testing for bleeding prevention in individuals who receive VKA therapy.
A polymorphism in the VKORC1 gene may affect the dose of vitamin K antagonists needed and the risk of bleeding after anticoagulation in some patients.
==== Body
Introduction
Vitamin K antagonists such as warfarin, acenocoumarol, and phenprocoumon are commonly used for the prevention and treatment of venous and arterial thrombosis. The molecular target of these anticoagulants is the vitamin K epoxide reductase complex of which the precise composition is largely unknown. Recently, one component of this complex, the vitamin K epoxide reductase complex subunit 1 (VKORC1), was identified by positional cloning [1]. This VKORC1 gene is mutated in individuals with combined deficiency of vitamin K-dependent clotting factors type 2 or with warfarin resistance [1,2].
The required dose of antagonist in vitamin K antagonist therapy (VKA) to achieve a target level of anticoagulation is variable, in particular between individuals, but also within a single individual, and depends on, for example, dietary intake and variations in pharmacokinetics. Earlier this year, polymorphisms in the VKORC1 gene have been reported that explain up to 30% of the variability in the pharmacological response to VKAs [3–5].
Bleeding is an important complication of treatment with VKA. In The Netherlands, intensive efforts by anticoagulation clinics are aimed at monitoring treatment with phenprocoumon and acenocoumarol, the two VKAs that are registered for use. These efforts are largely focussed on maintaining the level of anticoagulation, generally expressed as the international normalized ratio (INR), within a specified target range. In these efforts, individual patient characteristics such as those related to inherited determinants of the pharmacological response are only indirectly taken into account. We set out to estimate the contribution of a C1173T polymorphism in the VKORC1 gene to the acenocoumarol and phenprocoumon dose requirement and to the bleeding risk.
Methods
For the present analyses we used DNA from a previously reported case-control study [6]. This study includes 110 patients classified as “severe bleeders” and 220 “non-bleeders” (more than 96% of the participants are Caucasian) who were selected from two anticoagulation clinics. Major bleeding was defined as a bleeding that was clinically overt and met one of the following criteria: associated with a haemoglobin drop of ≥20 g/l, hospitalization, blood transfusion of two or more units, intracranial bleeding, intramuscular bleeding, intra-articular bleeding, or intraocular bleeding. Bleeders and non-bleeders were visited at home, during which a questionnaire was completed and blood was collected for the preparation of plasma and DNA. Genotyping for the C1173T polymorphism in the VKORC1 gene was performed using a simple restriction enzyme digestion of PCR-amplified DNA. Details of the protocol are available from the corresponding author upon request. Statistical analyses were performed for acenocoumarol and phenprocoumon separately and combined. We calculated odds ratios as measures of relative risks from the contingency table as the exposure odds ratios (OR), with 95% confidence intervals (CI) based on the assumption of a Poisson distribution, according to Woolf [7]. The institutional review boards of the Academic Medical Center and the Leiden University Medical Center approved the study protocol, and all patients gave written informed consent.
Results
Table 1 shows the dose requirements for the same target INR categorized for the different genotypes in controls. The data confirm the claims that were made in three earlier papers [3–5]. In comparison to CC genotype carriers, the average dose of acenocoumarol or phenprocoumon was 15%–30% less in CT heterozygotes, and about 40% less in homozygous TT individuals. There does not seem to be a major difference between acenocoumarol and phenprocoumon in this respect. The results are similar in the cases (data not shown).
Table 1 Relationship between C1173T Genotype and VKA Mean Daily Dose Requirement in Control Patients
Next we examined whether the C1173T genotype influenced the bleeding risk (Table 2). Overall the OR for major haemorrhage for carriers of at least one T-allele was 1.7 (95% CI: 1.1–2.5). When analysed separately, phenprocoumon seems to more strongly modify the bleeding risk of the C1173T genotype (OR = 2.6 [95% CI: 1.2–5.7 for T-allele carriers]), whereas in acenocoumarol users the genotype had little effect (OR = 1.2 [95% CI: 0.6–2.3]). When we add all controls together in the calculations of the odds ratios in order to obtain statistically more stable results (which is reasonable because genotype does not influence the prescription), the ORs for bleeding in T-carriers are 1.4 (95 % CI: 0.8–2.5) for acenocoumarol and 2.1 (95% CI: 1.1–4.2) for phenprocoumon.
Table 2 Numbers of Cases and Controls for the Different VKORC1 Genotypes and Crude ORs for Bleeding under VKA Treatment
We examined the quality of anticoagulation in the various groups of genotypes and VKAs. The percentage of time in range was considerably higher in phenprocoumon treated controls (CC genotype 60% in range (95% CI: 45–75, CT/TT genotype 66% in range (95% CI: 62–71) than in individuals treated with acenocoumarol (CC genotype 41% in range (95% CI: 35–47, CT/TT genotype 46% in range (95% CI: 42–51), whereas genotype had no appreciable effect on time in range.
Discussion
The results, although based on a small sample size of individuals with bleeding, support the suggestion that the bleeding risk for T-carriers is higher in phenprocoumon than in acenocoumarol users. If this finding is confirmed in additional studies and extended to the more frequently occurring and clinically relevant cases of nonmajor bleeding, it may imply that CT and TT carriers should be preferentially treated with acenocoumarol despite the fact that the genotype-related excess bleeding in phenprocoumon users is not mediated through unstable anticoagulation that is detected by the INR, because genotypes were associated with bleeding but not with unstable INR values. In fact, stability of treatment is better in phenprocoumon than in acenocoumarol users, which is in agreement with previous publications [8]. This paradox—apparently good INR control by all measures, but higher bleeding risks in phenprocoumon-treated patients—has been reported before, in particular in relationship to minor bleeding [8].
At present we have no clear explanation for risk differences between the two coumarin anticoagulants. It is unlikely that these drugs have differential effects on the carboxylation status of vitamin K–dependent clotting factors. In support of this, levels of factors IX (which do not influence the INR) are similar between the two treatment groups (data not shown). More likely, a difference in bleeding risk may find an explanation in the vastly different pharmokinetics of acenocoumarol (biological half-life = 11 h) and phenprocoumon (half-life = 140 h). Receivers of anticoagulants are often frail people with significant health problems. Perhaps alterations in their health status that affect coumarin sensitivity are not effectively managed with a long half-life product.
The increased bleeding risks give support to the notion that genotyping for this polymorphism in the VKORC1 gene may go beyond an academic interest and may become standard for every individual starting on acenocoumarol and phenprocoumon treatment. It may help in the identification of those individuals who are at the highest bleeding risk and thus should be monitored more intensely. Such VKORC1 testing may need to be complemented with testing for Cyp2C9 polymorphisms, which also influence the pharmacokinetics of VKAs and for which there is also increasing evidence for a relevant relationship between genotype and bleeding [9].
Patient Summary
Background
Patients who have experienced blood clots or who are at risk of getting clots are often put on anticoagulant treatment to reduce the ability of blood to clot efficiently. One group of these drugs works by interfering with the action of vitamin K, which is essential for blood clotting. The disadvantage of this type of drug is that the treatment has to be monitored closely to ensure that it does not work too well, in which case a patient may have bleeding, or not well enough, in which case the blood may clot.
Why Was This Study Done?
Previous work has suggested that a change in one of the genes involved in blood clotting, VKORCI, might affect how well these drugs work. The authors wanted to confirm this finding and see if changes in the gene also affect how likely patients are to bleed when given the drugs.
What Did the Researchers Do and Find?
They looked at 110 people who had bled after being given the anticoagulants and 220 who had not. They found that one particular variation in the gene resulted in people requiring less anticoagulation medicine. This same variation also meant that some people were more likely to bleed with one of the anticoagulant drugs, but not with another.
What Do These Findings Mean?
If these findings are confirmed in studies of other groups of patients, they suggest a reason why patients vary so much in their response to anticoagulation and why some people bleed whereas others do not. In the future, it may be possible to look at the genes in patients given anticoagulation to help make decisions about their treatment.
Where Can I Get More Information Online?
MedlinePlus has information on blood clots and the drugs used to treat them:
http://www.nlm.nih.gov/medlineplus/bleedingdisorders.html
Anticoagulation Europe is a site specifically for people taking anticoagulants:
http://www.anticoagulationeurope.org/
We thank Dr. F.J.M. van der Meer from the Thrombosis Service Leiden and Dr. M.G.H. Remkes from the Thrombosis Service Amsterdam for providing access to the patients under their care. The FACTORS study was supported by The Netherlands Heart Foundation (#99.165). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The corresponding author had access to all data in the study and had final responsibility for the decision to submit for publication.
Citation: Reitsma, PH, van der Heijden JF, Groot AP, Rosendaal FR, Büller HR (2005) A C1173T dimorphism in the VKORC1 gene determines coumarin sensitivity and bleeding risk. PLoS Med 2(10): e312.
Abbreviations
CIconfidence interval
INRinternational normalized ratio
ORodds ratio
VKAvitamin K antagonist therapy
==== Refs
References
Rost S Fregin A Ivaskevicius V Conzelmann E Hortnagel K Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2 Nature 2004 427 537 541 14765194
Li T Chang CY Jin DY Lin PJ Khvorova A Identification of the gene for vitamin K epoxide reductase Nature 2004 427 541 544 14765195
Bodin L Verstuyft C Tregouet DA Robert A Dubert L Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genotypes as determinants of acenocoumarol sensitivity Blood 2005 106 135 140 15790782
D'Andrea G D'Ambrosio RL Di Perna P Chetta M Santacroce R A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin Blood 2005 105 645 649 15358623
Rieder MJ Reiner AP Gage BF Nickerson DA Eby CS Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose N Engl J Med 2005 352 2285 2293 15930419
van der Heijden JF Rekke B Hutten BA van der Meer FJ Remkes MG Non-fatal major bleeding during treatment with vitamin K antagonists: Influence of soluble thrombomodulin and mutations in the propeptide of coagulation factor IX J Thromb Haemost 2004 2 1104 1109 15219193
Woolf B On estimating the relation between blood group and disease Ann Hum Genet 1955 19 251 253 14388528
Gadisseur AP van der Meer FJ Adriaansen HJ Fihn SD Rosendaal FR Therapeutic quality control of oral anticoagulant therapy comparing the short-acting acenocoumarol and the long-acting phenprocoumon Br J Haematol 2002 117 940 946 12060134
Visser LE van Schaik RH van Vliet M Trienekens PH De Smet PA The risk of bleeding complications in patients with cytochrome P450 CYP2C9*2 or CYP2C9*3 alleles on acenocoumarol or phenprocoumon Thromb Haemost 2004 92 61 66 15213846
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2021-01-05 10:39:14
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PLoS Med. 2005 Oct 11; 2(10):e312
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PLoS Med
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10.1371/journal.pmed.0020312
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==== Front
PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020352SynopsisHematologyUnderstanding Bleeding Risk after Anticoagulation Synopsis10 2005 11 10 2005 2 10 e352Copyright: © 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 C1173T Dimorphism in the VKORC1 Gene Determines Coumarin Sensitivity and Bleeding Risk
==== Body
One class of treatments commonly used for prevention and treatment of venous and arterial thrombosis is vitamin K antagonists (VKA), such as warfarin. The molecular target of these anticoagulants is the vitamin K epoxide reductase complex (VKORC) of which one component, VKORC1, was recently identified. The gene for this component is mutated in individuals with combined deficiency of vitamin K–dependent clotting factors type 2 or with warfarin resistance.
What makes VKA treatment so tedious for patients is that the dose of VKA required to achieve anticoagulation varies among patients and even changes over time in the same patient, so patients require regular monitoring to ensure that they are not over- or under-anticoagulated. The standard measure of anticoagulation is the international normalized ratio (INR). A recent study reported polymorphisms in the VKORC1 gene that explained up to 30% of the variation in the pharmacological response to VKAs, but few clinics take such inherited determinants of the pharmacological response into account.
In a paper published in PLoS Medicine, Dutch authors Pieter Reitsma and colleagues aimed to estimate the contribution of C1173T polymorphisms in the VKORC1 gene to dose requirement for the anticoagulants acenocoumarol and phenprocoumon and to bleeding risk. In this case-control study, the authors studied 110 patients who bled during VKA therapy and 220 controls free of bleeding under the same therapy. Patients in the study were being treated with anticoagulants for venous thrombosis or were receiving anticoagulants to prevent arterial thromboembolism related to their atrial fibrillation or mechanical heart valves.
What the authors found was that to achieve the same target INR, control patients with CT and TT genotypes required less phenprocoumon or acenocoumarol than control patients with the CC genotype. Also, compared with CC individuals, carriers of at least one T allele had increased bleeding risk in the phenprocoumon users but not in acenocoumarol users.
The results, although based on a small sample size of individuals, support the suggestion that bleeding risk for T carriers is higher in phenprocoumon users than in acenocoumarol users. If this suggestion is confirmed in additional studies and extended to more frequently occurring and clinically relevant nonmajor bleeding, it may imply that CT and TT carriers should be preferentially treated with acenocoumarol, despite the fact that this anticoagulant gives poorer quality of control of treatment intensity.
At present, there is no clear explanation for risk differences between the two coumarin anticoagulants, the authors noted, although it is possible that the long half-life of phenprocoumon—a 140-hour half-life versus an 11-hour half-life of acenocoumarol—is a factor.
Although this study needs to be repeated in other populations of patients, increased bleeding risk in some individuals supports the idea that genotyping for this polymorphism in the VKORC1 gene should be further explored. It may be that, in the future, genotyping will be a possibility for patients starting on acenocoumarol and phenprocoumon treatment in order to help identify those individuals who are at highest bleeding risk and who, thus, should be monitored more intensely.
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2021-01-05 10:38:16
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PLoS Med. 2005 Oct 11; 2(10):e352
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PLoS Med
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10.1371/journal.pmed.0020352
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==== Front
Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_04_0044
From the Editor in Chief
Summertime
Wilcox Lynne S. MD, MPH Editor in Chief
7 2004
15 6 2004
1 3 A012004
==== Body
Dandelion Wine is a lyrical coming-of-age story set in Green Town, Illinois, in the early 20th century (1). Douglas Spaulding is 12 years old, and summertime has arrived. When it’s a new summer, you need new shoes. In fact, you need “Royal Crown Cream-Sponge Para Litefoot Tennis Shoes.” But Doug doesn’t have the money. He bargains with Mr. Sanderson, the proprietor of the local shoe store:
“Soon as I get those shoes on, you know what happens? . . . Bang! I deliver your packages, pick up packages, bring your coffee, burn your trash, run to the post office, telegraph office, library! You’ll see twelve of me in and out, in and out, every minute.”
Many readers older than 50 will recognize Doug. He may have a different name, he may want a bike instead of tennis shoes, he may like root beer instead of lemonade. Still, it’s summertime, and he’s in 12 places all at once.
That story took place more than 75 years ago. Today, too few U.S. children enjoy the magic of an active, outdoor summer. They may not even recognize the season, because most of their entertainment is indoors and sedentary. Children spend up to four-and-a-half hours each day in front of a television or computer screen (2). I recently made a field trip to a large toy store. The front of the store was filled with music videos, computer games, DVDs, and other entertainment technologies that require little physical effort. Where were the bicycles, skate boards, and soccer balls? Along the back wall of the store.
It seems unlikely that toy manufacturers and store managers are plotting to keep kids sedentary. They are simply following good market practice by displaying their most popular merchandise in prominent locations. Meanwhile, obesity among individuals aged six to 19 years has tripled since the 1960s (3).
The July issue of Preventing Chronic Disease includes two articles on VERB™, a multiethnic campaign to promote physical activity among tweens, or children aged nine to 13 years (4,5). Congress appropriated $125 million in 2001 to the Centers for Disease Control and Prevention to develop a national media campaign to change children’s health behaviors. The VERB campaign features general media spots for all children, special spots directed toward ethnic and racial groups, and parent materials to encourage children’s physical activity. Campaign planners use process and outcome evaluations to assess the effectiveness of this mass communications approach to changing the attitudes and behaviors of tweens and their parents (4).
The design and operation of the VERB campaign is a remarkable achievement. If the evaluation results show changes in children’s physical activity levels over time, the campaign will be an even more noteworthy accomplishment. Both the successes and challenges of developing and implementing such campaigns in the United States are discussed in a commentary in this issue (6). While VERB targets only children aged nine to 13, the campaign hopes to shape the attitudes and behaviors of these individuals as they age: marketers of other products for tweens have found that children who become consumers of a product often are loyal to those products into adulthood (7).
VERB offers public health lessons beyond the nuts and bolts of this campaign. First, to affect the health habits of a generation, address the cohort moving from childhood to early adolescence — when kids begin to make their own decisions and to experience influences beyond the home. Second, to create major changes in public attitudes, commit serious resources. VERB demonstrates that with proper financial investment, public health messages can attract wide attention. Third, know your audience. Time spent researching the interests of a special group can make the difference between an effective campaign and an ineffective one. Fourth, to create a credible public health campaign, include evaluation of results as an essential component.
These statements seem simple and self-evident, but many public health professionals have spent their careers in underfunded programs. Lack of resources may cause audience research and results evaluation to take a back seat to the overwhelming pressures of launching a new campaign. VERB provides an excellent example of how to use social marketing principles, which include research and evaluation, to design a successful national public health campaign.
Back in Green Town, Doug persuades Mr. Sanderson to try a pair of Litefoot tennis shoes:
[Sanderson] laced the tennis shoes to his long narrow feet. They looked detached and alien down there next to the dark cuffs of his business suit. . . . [H]e began to sink deep in the shoes, to flex his toes, limber his arches, test his ankles. He rocked softly, secretly, back and forth in a small breeze from the open door.
Then, Mr. Sanderson hands Doug a pair of Litefoot shoes and listens wistfully to the boy running away down the street.
Dandelion Wine was set during a time when adults put their tennis shoes away with their youth. Over the last decade, U.S. adults as well as children have demonstrated sharp rises in obesity. All of us can benefit from lacing up our tennis shoes and joining Doug and the kids of VERB in running away into summertime.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Wilcox LS. Summertime. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0044.htm.
==== Refs
1 Bradbury R Dandelion wine New York (NY) Bantam Books (First published in its entirety by Doubleday & Company in 1957.) 1976 239
2 Woodard EH Media in the home 2000: the fifth annual survey of parents and children Philadelphia (PA) The Annenberg Public Policy Center of the University of Pennsylvania 2000
3 Ogden CL Flegal KM Carroll MD Johnson CL Prevalence and trends in overweight among U.S. children and adolescents, 1999-2000 JAMA 2002 288 1728 1732 12365956
4 Wong F Huhman M Heitzler C Asbury L Bretthauer-Mueller R McCarthy S VERB™ — a social marketing campaign to increase physical activity among youth Prev Chronic Dis [serial online] 2004 Jul [15 June 2004] Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0043.htm 2004 7 1 3 A10
5 Huhman M Heitzler C Wong F The VERB™ campaign logic model: a tool for planning and evaluation Prev Chronic Dis [serial online] 2004 Jul [15 June 2004] Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0033.htm 2004 7 1 3 A11
6 Bauman A Commentary on the VERB™ campaign — perspectives on social marketing to encourage physical activity among youth Prev Chronic Dis [serial online] 2004 Jul [15 June 2004] Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0054.htm 2004 7 1 3 A10
7 Sutherland A Thompson B Chapter 10, Kids as future purchasers Kidfluence: why kids today mean business 2001 Toronto (Ontario) McGraw-Hill Ryerson
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2022-01-24 23:45:21
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Prev Chronic Dis. 2004 Jun 15; 1(3):A01
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Prev Chronic Dis
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==== Front
Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_04_0054
Commentary
Commentary on the VERB™ Campaign — Perspectives on Social Marketing to Encourage Physical Activity Among Youth
Bauman Adrian PhD Center for Physical Activity and Health
Level 2, Medical Foundation Building, Sydney University, Sydney, Australia 2006 61 2 9036 3247 [email protected]
7 2004
15 6 2004
1 3 A022004
==== Body
The VERB™ campaign is a serious public health investment that aims to tackle the societal and health problems of inactivity and increasing obesity among young Americans (1,2). Worrisome trends in risk factors among young people throughout the developed world reflect the lack of clearly effective public health approaches. Effecting population-level change is difficult, given the ingrained societal acceptability of sedentary behaviors and over-nutrition. VERB is an innovative and expansive effort to improve the current state of affairs, commencing with a national paid mass media campaign designed to reframe beliefs and norms about being active among tweens — children aged nine to 13 years. Secondary campaign objectives are to identify and influence key stakeholders, such as parents and teachers, and to work within communities to support opportunities for youth physical activity (1).
A campaign to influence physical activity should focus first on affecting social norms (3). Short-term goals should include documentation of changes in proximal variables (i.e., awareness, beliefs, and attitudes). But media alone cannot change behavior, because it provides only a preliminary cue for action. Behavior change should be the long-term goal of a sustained campaign. Long-term change is likely to take place only after translating and disseminating programs developed to support the mass communication components (3,4).
Previous youth media campaigns have targeted tobacco use, illicit drugs, and sexual health (5-7). These campaigns have had some success in increasing awareness of an issue, changing social norms toward substance use or the risk of sexually transmitted diseases, and offering solutions for young people to prevent tobacco uptake, call or ask for help in reducing drug use, or practice safe sexual behavior (8). VERB is the first substantial youth campaign, however, to increase youth activity and encourage a healthy lifestyle. VERB targets proximal outcomes, such as beliefs about inactivity, and encourages tweens to “find their verbs” —activities they might try and enjoy. VERB promotes the notion that not only can activity be enjoyable but it also can foster friendships with peers, enhance curiosity, and generate positive feelings of autonomy. Creating and maintaining these values are essential prerequisites to adopting and maintaining physical activity throughout adolescence.
VERB is highly intense for a public sector campaign, but it remains modest amid the plethora of marketing messages targeting tweens. Public health campaigns that use paid media messages, including campaigns that promote physical activity, are often reported outside the United States (9-11), but within the United States, the costs of paid media generally prohibit their use for public health messages, and public service announcements (PSAs) are instead typically used. Although local media campaigns might rely on PSAs for effect or on local-level media, which is less expensive (12), national initiatives require a much greater investment to achieve recognition. Any amount invested, however, remains miniscule compared to the health and social costs of inactivity and obesity, or indeed to the amount spent on commercial marketing to tweens. Thus, VERB represents a strong commitment to improving youth health because it requires a large investment in paid media.
The public health challenge is to penetrate the commercial-marketing media morass with well-designed messages that reach their target population. Inducing change in beliefs and norms is only the first step, however. Subsequent challenges are to create physical environments and spaces for tweens to move, play, and be active. The challenge involves advocacy, support, and policy change at the local and state levels to provide resources to construct or redevelop activity-friendly environments, such as schools, parks, trails, and neighborhoods. VERB extends beyond a media campaign and emphasizes the need to form community partnerships and coalitions to reinforce the media component and initiate community events (1). Community commitment poses the greatest challenge: VERB sustainability will be determined not only by continued efforts to influence youth beliefs but also by persuading decision makers to deploy long-term resources at the community level.
VERB employs elements of a social marketing framework: it applies marketing techniques, including promotional strategies utilizing place (i.e., multiple channels and venues), with a clearly defined and branded product (i.e., encouraging youths to find their “verbs”) (1). Consistent with any social marketing effort (13), VERB proposes a voluntary exchange: tweens who take up activity, presumably in place of watching television or just sitting around, will derive the benefits of fun and social engagement. VERB clearly segments its audience; although mainstream VERB messages target all tweens, ethno-specific VERB messages target minority youth. If long-term sustainability of VERB is to be ensured, the initiative has the potential to develop into a formal social marketing campaign, which would require implementation of the environmental, policy, and regulatory supports suggested as essential elements of effective social marketing (13).
Comprehensive evaluation is an essential component of a mass media campaign. The first stages of evaluation include understanding the needs and motivations of the target audience and developing clear messages for them (4,14). This process results in a defined brand that is recognizable, seen across different initiatives, and deemed relevant by the target group. VERB evaluation commenced with a logic model to provide a conceptual framework for the intervention (2). Most importantly, VERB carried out substantial formative evaluation to develop relevant and acceptable messages for tweens (http://www.cdc.gov/youthcampaign/research/formative.htm). Often neglected in campaign development, formative research helps in producing messages and brands more likely to be acted upon by the target population. Then, evaluators seek short-term impact on campaign awareness, beliefs about being active, and social norms among tweens, while looking for long-term impact on physical activity behavior (2). VERB assesses these proximal and explanatory variables, as well as physical activity itself. Multiple measures of reported physical activity are required to overcome the methodological problems of self-report or parental report of physical activity in this age group. Campaign literature seldom explores dose-response relationships, but VERB developed high-dose media communities and compares their results with those of standard-dose communities.
The prevention of chronic disease cannot be modeled in a causal relationship to youth media campaigns, because the reduced risk of chronic conditions may not appear for decades. We can consider the VERB initiative a public health policy success if trends in childhood inactivity and obesity are reversed within a decade, consistent with Healthy People 2010 objectives. VERB-commissioned population surveys track proximal impact data; longer-term monitoring could occur through routine youth health surveys such as the Centers for Disease Control and Prevention’s Youth Risk Behavior Surveillance System (15). Process evaluation determines levels of VERB uptake by communities and minority populations in addition to measuring its impact on changing local policies and developing supportive community partnerships and sustainable physical environments.
Increases in rates of childhood obesity are not new, and declines in physical activity during adolescence are also well recognized in the scientific literature. Hence, it is timely that VERB was developed in an attempt to tackle these problems. VERB campaign efforts are not the end of the process but merely a well-resourced beginning upon which other efforts should build, synergize, and extend in partnership with community and state agencies to achieve population-level change. At the start of any such initiative, large-scale investment may be required as the spark plug to catalyze the first steps towards more active, healthier teenagers who have “found their verbs.”
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Bauman A. Commentary on the VERB™ campaign — perspectives on social marketing to encourage physical activity among youth. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0054.htm.
==== Refs
1 Wong F Huhman M Heitzler C Asbury L Bretthauer-Mueller R McCarthy S VERB™ — a social marketing campaign to increase physical activity among youth Prev Chronic Dis [serial online] 2004 Jul [15 June 2004] Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0043.htm 2004 7 1 3 A10
2 Huhman M Heitzler C Wong F The VERB™ campaign logic model: a tool for planning and evaluation Prev Chronic Dis [serial online] 2004 Jul [15 June 2004] Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0033.htm 2004 7 1 3 A11
3 Cavill N Bauman A Changing the way people think about health-enhancing physical activity: do mass media campaigns have a role? Journal of Sports Sciences Forthcoming.
4 Bauman A Precepts and principles of mass media campaign evaluation in Australia Health Promotion Journal of Australia 2000 10 89 92
5 Farrelly MC Niederdeppe J Yarsevich J Youth tobacco prevention mass media campaigns: past, present, and future directions Tob Control 2003 6 12 1 i35 i47 12773784
6 Kelder SH Pechmann C Slater MD Worden JK Levitt A The National Youth Anti-Drug Media Campaign Am J Public Health 2002 8 92 8 1211 1212 12144964
7 Brown JD Witherspoon EM The mass media and American adolescents' health J Adolesc Health 2002 31 6 Suppl 153 170 12470911
8 Randolph W Viswanath K Lessons learned from public health mass media campaigns — marketing health in a crowded media world Annu Rev Public Health 2004 25 419 437 15015928
9 Bauman A Bellew B Owen N Vita P Impact of an Australian mass media campaign targeting physical activity in 1998 Am J Prev Med 2001 21 1 41 47 11418256
10 Hillsdon M Cavill N Nanchahal K Diamond A White IR National level promotion of physical activity: results from England's ACTIVE for LIFE campaign J Epidemiol Community Health 2001 55 755 761 11553661
11 Bauman A McLean G Hurdle D Walker S Boyd J van Aalst I Evaluation of the national “Push Play” campaign in New Zealand — creating population awareness of physical activity N Z Med J 2003 116 1179 u535
12 Reger B Cooper L Booth-Butterfield S Smith H Bauman A Wootan M Wheeling Walks: a community campaign using paid media to encourage walking among sedentary older adults Prev Med 2002 8 35 285 292 12202072
13 Maibach EW Rothschild M Novelli W Glanz K Rimer B Lewis FM Social Marketing Health Behavior and Health Education: Theory, Research, and Practice 3rd ed. Indianapolis (IN) Jossey-Bass 2002 437 461
14 Barriers to children walking and biking to school — United States, 1999 MMWR Morb Mortal Wkly Rep 2002 8 16 51 32 701 704 12206284
15 Brener ND Kann L McManus T Kinchen SA Sundberg EC Ross JG Reliability of the 1999 youth risk behavior survey questionnaire J Adolesc Health 2002 31 4 336 342 12359379
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2022-01-26 23:17:59
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Prev Chronic Dis. 2004 Jun 15; 1(3):A02
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==== Front
Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_03_0020
Original Research
PEER REVIEWEDRecruiting Small Manufacturing Worksites That Employ Multiethnic, Low-wage Workforces Into a Cancer Prevention Research Trial
Barbeau Elizabeth M. ScD, MPH Department of Society, Human Development and Health, Harvard School of Public Health, Boston, Mass. Center for Community-Based Research, Dana-Farber Cancer Institute
44 Binney St, Boston, MA 02115 617-632-5390 [email protected]
Wallace Lorraine MPH Center for Community-Based Research, Dana-Farber Cancer Institute, Boston, Mass
Lederman Ruth MPH Center for Community-Based Research, Dana-Farber Cancer Institute, Boston, Mass
Lightman Nancy MM Center for Community-Based Research, Dana-Farber Cancer Institute, Boston, Mass
Stoddard Anne ScD Center for Community-Based Research, Dana-Farber Cancer Institute, Department of Biostatistics, University of Massachusetts, Amherst, Mass
Sorensen Glorian PhD, MPH Center for Community-Based Research, Dana-Farber Cancer Institute, Department of Society, Human Development and Health, Harvard School of Public Health, Boston, Mass
7 2004
15 6 2004
1 3 A042004
Introduction
Worksites, including those that employ multiethnic, low-wage workforces, represent a strategic venue for reaching populations at risk for developing cancer.
Methods
We surveyed 197 small manufacturing worksites prior to an effort to recruit their workforces into a randomized clinical trial designed to test the effectiveness of a cancer prevention intervention among multiethnic, low-wage manufacturing workers. This paper assesses the external validity of the trial based on three factors: the percentage of potential trial sites excluded from consideration, the percentage of eligible worksites that adopted the trial, and worksite characteristics associated with adoption.
Results
We found no statistically significant differences between worksites that adopted the trial and worksites that declined the trial with regard to employee demographics, anticipated changes in workforce size, and perceived importance and history of offering health promotion and occupational health and safety activities.
Conclusion
Small manufacturing worksites present a viable venue for reaching multiethnic, low-wage populations with cancer prevention programs, although program adoption rates may be low in this sector. Worksites that adopted the trial are likely to represent worksites deemed eligible for the trial.
==== Body
Introduction
Cancer risk associated with health behaviors and carcinogenic occupational exposures is concentrated among working-class employees, individuals with less education, and some racial and ethnic groups (1-14). Worksites are a strategic venue for reaching these at-risk populations to reduce cancer risk. Cancer prevention research in small manufacturing worksites is particularly important because small manufacturing worksites employ roughly 42% of all manufacturing workers (15), are less likely to offer health promotion programs and protection from occupational health and safety hazards (16-26), and have been largely understudied (27). Furthermore, according to national survey data, some subgroups of the workforce, including nonprofessionals, blacks, and individuals with less education, were least likely to work for companies that offer health promotion programs to employees (28). When programs are available, blacks report the highest participation levels among all racial and ethnic groups (28). These data highlight the importance of conducting cancer prevention research in small worksites to address excess cancer risk among workers of lower socioeconomic position and racial and ethnic minorities.
Within studies such as this one, it is critical that researchers assess and report on worksite-level consent to participate, also known as adoption rate. Glasgow et al recently introduced the RE-AIM (Reach, Efficacy or Effectiveness, Adoption, Implementation, and Maintenance) model to assess intervention impacts (29). This model includes a measure for adoption, in which adoption is measured as the percentage of eligible worksites that adopt or test a health promotion program.
Adoption rates also are assessed for representativeness, or how well worksites that elected to participate in a program represent all eligible worksites. Representativeness is measured by comparing the characteristics of eligible worksites that adopt a health promotion program to eligible worksites that decline to adopt. Both assessments are critical to establishing the external validity of worksite-based studies, that is, the extent to which worksites recruited into trials represent other worksites (30). This type of rigorous assessment of external validity, however, is rare.
Bull et al recently evaluated the external validity of worksite health promotion studies (30). They reviewed intervention studies on dietary change, smoking cessation, and physical activity published in 11 leading journals during the five years from 1996 through 2000. They discovered that, among the 24 published studies, only six (25%) reported the percentage of eligible worksites that elected to participate in a program; only two (8%) reported exclusion criteria; and none reported on representativeness. In the two studies that reported exclusion criteria (30-32), the number of employees determined exclusion, and one also excluded worksites based on turnover rates and non-English-speaking employees (31).
Using the RE-AIM measures of adoption, our paper overcomes shortcomings of prior worksite health promotion studies to report on the process and results of worksite recruitment and worksite characteristics associated with program adoption in Healthy Directions — Small Business (HD-SB), a randomized, controlled cancer prevention trial among small-sized manufacturing companies employing multiethnic, low-wage workforces. The purpose of this paper is to assess the external validity of the trial, based on the percentage of potential trial sites excluded from consideration, the percentage of eligible worksites that adopted the trial, and the characteristics associated with adoption.
Methods
Overview
To assist the reader in interpreting the results of this report, we begin with an overview of the HD-SB cancer prevention trial itself and then focus on how we recruited worksites. The main question under investigation in HD-SB is whether or not a cancer prevention intervention that integrates health promotion and occupational health protection leads to significant mean improvements in workers’ consumption of fruits and vegetables, levels of physical activity, smoking cessation, and reductions in workers’ exposure to occupational carcinogens in small manufacturing worksites that employ multiethnic, low-wage workforces. Participating worksites are randomly assigned to either an intervention or a minimal intervention control condition. The intervention worksites receive an 18-month intervention focused on physical activity, diet, smoking cessation, and occupational health and safety. The control worksites receive only smoking cessation programs. Our institutional review board approved the trial protocol; employee participation in the trial is voluntary.
The intervention is an integrated health-promotion/health-protection model (33) based on social ecological theory (34,35). This model addresses both workers’ personal behaviors and the hazards of their work environments. Interventions are conducted at three levels: individual workers (e.g., health education about diet, physical activity, occupational health and safety), organization (e.g., worksite food options, programs to support worker physical activity such as lunchtime walking groups, occupational health and safety policies), and physical environment (e.g., reduction of carcinogenic exposures).
Study population
The study population for this report is manufacturing worksites. We used the Dun and Bradstreet database to identify worksites with Standard Industrial Classification codes in the manufacturing group (Group D) that are located in and around a large northeast urban area in the United States and that employ between 50 and 150 workers. We selected manufacturing worksites because they are more likely than other worksites, such as those in the service sector, to use potential carcinogens in work processes. The worksite use of potential carcinogens allows us to intervene on cancer risks related to individual health behaviors as well as occupational exposures. We identified 224 companies in the Dun and Bradstreet database.
Pre-recruitment survey measures
After identifying the 224 companies, we conducted a pre-recruitment worksite survey to determine eligibility for participation in the HD-SB trial. The pre-recruitment survey took place from March through August 1999. Our study eligibility requirements included the following:
Employing a multi-cultural or multiethnic population (defined as 25% of workers being first- or second-generation immigrants or people of color).
Having an employee turnover rate of less than 20% in the previous year.
Being autonomous in decision-making power to participate in a study (if part of a larger parent company).
The survey asked respondents to indicate the total number of employees, the percentage of their workforce that was white and American-born, and the percentage of employee turnover within the last three years. To determine degree of autonomy, the survey asked respondents if they were able to make their own decision on program participation. In addition, the survey collected information about worksite characteristics (36) that we hypothesized would be positively associated with adoption, including perceived importance of and prior experiences with health promotion and protection programs and a positive financial outlook. The survey also asked respondents to rate their perception of the importance of health promotion and occupational health and safety activities on a 5-point Likert scale, to indicate if their worksite had previously offered such programs, and to say whether they anticipated increases, decreases, or no changes in workforce size in the next year (as an indicator of financial outlook).
Data collection
Research staff placed phone calls to the 224 companies identified in the Dun and Bradstreet database to verify contact information. We then mailed the pre-recruitment survey to the CEO and director of personnel/human resources with a cover letter requesting their assistance in completing the survey as part of a research project to develop educational health promotion and health protection programs for manufacturing businesses. The letter contained no additional information about the research project. We contacted non-responders by telephone within two weeks, and research staff conducted the survey over the telephone. We attempted to reach non-responders at least 10 times by telephone. We attempted to reach both the CEO and director of personnel/human resources to maximize the potential for response. If both responded, we accepted the responses of the CEO only, thereby standardizing this aspect of data collection.
The mailed survey administration method yielded an unacceptably low response rate (11%; n = 24). As a result, we shortened the pre-recruitment survey and attempted to reach non-responders by telephone. The longer version of the survey asked about factors that would assist us in planning for intervention implementation, such as shift schedules, estimated percentage of employees who speak specified languages, and barriers and facilitators to worksite health promotion. We eliminated these questions to create the shortened survey (Appendix), which focused only on the measures, reported herein, that we hypothesized would relate to adoption. Research staff re-contacted non-responders and administered the shortened survey by telephone to either the worksite's CEO or director of personnel/human resources, increasing the response rate to 88%.
Worksite recruitment
Once we deemed a worksite eligible to participate in the HD-SB trial based on the pre-recruitment survey, a member of the research staff contacted the survey respondent by telephone to describe the research trial and to assess interest in participating. If a company expressed interest, we conducted an in-person, on-site recruitment meeting to describe what would be required of participating companies, the specifics of the intervention condition, and the process of randomization to intervention or control condition. To participate, companies had to consent to allow employees to take baseline and final surveys, to allow research staff to conduct an industrial hygiene walk-through assessment of the worksite, and to conduct additional surveys with management on occupational health and organizational characteristics. If randomized to the intervention condition, worksites also were asked to
Permit between five and 10 employees to meet monthly as part of an employee team designated to assist project staff with program implementation.
Allow all employees at least 15 minutes per month during work time to attend project intervention activities.
Have a HD-SB staff industrial hygienist consult with management to make plans for improving occupational health and safety conditions.
Once a company had agreed verbally to participate in the trial, a research staff member and company representative signed a letter of agreement stating participation requirements and indicating informed consent, or adoption. Recruitment took place from September 1999 through December 2000, with the first company beginning its 18-month intervention in September 2000 and the last company beginning its intervention in December 2000. All interventions were concluded by June 2002.
Data analysis
Using data from the pre-recruitment survey, we determined the percentage of worksites that did not meet eligibility criteria and the percentage of worksites that met eligibility criteria and that adopted the program, and we compared the characteristics of companies that chose to participate in the trial with the companies that declined to participate. We calculated means and proportions to describe the sample and conducted Student t-tests (two-tailed) and chi-square tests for significance, with an alpha level of 5%.
Results
Of the 224 worksites, 197 (88%) completed the pre-recruitment survey and 131 (66%) of these met the trial eligibility criteria. Among the 66 (34%) worksites deemed ineligible, reasons for ineligibility included not being engaged in manufacturing (n = 15), size of workforce (n = 23), lack of autonomy in decision making (n = 9), or insufficient percentage of workers being first- or second-generation immigrants or people of color (n = 19). Of the 131 worksites that met eligibility criteria, 26 consented to participate in the trial, for an adoption rate of 20%. The worksites recruited to the trial manufacture a range of products, including medical equipment, dog food, specialty pumps, textiles for the automobile industry, and electronics. Three of the worksites provide services to other businesses (laundry and printing).
Characteristics of eligible companies (n = 131) that adopted the intervention (n = 26) are compared with companies that declined (n = 105) (Table). On average, among all eligible companies, about half of all employees were persons of color and/or first- or second-generation immigrants to the United States; approximately one half of the worksites anticipated increasing the size of their workforce in the next year; approximately one quarter had a history of offering health promotion activities; approximately one quarter perceived such programs to be important (mean scores of 3.0 and 3.3 out of possible 5); most had a history of occupational health and safety activities; and most perceived these to be very important (mean score of 4.5 and 4.4 out of possible 5). Worksites that adopted the program were slightly more likely (differences not statistically significant) to have a larger percentage of white and American-born workers; to anticipate an increase in workforce size in the next year; to have offered health promotion and safety programs in the past year; and to perceive health promotion as important. We have no meaningful data on the small number of worksites that declined to complete the pre-recruitment survey (n = 27) and so cannot compare them to those that did.
An additional seven worksites consented to participate but withdrew prior to the start of the intervention (categorized as decliners in presented data), citing concern about lack of time to participate in the trial given increasingly tight production schedules. These seven companies were also slightly more likely to perceive health promotion as important and to have offered health promotion programs in the past, compared to other eligible worksites (differences not statistically significant). Later in the trial, one worksite withdrew from the intervention condition and another withdrew from the control condition; both cited lack of time as reason for withdrawal.
Discussion
This paper reports on the process and outcome of our efforts to recruit small manufacturing worksites employing multiethnic, low-wage workforces into a cancer prevention intervention trial. Trial eligibility criteria excluded about 34% of worksites responding to our survey. Among eligible sites, 20% (26 of 131) adopted the program, a rate similar to other cancer prevention studies (13,33,37). An additional seven worksites initially consented but withdrew very early in the trial. Among worksites eligible to participate, we observed no statistically significant differences between those that consented and those that declined to participate in the trial with regard to workforce composition, anticipated expansion of the workforce (financial outlook), and perceived importance and history of heath promotion activities and occupational health and safety programs. In sum, we found that the racial and ethnic composition of the workforce, financial outlook, and perceived importance and experience with health programs were not barriers to adoption in cancer prevention trials in this sample of worksites.
The study had a few limitations. First, the survey relied on self-reports by a worksite representative, and we did not attempt to validate the information provided. Second, using the RE-AIM measures, we attempted to assess worksite participation in a cancer prevention research trial as a proxy measure for adoption of a cancer prevention program. Participation in a research trial is not the same as adoption of a program. And finally, our pre-recruitment survey did not contain measures that allowed us to characterize differences between adopters and decliners, suggesting that additional measures may be needed, the development of which might rely on qualitative, open-ended questions on factors that promote or inhibit adoption. The survey administrators noted anecdotally that employer reasons for adoption included having a family member with a history of cancer; viewing participation as a low-cost, value-added benefit for employees during a time of tight labor markets; wanting to take advantage of our occupational health and safety expert consultations; and believing that a healthy workforce is a more productive one. Common reasons noted by employers for declining to participate were lack of time and poor labor-management relations. These reasons may form the basis for distinguishing adopters and decliners in recruitment surveys for future trials.
Our findings have several important implications for the HD-SB trial and for other future worksite-based trials. First, although our adoption rate was 20%, a systematic assessment of the adoption rate using the RE-AIM framework indicates strong external validity for HD-SB trial findings: we found no significant differences between eligible worksites that adopted the cancer prevention trial and those that declined. We may generalize the findings of our main trial to other small manufacturing businesses that are located in urban areas and employ multiethnic, low-wage workers. The application of the RE-AIM measures for worksite adoption used here represents a key strength of our trial: few prior studies have reported explicitly on the percentage and representativeness of worksites that are willing to adopt or try a health promotion program (32). Second, the results provide guidance to future researchers and practitioners in estimating likely rates of adoption and early withdrawals. When recruiting small manufacturing worksites, which may be particularly vulnerable to volatile economic conditions and production schedules, it may be necessary to recruit additional worksites to allow for early withdrawals and avoid threatening the trial’s statistical power. A related point is that when attempting to reach worksites to assess eligibility for recruitment, researchers ought to use a short survey instrument that they can administer conveniently, preferably by telephone. Third, the high mean level of reported importance of occupational health and safety programs among all eligible worksites is noteworthy, suggesting that these programs may represent an attractive intervention component for small manufacturing businesses. This level of interest in health and safety has not been evident in studies of larger manufacturing worksites (33,37).
Recruitment for this trial took place within a larger social context: the decline of the U.S. manufacturing sector. U.S. manufacturing companies often are in precarious financial situations, or they may perceive that they have too little time to commit to a health promotion trial. On the other hand, they may view such an endeavor as a “free” resource. Our anecdotal data support both of these hypotheses, which can be subjected to rigorous assessment in future trials.
Reducing racial/ethnic and class-based health disparities is a major focus for the U.S. Public Health Service (12,38). Intervention research is essential to developing effective methods for reducing the disproportionate cancer risk associated with health behaviors and occupational exposures among immigrant, multiethnic and multi-racial, less-educated, and low-wage workers. Our results indicate that small manufacturing worksites are a viable community-based channel for reaching low-wage, multiethnic populations with cancer prevention programs, but that we can expect low adoption rates within this sector. Future intervention studies in these settings need to address the concerns of small businesses and to assess systematically the worksite characteristics that promote participation in trials and, ultimately, program adoption.
Funding for this study is provided by National Cancer Institute grant number 5 P01 CA75308-02 and Liberty Mutual Insurance Company. The authors thank Kathleen Yaus for her assistance with literature reviews, Cora Roelofs for her helpful comments on an earlier version of this paper, and Richard Martins for administrative assistance.
Appendix
Telephone Survey of Small Manufacturing Worksites That Employ Multiethnic, Low-wage Workforces, Northeastern United States, 1999
Hello, my name is ______________________. I am calling from Dana-Farber Cancer Institute. We recently sent your company a questionnaire for a project we are conducting with small businesses in the Boston area. The questionnaire was called the “Health Survey of Small Businesses in Massachusetts.” We have reviewed the survey and have made changes to shorten it. Since we did not receive a completed survey from your company, would you be able to take about five minutes now to answer a few questions?
Today’s Date:
Your Company’s Name:
Your Name:
Your Title:
Your Phone Number:
Your Fax Number:
Do manufacturing or production operations go on at this worksite? (Yes/No)
About how many permanent employees working 20 hours or more per week are there in your company? Do not include temporary workers. (Total number)
About how many of those employees would you say are blue collar or directly involved in the manufacturing or production process? (Number)
About how many are piece workers? (Number)
Approximately what percentage of your workforce is represented by union(s)? (Percentage)
About what percentage or your workforce is white/American-born? (Percentage)
Do you anticipate your workforce will increase, downsize, or have no change in the next year? (Check one only)
In the past year, has your company offered any health promotion programs? (Yes/No) Check all that apply. Use the following list as prompts:
Nutrition classes
Exercise classes
Weight control classes
Health fairs
Smoking cessation classes
Safety Programs
Other (text)
In the past year, has your company offered any safety programs? (Yes/No)
About what percentage of your employees are currently covered by any amount of company paid health insurance? (Percentage)
If you are talking to the Human Resource Director, skip to Question #12.
How important do you think it is to have worksite health promotion programs in your company? For example, nutrition, exercise classes, smoking cessation programs or material.
Not at all important Very Important
1 2 3 4 5
How important do you think it is to have worksite safety programs in your company?
Not at all important Very Important
1 2 3 4 5
In your opinion, how important does your company management think it is to have worksite health promotion programs in your company? For example, nutrition, exercise classes, smoking cessation programs or material.
Not at all important Very Important
1 2 3 4 5
In your opinion, how important does your company management think it is to have worksite safety programs in your company?
Not at all important Very Important
1 2 3 4 5
I would like to thank you for your participation in the Health Survey of Small Businesses. One of the purposes of this survey is to identify potential participants for the Cancer Prevention in Small Businesses project, funded by the National Cancer Institute. The goal of the project is to develop a national model for worksite cancer prevention. The study offers two years of health programming provided by experienced staff at no cost to you. We will focus on healthy eating, increased physical activity, and safety issues.
Are you able to make the decision to participate in a program like this one on your own? (Yes/No) Who else would have to be consulted?
Thank you for your participation in this survey.
Figures and Tables
Table Comparison of Characteristics of 131 Eligible Worksites That Adopted or Declined Cancer Prevention Intervention for Employees, Northeastern United States, 2000a
Worksite Characteristic Declined Intervention
n = 105 Adopted Intervention
n = 26
Mean percentage of workforce white and American-born 52.2% 60.6%
Proportion that anticipate increase in number of employees in next year 49.0% 53.9%
Proportion that offered health promotion programs in past year 24.8% 26.9%
Proportion that offered safety programs in past year 84.6% 88.5%
Mean perceived importance of worksite health promotion programs in company (1 = low; 5 = high) 3.0 3.3
Mean perceived importance of worksite safety programs in company (1 = low; 5 = high) 4.5 4.4
a No differences were found to be statistically significant, based on Student t-tests (two-tailed) and chi-square tests.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Barbeau EM, Wallace L, Lederman R, Lightman N, Stoddard A, Sorensen G. Recruiting small manufacturing worksites that employ multiethnic, low-wage workforces into a cancer prevention research trial. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/03_0020.htm.
==== Refs
1 Sorensen G Pechacek T Pallonen U Occupational and worksite norms and attitudes about smoking cessation Am J Public Health 1986 76 5 544 549 3963283
2 Sorensen G Stoddard A Hammond SK Hebert JR Avrunin JS Ockene JK Double jeopardy: workplace hazards and behavioral risks for craftspersons and laborers Am J Health Promot 1996 10 5 355 363 10163305
3 Siegel PZ Frazier EL Mariolis P Brackbill RM Smith C Behavioral Risk Factor Surveillance, 1991: monitoring progress toward the nation's Year 2000 Health Objectives MMWR Surveill Summ 1993 42 SS-4 1 30
4 Pierce JP Fiore MC Novotny TE Hatziandreu EJ Davis RM Trends in cigarette smoking in the United States. Educational differences are increasing JAMA 1989 261 1 56 60 2908995
5 Patterson BH Block G Food choices and the cancer guidelines Am J Public Health 1988 78 3 282 286 [published erratum in: Am J Public Health 1988 Jun;78 (6):620] 3341498
6 Sorensen G Hunt MK Cohen N Stoddard A Stein E Phillips J Worksite and family education for dietary change: the Treatwell 5-a-Day program Health Educ Res 1998 12 13 4 577 591 10345908
7 Blair SN Kohl HW Barlow CE Physical activity, physical fitness, and all-cause mortality in women: do women need to be active? J Am Coll Nutr 1993 8 12 4 368 371 8409097
8 Covey LS Zang EA Wynder EL Cigarette smoking and occupational status: 1977 to 1990 Am J Public Health 1992 9 82 9 1230 1234 1503163
9 Miller BA Kolonel LN Bernstein L Young JL Swanson GM West DW Racial/ethnic patterns of cancer in the United States 1988-1992 Bethesda (MD) National Cancer Institute
10 Nelson DE Emont SL Brackbill RM Cameron LL Peddicord J Fiore MC Cigarette smoking prevalence by occupation in the United States. A comparison between 1978 to 1980 and 1987 to 1990 J Occup Med 1994 36 5 516 525 8027876
11 Leigh J Occupations, cigarette smoking, and lung cancer in the epidemiologic follow-up to the NHANES I and the California Occupational Mortality Study. Bull NY Acad Med 1996 73 2 370 397
12 President's initiative to eliminate racial and ethnic disparities in health [Internet] Washington (DC) U.S. Department of Health and Human Services, HHS Data Council Working Group on Racial and Ethnic Data 1999 12 [cited 2003 Sept 23] Available from: URL: http://aspe.hhs.gov/DATACNCL/racerpt/execsumm.htm
13 Jason LA Salina D McMahon SD Hedeker D Stockton M A worksite smoking intervention: a 2 year assessment of groups, incentives and self-help Health Educ Res 1997 3 12 1 129 138 10166900
14 Barbeau E Krieger N Soobader M Working class matters: socioeconomic disadvantage, race/ethnicity, gender and smoking in NHIS 2000 Am J Public Health 2004 2 94 2 269 278 14759942
15 1996 U.S. Department of Commerce, Economics and Statistics Administration, Bureau of the Cenus. County business patterns 1994, United States Washington (DC) U.S. Government Printing Office
16 Fielding JE Piserchia P Frequency of worksite health promotion activities Am J Public Health 1989 79 16 20 2909175
17 Eakin JM Leaving it up to the workers: sociological perspective on the management of health and safety in small workplaces Int J Health Serv 1992 22 4 689 704 1399176
18 MacKinnon B Work-related injuries and illnesses: small employers, 1977-1985 Edmonton (Alberta) Research Branch, Occupational Health and Safety Division, Government of Alberta, Edmonton 1987
19 Tolonen M Hassi J Arrangement of municipal occupational health services for small work places: conclusions of a survey of 163 small workplaces Scand J Work Environ Health 1979 5 2 50 59 524093
20 1995 Research Division, Rhode Island Department of Economic Development. Rhode Island jewelry industry Providence (RI) Research Division Rhode Island Department of Economic Development
21 Pedersen DH Sieber W 1988 National occupational exposure survey volume 3: analysis of management, interview responses Cincinnati (OH) Department of Health and Human Services, National Institute for Occupational Safety and Health
22 1993 Office of Regulation Analysis. Description and evaluation of medical surveillance programs in general industry and construction. Final report Washington (DC) U.S. Department of Labor, Occupational Safety and Health Administration, Office of Regulation Analysis, Directorate of Policy
23 Eakin JM Lamm P Limborg HJ Frick K J PL Quinlan M Wilthagen T International perspectives on the promotion of health and safety in small workplaces Systematic OHS management: perspectives on an international development Amsterdam (The Netherlands) Elsevier 2000 227 247
24 1993 U.S. Department of Health and Human Services. National survey of worksite health promotion Washington (DC) U.S. Public Health Service
25 Hollander RB Lengermann JJ Corporate characteristics and worksite health promotion programs: survey findings from Fortune 500 companies Soc Sci Med 1988 26 5 491 501 3127893
26 2002 National Institute for Occupational Safety and Health. Safety and health resource guide for small businesses Cincinnati (OH) U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention
27 Janer G Sala M Kogevinas M Health promotion trials at worksites and risk factors for cancer Scand J Work Environ Health 2002 28 3 141 157 12109553
28 Grosch J Alterman T Petersen M Murphy L Worksite health promotion programs in the U.S.: factors associated with availability and participation Am Journal Health Promot 1998 13 1 36 45 10186933
29 Glasgow RE Vogt TM Boles SM Evaluating the public health impact of health promotion interventions: the RE-AIM framework Am J Public Health 1999 89 9 1322 1327 10474547
30 Bull SS Gillette C Glasgow RE Estabrooks P Work site health promotion research: to what extent can we generalize the results and what is needed to translate research to practice? Health Educ Behav 2003 30 5 537 549 14582596
31 Sorensen G Stoddard A Ockene JK Hunt MK Youngstrom R Worker participation in an integrated health promotion/health protection program: results from the WellWorks project Health Educ Q 1996 23 2 191 203 8744872
32 Willemsen MC de Vries H van Breukelen G Genders R Long-term effectiveness of two Dutch work site smoking cessation programs Health Educ Behav 1998 25 4 418 435 9690101
33 Sorensen G Himmelstein JS Hunt MK Youngstrom R Hebert JR Hammond SK A model for worksite cancer prevention: integration of health protection and health promotion in the WellWorks project Am Journal Health Promot 1995 10 1 55 62 10155659
34 McLeroy KR Bibeau D Steckler A Glanz K An ecological perspective on health promotion programs Health Educ Q 1988 15 4 351 377 3068205
35 Bandura A Prentice Hall Englewood Cliffs (NJ) Social foundations of thought and action: a social cognitive theory 1986 544
36 Biener L DePue JD Emmons KM Linnan L Abrams DB Recruitment of worksites to a health promotion research trial. Implications for generalizability J Occup Med 1994 36 6 631 636 8071725
37 Sorensen G Stoddard A LaMontagne A Emmons K Hunt MK Youngstrom R A comprehensive worksite cancer prevention intervention: behavior change results from a randomized controlled trial (United States) Cancer Causes Control 2002 13 6 493 502 12195637
38 2000 U.S. Department of Health and Human Services. Healthy people 2010: understanding and improving health Washington (DC) U.S. Government Printing Office 76
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Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_03_0013
Original Research
PEER REVIEWEDSun Protection Policy in Elementary Schools in Hawaii
Maddock Jay PhD Assistant Professor Department of Public Health Sciences, University of Hawaii
1960 East-West Rd D104, Honolulu, HI 96822 [email protected]
808-956-5779
Eakin Paul MD Department of Pediatrics, John A. Burns School of Medicine University of Hawaii at Manoa
Techur-Pedro Angela Department of Public Health Sciences, University of Hawaii at Manoa
Kaliko Raphael Department of Public Health Sciences, University of Hawaii at Manoa
Derauf D. Christian MD Department of Pediatrics, John A. Burns School of Medicine, University of Hawaii at Manoa
7 2004
15 6 2004
1 3 A052004
Introduction
Childhood sun exposure is a major risk factor for skin cancer, the most common form of cancer in the United States. Schools in locations that receive high amounts of ultraviolet radiation have been identified as important sites for reducing excessive sun exposure.
Methods
The objective of this study was to determine the prevalence of sun protection policies, environmental features, and attitudes in public elementary schools in Hawaii. Surveys were sent to all (n = 177) public elementary school principals in Hawaii. Non-respondents were called three weeks after the initial mailing. The survey asked about sun protection policies, environmental features, and attitudes toward sun protection. The survey was designed to measure all seven components of Guidelines for School Programs to Prevent Skin Cancer, issued by the Centers for Disease Control and Prevention.
Results
Seventy-eight percent of schools responded to the survey. Only one school had a written school policy. Almost all schools (99.3%) scheduled outdoor activities during peak sun hours. School uniforms rarely included long pants (6.5%), long-sleeved shirts (5.1%), or hats (1.5%). Current policies did not support or restrict sun protection habits. Almost one third of those surveyed were in favor of a statewide policy (28.1%), and most believed excessive sun exposure was an important childhood risk (78.9%), even among non-white students (74.5%).
Conclusion
Results of this study suggest the following: 1) school personnel in Hawaii are concerned about childhood sun exposure; 2) current school policies fail to address the issue; 3) most schools are receptive to developing sun protection policies and programs; and 4) students appear to be at high risk for sun exposure during school hours.
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Introduction
In the United States, the incidence of skin cancer is increasing faster than that of any other type of cancer (1). More than 1 million cases of skin cancer occur every year in the United States, nearly the same number as all other cancers combined (2). Basal cell carcinoma and squamous cell carcinoma are the most prevalent types of skin cancer but are the most curable. These two cancers accounted for almost 1.3 million new cases in the United States in 2002 (3). Melanoma, while much less prevalent, has a higher mortality rate, accounting for 75% of all skin cancer deaths (3). In Hawaii, melanoma incidence rates are similar to the nation at 20.5 per 100,000 for males (compared to 19.0 per 100,000 for the United States) and at 10.3 per 100,000 for females (compared to 12.0 per 100,000 for the United States) (4). Melanoma incidence rates among white males in Hawaii are rising rapidly from less than 30 per 100,000 between 1975 and 1979 to 62.8 per 100,000 from 1995 to 2000 (4).
Estimates show that the majority of lifetime sun exposure takes place during youth and that 50% to 80% of lifetime cumulative sun exposure occurs prior to age 18 (5). In addition, blistering sunburns prior to age 20 have been associated with an increased risk of developing malignant melanoma (6). Exposure early in childhood appears to be particularly important, with one study showing that children aged nine to 10 sustain more sun exposure than adolescents aged 14 to 15 (5). Another study using ultraviolet (UV) light-sensitive badges in six schools in England showed that primary school students had higher levels of exposure than secondary school students (7). Furthermore, consistent use of sunscreen with a sun protection factor of 15 throughout childhood and adolescence may reduce the lifetime incidence of basal and squamous cell carcinomas by 78% (5).
In the United States, Hawaii is the only state located in the tropics. Additionally, most Hawaii residents have a lifestyle that emphasizes outdoor activities. Hawaii's tropical location results in more direct UV radiation from the sun than non-tropical locations; research has shown that as latitude decreases, measured UVB radiation and melanoma incidence increases (8). Hawaii has a multiethnic population, but skin cancer occurs in all ethnic groups, especially in tropical climates (9). Also, due to Hawaii’s warm climate, schools often are built with sprawling, open layouts to take advantage of cooling trade winds. Because of this, students are frequently exposed to direct sunlight when walking between classes or during recess and physical education class. Several studies have been conducted evaluating institutional sun protection policy, including observational studies of children at daycare (10) and a national survey of elementary schools (11). In Hawaii, skin cancer prevention practices have been studied (12), and public health interventions in outdoor recreational environments have been able to show modest improvements in children’s sun protection behaviors (13-15), but no studies have been published on sun exposure or protection in schools. In the United States, only 12 states require education about skin cancer prevention at the elementary school level; Hawaii is not one of these states (16).
Sun exposure among children in the United States is common. In a national study of children aged six months to 11 years, children spent a median of 20 hours per week outdoors, including 10 hours while at school (17). Sunscreen (61.8%) and shade (26.5%) were the most common methods of sun protection in this study (17). Parents of children aged five to 10 at pools in Boston and Hawaii reported similar incidence of sunburn (40%) among their children during one summer (18). In this sample, sunscreen was the most widely used form of sun protection, with very few children wearing sunglasses or hats (18).
In April 2002, the Centers for Disease Control and Prevention (CDC) released Guidelines for School Programs to Prevent Skin Cancer (19). This document outlines seven recommendations for skin cancer prevention in schools:
Establish policies to reduce sun exposure.
Provide environmental supports for sun protection.
Provide health education on sun safety.
Involve the family in sun safety.
Provide professional development for staff for sun protection.
Support sun safety with health services.
Evaluate the effectiveness of these efforts.
With these seven guidelines in mind, we developed a survey to create a baseline measure of how well public schools in Hawaii were meeting these recommendations. We hypothesized that 1) elementary school children in Hawaii receive a significant amount of sun exposure during school hours, and 2) school policies rarely attempt to limit sun exposure or teach about the dangers inherent to sun exposure.
Methods
In September 2002, a list of all public elementary schools (n = 177) in the state of Hawaii was obtained from the State Department of Education. Elementary schools were defined as schools that contained the first through fifth grades. Eight schools (4.5%) also included the seventh and eighth grades and were included in the sample. Students in sixth grade and kindergarten were included in approximately half of the schools, depending on the school complex. Private schools were not included in the study because of the difficulty in obtaining a sample that adequately represented charter schools, home schools, and other small schools (with less than 50 students), which are prevalent in Hawaii.
The 26-item survey queried current sun protection policies, amount of time students spent outside during peak sun hours, the use of sunscreen and sun-protective clothing by students and staff, and attitudes about the importance of sun protection. It was designed to measure all seven components of the CDC guidelines: policy; environmental change; education; family involvement; professional development; health services; and evaluation (19). Survey items were based on previous studies in the United States (11) and Australia (20). Additional items were generated by the study team. The survey instrument is available in the Appendix. All procedures were approved by the University of Hawaii's Institutional Review Board and the Hawaii Department of Education.
The survey was pre-tested with a convenience sample of seven elementary school administrators to determine readability and face validity of the instrument. Seven school administrators who had agreed to participate in a larger study to develop observational methods for sun protection among elementary school children were mailed a survey and then visited by a trained research staff member one to two weeks later. The staff member completed a structured interview with each school administrator following the written survey and assessed the comprehension of each question. None of the administrators reported any problems in understanding or completing any of the questions. Since no data were changed on these forms, the survey responses were pooled with all other responses.
During October 2002, cover letters explaining the study, survey, and return postcards were mailed to all remaining public elementary schools (n = 170) in the state of Hawaii. The surveys were returned anonymously; a return postcard sent by participants under separate cover identified schools that had completed the survey. Non-respondents were contacted by telephone three weeks after the initial mailing and encouraged to participate.
Data analysis was conducted using SPSS 11.5. Data were analyzed primarily by calculating percentages. For questions using a 5-point Likert scale, respondents who endorsed an item with a 4 or 5 were coded as agreeing with the statement. Schools were also grouped into either having a high enrollment (more than 40%) or a low enrollment (less than of 40%) of white students. Mean differences in the endorsement of items were assessed using t-tests.
Results
Overall, 78% (n = 138) of the schools responded to the survey. Most of the respondents were principals (59%), vice principals (9%), or administrators (14.9%). Schools reported substantial ethnic diversity of their student bodies, with an average of 21.2% white, 26.9% Asian, 26.7% Native Hawaiian, and 10.7% Pacific Islander. Only 15.9% of schools that reported the ethnic composition of the student body reported 40% or more white students. The postcard was returned by 110 (62.1%) of the schools. Return of postcards was similar for Oahu (61.8%) and the neighbor islands (64.8%), (χ2[1] = .147, P = .70).
Table 1 presents results of the school survey. Only one school (0.7%) reported having a written policy to limit student sun exposure. However, 28.1% of schools believe that a statewide policy is needed. Of the 14.5% of schools with uniforms, only 1.5% include hats, 6.5% long pants, 5.1% long skirts, and 5.1% long sleeves as protective clothing options. Many schools allow students to wear protective clothing when outside. Hats (86.9%), sunglasses (72.9%), and sunscreen (98.5%) are allowed by most schools, and only 6.7% of schools require a doctor’s note to bring sunscreen to school. Very few schools provide sunscreen on field trips (4.3%).
Almost all (99.3%) of schools schedule outdoor activities between 10 AM and 2 PM, with 28.3% of schools scheduling at least half of their outdoor activities during this time. Shade-producing structures are common among schools (75.4%), but most (81.8%) cover less than 25% of play areas. Less than one quarter (22.2%) of school personnel reported that they often or always practice sun protection behaviors when supervising outdoor activities.
Less than half (47.7%) of schools teach sun protection as part of the health education curriculum. Almost half (48.9%) of schools have some students expressing concern about excessive sun exposure. Most schools (85.2%) are interested in interactive training sessions on the dangers of sun exposure. Only one fifth (20.1%) of schools send information home to parents about keeping their children sun-safe.
Less than 30% of schools reported that they provide any training in sun protection practices to physical education (PE) teachers, school administrators, and teachers. More than half of schools (53.6) reported having school nurses trained or knowledgeable about sun protection behaviors, but only 12.1% of schools reported that these nurses always or often instruct students about practicing sun protection behaviors. Only 6.6% of respondents had seen the CDC school guidelines, while 87.4% were interested in receiving a copy.
Most respondents (78.9%) believed that excessive sun exposure during childhood is an important health concern, and 74.4% believed this is also true for non-white students. However, only 19.8% of schools believed that they have better than average measures to protect their children from the sun.
No significant differences were seen between schools with a high proportion of white students (more than 40%) and schools with a low proportion (less than 40%) with regard to 1) the need for a statewide policy, instruction on sun protection behaviors, or information sent home to parents; 2) the perception that the school has adequate measures in place; and 3) the number of students expressing concern about sun exposure. A non-significant trend (P = .06) exists with schools with a higher percentage of white students believing that excessive sun exposure is an important health concern (Table 2).
Discussion
Among public elementary schools in Hawaii, we found an absence of policies to reduce sun exposure and a lack of knowledge about the CDC guidelines to prevent skin cancer. We found that few teachers receive professional development in sun protection practices, and that uniform policies do not usually require protective clothing. Most current school policies do not prohibit or encourage sun protection behaviors, and most administrators stated that they had never thought about the effects of sun exposure on students during school time. Despite these results, administrators are largely in favor of stronger policies and believe sun exposure is an important health issue. Our results suggest the need for state education departments to develop sun protection policies, environmental supports, and a sun protection curriculum.
Limitations of our study include the selection bias inherent to survey studies and the possibility that the person responding to the survey is not well-informed about school conditions. A further study is currently underway that will address some of these issues through site visits and direct observation of students’ sun protection behaviors.
Our results are similar to the study done in 1997 by Buller et al in which only 3.4% of schools in the United States reported written sun protection policies, while 76.4% of principals were willing to make environmental changes (11). The Buller study, however, had several methodological limitations, including a low response rate (41%) and a high proportion of schools in cities with low UV intensity (63%). Our findings are likely to be similar to findings in other states that do not have a comprehensive statewide or district-wide policy, because administrators appear to be largely in favor of sun protection measures but have not made them a priority for their schools.
The results from Hawaii and the United States contrast starkly with results from Australia. In 1993, the Victoria Anti-Cancer Council developed a SunSmart school accreditation program to recognize schools for having comprehensive sun protection policies (20). From 1992 to 1997, sun protection policies in Victoria increased from 17% of schools to 76% (20,21). Sun protection practices are also much more rigorous in Victoria, with 78% of schools recommending broad-brimmed hats, 96% providing sunscreen or encouraging parents to supply it, and 93% teaching sun protection in classes (20). The CDC has recommended that school districts conduct periodic evaluations to assess how well schools are meeting the guidelines (19). To our knowledge, this study represents the first attempt to evaluate the sun protection policy and environment of all public elementary schools in a state. Given the substantial lifetime sun exposure burden encountered during the elementary school years, sun protection policies have the potential to significantly alter an individual's risk for later development of skin cancers as adults. Results from this study and from programs in Australia are encouraging because most school principals have a positive outlook on sun protection policies. The development of an accreditation program by a national or state group could lead to great changes in elementary school sun protection practices. The Guidelines for School Programs to Prevent Skin Cancer should also be widely distributed along with model policies to schools — especially to district- and state-level administrators — to encourage their adoption.
This study was funded by a grant from the Hawaii Community Foundation.
Appendix
For each question please mark the one answer that most closely represents the situation for your school.
1. Is your school public or private?
Public
Private
2. What is your position at your school? ______________________________
3. Does you school have a written policy that attempts to limit your students' sun light exposure?
Yes
No
4. Does your school have outdoor activities scheduled between the hours of 10 AM and 2 PM? (e.g., PE classes, recess)
Yes
No
If yes, what percent of outdoor activities fall during this time?
A. Less than 25%
B. Between 26-50%
C. Between 51-75%
D. More than 75%
If no, please go to number 3.
5. Do your outdoor activity areas contain shade-producing structures such as trees or roof overhangs?
Yes
No
6. What percentage of your play/activity area is covered by shade?
A. Less than 25%
B. Between 26-50%
C. Between 51-75%
D. More than 75%
7. Does your school utilize mandatory uniforms?
Yes
(please answer number 8)
No
(skip to question 9)
8. Does the school uniform include any of the following options?
(Please circle all that apply).
Long sleeves
Hats
Long pants
Long skirts
9. Do the school personnel who supervise outdoor activities use sun protection behaviors such as wearing a hat, long sleeves or umbrellas the majority of the time?
Always Sometimes Never
5 4 3 2 1
10. Does your school allow students to wear hats when outdoors?
Yes
No
If not, why not?
11. Are students allowed to wear sunglasses during outdoor activities at school?
Yes
No
If not, why not?
12. Does your school allow students to wear sunscreen when outdoors?
Yes
No
13. Does your school provide sunscreen for students to use during school field trips?
Yes
No
14. Would the use of sunscreen by a student require a note from their doctor?
Yes
No
15. To what extent do you feel that excessive sun exposure during childhood is an important health concern?
Very much Average Not at all
5 4 3 2 1
16. To what extent do you feel that excessive sun exposure during childhood is an important health concern in non-white students?
Very much Average Not at all
5 4 3 2 1
17. Do you feel that your school has adequate measures in place to limit the amount of sun exposure students receive during the school day?
Very much Average Not at all
5 4 3 2 1
Please explain why or why not you have these measures in place:
18. Are you aware of students expressing concern about excessive sun exposure during school activities to you or to other faculty?
Very much Average Not at all
5 4 3 2 1
19. Does your school teach sun protection as part of the health education curriculum?
Yes
No
If yes, in which grades?
20. Does your school send home information to parents about keeping their children sun safe?
Yes
No
21. Are any of the following staff members routinely trained in sun protection practices? (Circle all that apply)
Teachers
School administrators
PE teachers
School nurses
Others
22. Do the school health nurses routinely instruct students on sun protection behaviors?
Always Sometimes Never
5 4 3 2 1
23. Would you be interested in having us give an interactive session with your students about the dangers of sun exposure?
Very much Average Not at all
5 4 3 2 1
24. Do you feel that a statewide policy attempting to limit sun exposure to children during school is needed?
Very much Average Not at all
5 4 3 2 1
25. The Centers for Disease Control and Prevention recently published guidelines for schools to help prevent skin cancer. Have you seen this document?
Yes
No
26. Would you like to receive a copy of this document?
Yes
No
This is the end of the survey. Thank you very much for your time and effort in completing this survey.
Figures and Tables
Table 1 Existing School Policies and Education on Sun Protection, Public Elementary Schools, Hawaii, 2002
Policies % Respondents (n = 138)
Schools with written policy that limits students' exposure to sunlight during outdoor activities 0.7
Schools that feel statewide policy to limit sun exposure to children during school is needed
5 - Very much 11.1
4- 17.0
3 - Average 28.9
2- 25.2
1 - Not at all 17.8
Schools that allow the use of sunscreen during outdoor activities 98.5
Schools that require doctor's prescription or note for student to use sunscreen lotion 6.7
Schools that allow the use of hats during outdoor activities 86.9
Schools that allow students to wear sunglasses when outdoors 72.9
Schools that enforce a uniform policy 14.5
Uniform options among schools with uniform policies
Hats 1.5
Long pants 6.5
Long skirt 5.1
Long sleeves 5.1
Environmental Support
Schools have outdoor activities between 10 AM and 2 PM 99.3
Percent of activities that fall between 10 AM and 2 PM
< 25% 51.5
26-50% 20.1
51-75% 13.4
> 75% 14.9
Percent of play or activity covered by shade
< 25% 81.8
26-50% 12.4
51-75% 3.6
> 75% 2.2
Schools that provide sunscreen for students during school field trips 4.3
Schools that provide shade-producing structures 75.4
Schools who claim that students are concerned about excessive sun exposure during outdoor activities 48.9
School personnel who practice sun protection behaviors while supervising outdoor activities
Always 3.7
Often 18.5
Sometimes 52.6
Seldom 21.5
Never 3.7
Education
Percent of schools that teach sun protection as part of the health education curriculum
Yes 47.7
No 47.7
Not sure 4.7
Family Involvement
Schools that send home information to parents about sun safety 20.1
Professional Development
Staff/teachers trained or knowledgeable about sun damage and sun protection practices
Physical education teachers 27.5
Administrators 21.7
School nurses 53.6
Teachers 24.6
Others 8.0
School staff had seen CDC sun protection guidelines
Yes 6.6
No 92.7
Don't Know 0.7
Health Services
School health nurses routinely instruct students on sun protection behaviors
Always 3.0
Often 9.1
Sometimes 50.0
Seldom 25.0
Never 12.9
Attitudes toward sun exposure
Schools that believed that excessive sun exposure during childhood is an important health concern 78.9
Schools that believed that excessive sun exposure during childhood is an important health concern for non-white children 74.4
Schools that believed they have better than average measures to protect their children from the sun 19.8
Table 2 Results of Survey on Sun Protection Policies Among Public Elementary Schools With a High and Low Proportion of White Students, Hawaii, 2002
Low
(< 40%) White Population
(n = 111)a High
(> 40%) White Population
(n = 21)
Survey Question Mean (SD) Mean (SD) t value
A statewide policy to limit sun exposure in schools is needed 2.84 (1.24) 2.85 (1.31) t127 = -0.02, P = .98
School personnel instruct students on sun protection behaviors 2.68 (0.90) 2.81 (0.98) t124 = -0.61, P = -.54
School sends information home to parents on sun protection behaviors 1.79 (0.41) 17.6 (0.44) t126 = 0.33, P = .75
Schools teach sun protection as part of the curriculum 1.58 (0.59) 1.55 (0.60) t122 = 0.19, P = .86
Amount of concern that students express about sun exposure 1.65 (0.71) 1.65 (0.81) t126 = -0.01, P = .99
Perception that school has adequate measures to limit students' sun exposure 2.86 (1.00) 2.62 (1.12) t129 = 0.99, P = .32
Perception that excessive sun exposure is an important health concern 4.05 (0.91) 4.36 (0.73) t129 = -0.15, P = .14
Perception that excessive sun exposure is an important health concern among non-white students 4.13 (0.85) 4.50 (0.74) t130 = -1.88, P = .06
School personnel practice sun protection behaviors while outside 3.03 (0.82) 3.10 (0.64) t127 = -0.37, P = .71
a Six schools did not report student body ethnicity.
b All survey questions used a 5-point Likert scale, with 4 or 5 coded as agreeing with the survey statement.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Eakin P, Maddock J, Techur-Pedro A, Kaliko R, Derauf DC. Sun protection policy in elementary schools in Hawaii. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/03_0013.htm.
==== Refs
1 Mackie RM Ruiter DJ Welvaart K Ferrone S The epidemiology of melanoma Cutaneous melanoma and precursor lesions Boston (MA) Academic Publishers 1984 1 8
2 Miller DL Weinstock MA Nonmelanoa skin cancer in the United States: incidence J Am Acad Dermatol 1994 30 774 778 8176018
3 2002 American Cancer Society. Cancer prevention and early detection — cancer facts and figures 2002 Atlanta (GA) American Cancer Society Available from: URL: http://www.cancer.org/docroot/STT/stt_0_2002.asp?sitearea=STT&level=1
4 American Cancer Society, Hawaii Pacific Inc. Hawaii cancer facts & figures 2003-2004 Honolulu (HI) American Cancer Society 2003 37 38
5 Stern RS Weinstein MC Baker SG Risk reduction for nonmelanoma skin cancer with childhood sunscreen use Arch Dermatol 1986 122 5 537 545 3707169
6 Weinstock MA Colditz GA Willett WC Stampfer MJ Bronstein BR Mihm MC Nonfamilial cutaneous melanoma incidence in women associated with sun exposure before 20 years of age Pediatrics 1990 85 4 626 627
7 Diffey BL Gibson CJ Haylock R McKinlay AF Outdoor ultraviolet exposure of children and adolescents Br J Dermatol 1996 134 6 1030 1034 8763419
8 Elwood JM Melanoma and ultraviolet radiation Clin Dermatol 1992 10 1 41 50 1504927
9 Goldstein N Skin cancers in Hawaii (1993) Hawaii Med J 1993 52 126 128 8320090
10 Grin CM Pennoyer JW Lehrich DA Grant-Kels JM Sun exposure of young children while at day care Pediatric Dermatol 1994 11 4 304 309
11 Buller DB Geller AC Cantor M Buller MK Rosseel K Hufford D Sun protection policies and environmental features in U.S. elementary schools Arch Dermatol 2002 138 771 774 12056958
12 Glanz K Lew RA Song V Cook VA Factors associated with skin cancer prevention practices in a multiethnic population Health Educ and Behav 1999 26 3 344 359
13 Glanz K Chang L Song V Silverio R Muneoka L Skin cancer prevention for children, parents and caregivers: a field test of Hawaii's SunSmart Program J Am Acad Dermatol 1998 38 3 413 417 9520022
14 Glanz K Maddock JE Lew RA Murakami-Akatsuka L A randomized trial of the Hawaii SunSmart Program's impact on outdoor recreation staff J Am Acad Dermatol 2001 44 973 978 11369909
15 Glanz K Lew RA Song V Murakami-Akatsuka L Skin cancer prevention in outdoor settings: effects of the Hawaii SunSmart Program Eff Clin Pract 2000 3 2 53 61 10915324
16 Centers for Disease Control and Prevention. School health policies and programs study, 2000 2000 [cited 2003 Oct 29] Available from: URL: http://www.cdc.gov/nccdphp/dash/ shpps/summaries/index.htm
17 Hall HI Jorgensen CM McDavid K Kraft JM Breslow R Protection from sun exposure in U.S. white children ages 6 months to 11 years Public Health Rep 2001 116 4 353 361 12037264
18 Glanz K Geller AC Shigaki D Maddock JE Isnec MR A randomized trial of skin cancer prevention in aquatics settings: the Pool Cool program Health Psychol 2002 21 6 579 587 12433010
19 Centers for Disease Control and Prevention. Guidelines for school programs to prevent skin cancer MMWR Recomm Rep 2002 4 51 4 1 16
20 Dobbinson SJ Peipers AM Borland R Nolan KM Are Victorian primary schools SunSmart? Health Promotion J 2000 10 43 50
21 Seagan C Evaluation of SunSmart policies, practices and resource usage in Victorian primary schools: telephone interviews with school principals SunSmart Evaluation Studies No. 3 Anti-Cancer Council of Victoria Melbourne 1994
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Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_04_0001
Original Research
PEER REVIEWEDUsing Focus Groups to Develop a Bone Health Curriculum for After-school Programs
Economos Christina D PhD Associate Director John Hancock Center on Physical Activity and Nutrition, Gerald J. and Dorothy R. Friedman School of Nutrition Science and Policy, Tufts University
150 Harrison Ave, Boston, MA 02111 Tufts University 617-636-3784 [email protected]
Folta Sara C MS Gerald J. and Dorothy R. Friedman School of Nutrition Science and Policy, Tufts University, Boston, Mass
Goldberg Jeanne P PhD, RD Gerald J. and Dorothy R. Friedman School of Nutrition Science and Policy, Tufts University, Boston, Mass
Marcotte Lori P MPH, MS, RD Gerald J. and Dorothy R. Friedman School of Nutrition Science and Policy, Tufts University, Boston, Mass
7 2004
15 6 2004
1 3 A062004
Introduction
Childhood behaviors influence peak bone mass and osteoporosis risk in later life. The after-school environment provides an opportunity to enrich a child’s learning and experience. Our objective was to gain a better understanding of the knowledge of, attitudes and beliefs about, and barriers to achieving bone health among children, parents, and after-school program leaders from low-income, ethnically diverse communities. Findings led to the development, implementation, and evaluation of a bone health curriculum in the after-school setting.
Methods
Eight focus groups were conducted in three representative communities. Focus group participants included children aged six to eight years, parents of children aged six to eight, and after-school program staff. Transcripts and written notes from each session were reviewed and common themes were identified within each group.
Results
Most adults had some understanding of osteoporosis, but did not recognize that childhood behaviors had a role in developing the disease. Program leaders raised concerns about their ability to implement a health program and recommended a flexible format. Parents and program leaders recognized the importance of maintaining a fun atmosphere.
Conclusion
It is feasible to create a curriculum for a bone health program that meets the unique needs and interests of children and program leaders in the after-school setting. Addressing the needs, interests, and common barriers of the target population is an essential first step in curriculum development.
==== Body
Introduction
Osteoporosis is a childhood disease with adult consequences. Childhood behaviors, including diet and physical activity (1-4), have a major influence on the attainment of peak bone mass and the primary prevention of osteoporosis (5-11). The higher the peak bone mass in childhood, the more an individual can afford to lose in adulthood (12-14). The long-term benefits of increasing bone mineral density during childhood are compelling (15,16). A change in one negative standard deviation in bone mass may double fracture risk (17,18).
In the United States, there is a large gap between childhood behaviors known to help maximize bone health and what children actually do. National survey data estimate that more than half of girls aged six to 11 years are not meeting 100% of the 1989 Recommended Dietary Allowance for calcium, and nearly half of boys are not meeting this requirement (19). The gap between recommendations and intakes is difficult to reverse as children age (20,21). Of equal importance is that children of all ages do not obtain adequate levels of physical activity (22-25). Studies show that sedentary behavior increases and moderate physical activity decreases as children advance through elementary school (26,27) and that this decline continues into adolescence (27,28). Furthermore, girls are less likely to engage in physical activity than boys (27-29), and black children are less active than white children (28,29). The gap between the long-term effect of modifiable influences on bone health and the behaviors of millions of children suggests that cost-effective interventions to promote bone health in children are urgently needed.
After-school programs are ideal for complementing the school day with health education and physical activity. Several million children participate in after-school programs, and demand outstrips supply by a rate of approximately two to one (30). Furthermore, many programs lack adequate funding, and quality is highly variable (30). Curriculum-based interventions may enhance existing enrichment activities and provide structure to programs that are not highly developed. Reviews of nutrition and physical activity education curricula indicate that they can contribute to significant improvements in students’ knowledge, skills, and behavior, but that they must have certain characteristics to be effective (31-33). A health curriculum should be theory driven and should address children’s needs, interests, and concerns, in addition to their knowledge, attitudes, and beliefs (31-33). Addressing barriers to change is also important. This paper describes the design of a curriculum to promote bone health based on data obtained from focus group research to identify motivating factors, preferences, and barriers to change among children, parents, and after-school program leaders.
Methods
Eight focus groups were conducted in three low- to moderate-income, multiethnic Massachusetts communities in the three months from November 1999 through January 2000. In total, 66 individuals participated. Participants included three groups of children aged six to eight years (N = 26; 70% white, 30% African American; 61% male); three groups composed of parents with children aged six to eight (N = 24; 80% white, 20% African American; 8% male); and two after-school program staff groups (N = 16; race and ethnicity not specified; 19% male). Of the 16 program staff who participated, two oversaw staff and program development and 14 taught. Focus groups took place at the after-school program sites and were led by two professional focus-group facilitators with expertise in conducting groups with children. Sessions typically lasted two hours and included six to 11 participants. Each adult participant received $30 and each child received a $20 gift certificate to a local toy store. Each session was recorded on audiotape for subsequent transcription; focus-group facilitators took additional notes.
Focus-group facilitators provided a brief introduction and invited parents and leaders to offer general opinions and comments about health education and strategies for engaging children in desired behaviors in after-school programs. Facilitators told children that the purpose of the meeting was to learn about what children like to eat and play. All groups were told there were no right or wrong answers. Facilitators explored knowledge, attitudes, beliefs, preferences, and barriers related to bone health and to the potential implementation of a curriculum that focused on bone health in the after-school environment.
The two facilitators systematically analyzed transcripts. Each one read the original transcripts to identify themes of each topic of discussion before collaborating on the summary report and submitting the report to an independent investigator. The investigator reviewed the transcripts and final report and recoded key phrases into a matrix constructed to conform to the project’s conceptual framework. Recoding key phrases into the matrix allowed for a more detailed understanding of the key themes identified by the facilitators and provided the ability to incorporate these themes into the project development.
The Institutional Review Board at Tufts University gave human subjects research approval for this project.
Results
Knowledge and awareness
As expected, the children had limited knowledge about bone health and the factors that affect it. Some understood the connection between bone health and drinking milk. Not surprisingly, they were generally aware of something called “calcium” but did not understand that it is a mineral or know where it is found in the diet: “It’s a kind of vitamin and cereal has it” was a typical response. After calcium was defined for them, many children commented, “Calcium makes you stronger, smarter, and helps you learn.”
As expected, none of the children understood “osteoporosis.” Among parents, knowledge of osteoporosis was mixed, whereas most after-school program leaders had a basic knowledge of what osteoporosis is and how to prevent it. In general, both parents and after-school program staff were aware of the effect of calcium and exercise on bone health and development.
Attitudes, perceptions, and beliefs
Children showed little interest in understanding osteoporosis, but some interest in knowing how to make bones healthy and strong. Children appropriately associated bones with certain foods: “Bones make you think about dairy products.” Parents felt that nutrition played a critically important role in their child’s development. Among their chief concerns were getting their children to eat enough fruits and vegetables and limiting their intake of sweets and other “junk foods”: “I worry about the long-range effect of nutrition on them in their twenties, what will have been done by then.” “I like to make sure my kids get their vitamins every day…because I know they don’t eat right. They don’t eat enough vegetables.” “Other than genetics, nutrition is the number-one thing for your child’s health.”
Parents were less concerned that their children’s diets had enough calcium and did not consider osteoporosis a major health threat: “As long as they’re eating from the basic four food groups, I’m not worried.” “I always think of osteoporosis as an adult issue.” “I think I need the bone help more than them.”
Parents and after-school program leaders were both concerned about the amount of physical activity the children were getting. One parent commented, “He doesn’t get enough exercise — never. He’s healthy, but he has an interest in video games and anything electronic. I’m worried about down the road.” An after-school program leader observed, “If you talk to a gym teacher or watch a class, these kids aren’t in any shape at all. In my class, there are four or five kids who can’t run around the bases without stopping and huffing and puffing.”
Preferences
Most children said they liked or drank milk. The perspectives of children and parents differed on the subject of physical activity. Children said that if given a choice, they would prefer physical activity, games, or sports during their free time: “I like to play tag and play games like when you pretend to be monsters and things…I’d rather play outside.” In contrast, parents consistently said that if left on their own, children would choose television and video games rather than physical activity. Parents demonstrated an awareness of the importance of physical activity and of their role in promoting it: “I do make them go outside, but it’s like kicking and screaming — they don’t like to go.”
Barriers
After-school program leaders were concerned about the amount of planning required to implement a curriculum. Said one participant, “I’m a second-grade teacher. I have enough planning to do all day long. I don’t have the time.” Some after-school program leaders also expressed a desire for flexibility: “I think ideas would be better, because if you disagree with the format, then you’re going to come to some conflict with ‘Oh, I have to do this?’ Make it more optional. ‘You may want to do this, or you could do A, B, and C.’” “[It] just depends on the mood of the children what I’m going to do that day. If they’re fidgety, we go out and run around the park.”
While after-school program leaders recognized the need to provide guidance for children about healthy eating and exercise, they did not perceive health education as a priority: “I think health education is important but as [another participant] said, they get a lot of it during the day at school, and we’re more geared toward their social and emotional growth, socializing with other children and interacting with adults.”
Both parents and after-school program leaders expressed some concerns about the nutrition education component of the curriculum. They worried that the activity would replace the children’s already limited time for play and fun. One after-school program leader stated, “I don’t want it to be a bore for them. Especially since they’ve been in school all day long. I do think it’s important, but when they come to us, it’s time to let loose some steam.” A parent said, “I’m hoping they’ll come home and say ‘I had fun doing this and that today.’ If he says, ‘I have to go here,’ then he’s in the wrong place.”
Parents also expressed a concern that the nutrition education activity might be too academic: “It needs to be addressed for children as not so medical. It needs to be presented as fun.” “I think the calcium-focused activity would get old fast. You know: ‘Calcium again, I’m so sick of calcium.’”
Despite wanting their children to enjoy a break from academics, getting homework done during the after-school time was a high priority for parents. After-school program leaders felt pressure to make sure homework was complete by the time parents arrived to pick children up. One parent commented, “Homework has to be the first priority. I get home too late to get it done with him.” An after-school leader said, “I know my parents: they want [their children] to get their homework done.”
After-school program leaders consistently and poignantly expressed their concern that they might not know enough to effectively teach the bone-health curriculum. They were afraid they would be embarrassed if they could not answer a child’s question. “I’m not saying I’m ignorant about osteoporosis, but I’m not as knowledgeable as I’d like to be.” “[I would want] more knowledge about osteoporosis, questions the kids would ask us, so we could have answers for them.” Parents also expressed concern about the ability of the after-school program leaders to implement the curriculum: “The after-school teachers would need training. They’re capable, but need training.” While an extensive training program was proposed, most after-school program leaders suggested that only minimal training would be possible because of limited available time. Because of high staff turnover rates in the after-school setting, they also voiced an interest in ongoing oversight and support so the curriculum could continue even if trained leaders left the after-school program.
Shaping the curriculum
Curriculum development relied heavily on information obtained in the focus groups. To respond to the needs of program leaders in the after-school setting, short and simple lessons were designed with alternate activity options, tips for implementation, and ideas for modifying games. Curriculum components could complement regular program activities without interfering with priorities such as homework. Ongoing support was offered via newsletters, and research staff were available to assist new leaders during the year.
From the outset of the project, the objective was to package both a physical-activity component and a nutrition education component so that the children would have fun while learning. The children know the project as “The Bones Club.” To address the desire expressed in focus groups to allow children to use after-school time for fun activities that would enable them to socialize and “let off some steam” and to fulfill the objective of offering simple, non-academic language, the physical activity component was named “Let’s Play.” Activities identified as favorites with the children were adapted to weight-bearing activities (similarly titled “Let’s Run” and “Let’s Jump”). Because after-school program leaders indicated that they operate in a wide variety of physical environments, all games included simple modifications to accommodate play in different environments.
Likewise, the nutrition education component was named “Let’s Explore” to reflect some of the preferred activities of children and to emphasize both teamwork and fun. During the focus groups, children indicated an interest in reading, and after-school program leaders reported “circle time” as a common component of the after-school day. Age-appropriate books were provided to support the learning themes of the “Let’s Explore” lessons. Many after-school program leaders expressed concern that they may not know enough about bone health to teach the curriculum effectively. To begin to address this, an appendix was included with each section of the written curriculum that answered commonly asked questions and provided a quick reference guide for additional resources.
Evidence shows that nutrition education programs and curricula targeted at elementary-aged children are more effective when they include a family component (33). Some parents received newsletters that corresponded to curriculum units to reinforce after-school program lessons at home. Newsletters included quick and easy recipes and physical activity tips that took into account the time constraint that was mentioned as a barrier in the focus groups. Parents also were given a directory that allowed them to leverage their own limited resources by using nutrition, physical activity, and health resources available in their communities.
Discussion
This study demonstrates how focus groups can be used to shape a curriculum to meet the needs of after-school program leaders, parents, and children so that maximum buy-in and learning can occur. Of particular importance, focus groups can identify key barriers to implementing the curriculum that might otherwise go unnoticed. Perhaps the most important barrier was that health education was not considered a priority by either parents or after-school program leaders. To succeed, the curriculum would need to focus on fun for the children and ease of implementation for the program leaders. The curriculum was designed to be short and flexible so it would not replace activities that were considered a priority.
Parents and program leaders indicated limited confidence in promoting health, particularly nutrition, to children. Still, program leaders believed they could incorporate such a program into their existing after-school program structure and implement it as long as they are given adequate support.
Not surprisingly, children were not interested in osteoporosis the disease, but they did want to learn about how bones move and what they could do to grow big and strong. This perception confirmed that it is possible to engage even very young children in a health topic if the topic is presented at their level of comprehension and if it appeals to their interests.
The children who participated in the focus groups were young. Sometimes they were wonderfully direct and open, and at other times their responses were colored by the need for peer acceptance. In this series of focus groups, their responses about likes and dislikes differed from those of their parents. For instance, children overwhelmingly expressed a preference for active games or sports over video games, but parents reported difficulty in engaging children in outdoor play. This observation confirms the need to conduct focus groups that include both children and parents to obtain a more balanced picture of preferences and behaviors.
The inconsistency between children’s reported desire for physical activity and parents’ reports that children engage in sedentary behaviors if given a choice is difficult to reconcile. Possibly, while children may like the idea of physical activity, they are reluctant to engage in it once they have started other activities. Several factors may draw children to activities that are more sedentary. In the focus groups, parents noted the ubiquity of televisions and computer games in their homes. In addition, cold weather and early darkness were also mentioned as serious barriers to outdoor play. Regardless of these perceived barriers, children participated willingly when provided with the types of physical activities in the after-school programs that both the children and the program leaders agreed were fun.
Focus groups do not provide data that are generalizable to other populations, but they can be a time-efficient and cost-effective method for identifying attitudes, beliefs, and barriers toward health behaviors among defined target populations. Through an interactive discussion led by trained professionals, it is possible to identify information that is critical to program success and that might not be uncovered in survey research. For example, the permissive environment allowed after-school leaders to openly describe their perceptions of their limited knowledge about osteoporosis and bone health, which, if not addressed, could limit their ability to implement the curriculum and could consequently hinder the success of the program.
Response to the bone-health curriculum has been enthusiastic. More than 50 after-school programs in Massachusetts and Rhode Island have implemented it successfully, and it has been well-accepted by after-school program leaders, parents, and children. After-school program leaders report that the curriculum has enhanced their programs and has had the unexpected benefit of improving their relationships with the children. They indicate that children enjoy being in the “Bones Club” and having something to call their own. Participation is optional, but remains at a high level, and dropout rates related to dissatisfaction are extremely low (less than 1%). Dropout is linked almost exclusively to children leaving the after-school program or the school district itself.
An environment that fosters the development of behaviors to promote bone health can contribute to positive habits that children will adopt before entering their preteen years, when peer influences gain power. After-school programs have been an underused setting for health interventions. As they grow in number, they provide an opportunity to use time that traditionally has been difficult to fill consistently with appropriate physical and cognitive activities for all children who attend them. Health interventions that include an academic and a physical-activity component are difficult to implement given the varied experience of leaders and the lack of funds for training and technical support. Limited staff, high turnover rates, and competing demands on program time are major barriers. Curricula based on formative research can overcome these barriers, help to improve the health of children, and prevent chronic disease later in life.
This research was supported by a grant (NIH/1RO1 HD 37752-01) and is based on work supported by Grant P01-DK42618 from the National Institutes of Health, and the U.S. Department of Agriculture (USDA), under agreement number 58-1950-9-001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the USDA.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Folta SC, Goldberg JP, Marcotte LP, Economos CD. Using focus groups to develop a bone health curriculum for after-school programs. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0001.htm.
==== Refs
1 Bonjour J Theintz G Law F Slosman D Rizzoli R Peak bone mass Osteoporosis Int 1994 4 1 7 13
2 Deal C Osteoporosis: Prevention, diagnosis, and management Am J Med 1997 102 1A S35 S39
3 Lysen V Walker R J Sch Health Osteoporosis risk factors in eighth grade students 1997 67 8 317 321 9425605
4 Anderson J Rondano P Holmes A Roles of diet and physical activity on the prevention of osteoporosis Scand J Rheumatol Suppl 1996 103 65 74 8966493
5 Teegarden D Proulx WR Martin BR Zhao J McCabe GB Lyle RM Peak bone mass in young women J Bone Miner Res 1995 10 5 711 715 7639106
6 Recker RR Davies KM Hinders SM Heaney RP Stegman MR Kimmel DB Bone gain in young adult women JAMA 1992 268 17 2403 2408 1404797
7 Slemenda C Reister TK Hui SL Miller JZ Christian JC Johnston CC Jr Influences on skeletal mineralization in children and adolescents: evidence for varying effects of sexual maturation and physical activity J Pediatr 1994 125 201 207 8040762
8 Matkovic V Nutrition, genetics and skeletal development J Am Coll Nutr 1996 15 6 556 569 8951732
9 Bailey D The Saskatchewan Pediatric Bone Mineral Accrual Study: bone mineral acquisition during the growing years Int J Sports Med 1997 18 3 S191 S194 9272847
10 Barr S McKay H Nutrition, exercise, and bone status in youth Int J Sports Med 1998 8 124 142
11 French SA Fulkerson JA Story M Increasing weight-bearing physical activity and calcium intake for bone mass growth in children and adolescents: a review of intervention trials Prev Med 2000 31 722 731 11133340
12 Ribot C Tremollieres F Pouilles JM Late consequences of a low peak bone mass Acta Paediatr Suppl 1995 411 31 35 8563066
13 Riis B Hansen MA Jensen AM Overgaard K Christiansen C Low bone mass and fast rate of bone loss at menopause: equal risk factors for future fractures: a 15-year follow-up study Bone 1996 19 9 12 8830981
14 Seeman E Tsalamandris C Formica C Hopper JL McKay J Reduced femoral neck bone density in daughters of women with hip fractures: the role of low peak bone density in the pathogenesis of osteoporosis J Bone Miner Res 1994 9 739 743 8053404
15 Johnston CC Jr Slemenda CW Risk assessment: theoretical considerations Am J Med 1993 95 5A S2 S5
16 Matkovic V Kostial K Simonovic I Buzina R Brodarec A Nordin BE Bone status and fracture rates in two regions of Yugoslavia Am J Clin Nutr 1979 32 540 549 420146
17 Hui SL Slemenda CW Johnston CC Jr Baseline measurement of bone mass predicts fracture in white women Ann Intern Med 1989 111 355 361 2764403
18 Wasnich R Ross PD Davis JW Vogel JM A comparison of single and multi-site BMC measurements for assessment of spine fracture probability J Nucl Med 1989 30 1166 1171 2738698
19 Food and nutrient intakes by children 1994-96, 1998 U.S. Department of Agriculture, Agricultural Research Service ARS Food Surveys Research Group Beltsville (MD) 1999
20 Albertson AM Tobelmann RC Marquart L Estimated dietary calcium intake and food sources for adolescent females: 1980-92 J Adolesc Health 1997 20 20 26 9007655
21 Zive MM Nicklas TA Busch EC Myers L Berenson GS Marginal vitamin and mineral intakes of young adults: The Bogalusa Heart Study J Adolesc Health 1996 19 39 47 8842859
22 Simons-Morton BG O’Hara NM Parcel GS Huang IW Baranowski T Wilson B Children's frequency of participation in moderate to vigorous physical activities Res Q Exerc Sport 1990 61 4 307 314 2132887
23 Pate R Long B Heath G Descriptive epidemiology of physical activity in adolescents Pediatr Exerc Sci 1994 6 434 447
24 Sallis JF Epidemiology of physical activity and fitness in children and adolescents Crit Rev Food Sci Nutr 1993 3 33 4-5 403 408 8357503
25 Duke J Huhman M Heitzler C Physical activity levels among children aged 9-13 years — United States, 2002 MMWR Morb Mortal Wkly Rep 2003 52 33 785 788 12931076
26 Myers L Strikmiller PK Webber LS Berenson GS Physical and sedentary activity in school children grades 5-8: the Bogalusa Heart Study Med Sci Sports Exerc 1996 28 7 852 859 8832539
27 Trost SG Pate R Sallis JF Freedson PS Taylor WC Dowda M Age and gender differences in objectively measured physical activity in youth Med Sci Sport Exerc 2002 34 2 350 355
28 Grunbaum J Kann L Kinchen SA Williams B Ross JG Lowry R Youth risk behavior surveillance — United States, 2001 MMWR Surveill Summ 2002 51 4 1 62 12102329
29 Andersen RE Crespo CJ Bartlett SJ Cheskin LJ Pratt M Relationship of physical activity and television watching with body weight and level of fatness among children: results from the Third National Health and Nutrition Examination Survey JAMA 1998 279 12 938 942 9544768
30 Working for children and families: safe and smart after-school programs U.S. Department of Education. U.S. Department of Justice Washington (DC) 2000
31 Contento I Balch GI Bronner YL Paige DM Gross SM Lytle LA Nutrition education for school-age children J Nutr Ed 1995 27 298 311
32 Stone EJ McKenzie TL Welk GJ Booth ML Effects of physical activity interventions in youth. Review and synthesis Am J Prev Med 1998 15 4 298 315 9838974
33 Lytle L Achterberg C Changing the diet of America's children: what works and why J Nutr Ed 1995 27 5 250 260
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Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_03_0029
Original Research
PEER REVIEWEDIncreasing Employee Awareness of the Signs and Symptoms of Heart Attack and the Need to Use 911 in a State Health Department
Harwell Todd S MPH Montana Department of Public Health and Human Services
Cogswell Building, C-317, PO Box 202951, Helena, MT 59620-2951 [email protected]
406-444-1437
Fogle Crystelle C RD, MS, MBA Montana Department of Public Health and Human Services, Helena, Mont
Oser Carrie S MPH Montana Department of Public Health and Human Services, Helena, Mont
Blades Lynda L MPH Montana Department of Public Health and Human Services, Helena, Mont
Helgerson Steven D MD, MPH Montana Department of Public Health and Human Services, Helena, Mont
Gohdes Dorothy MD Montana Department of Public Health and Human Services, Helena, Mont
Spence Michael R MD, MPH Montana Department of Public Health and Human Services, Helena, Mont
Dawson Drew E Montana Department of Public Health and Human Services, Helena, Mont
7 2004
15 6 2004
1 3 A072004
Introduction
Early recognition of the signs and symptoms of a heart attack can lead to reduced morbidity and mortality.
Methods
A workplace intervention was conducted among 523 Montana state health department employees in 2003 to increase awareness of the signs and symptoms of heart attack and the need to use 911. All employees received an Act in Time to Heart Attack Signs brochure and wallet card with their paychecks. Act in Time posters were placed in key workplace areas. A weekly e-mail message, including a contest entry opportunity addressing the signs and symptoms of heart attack, was sent to all employees. Baseline and follow-up telephone surveys were conducted to evaluate intervention effectiveness.
Results
Awareness of heart attack signs and symptoms and the need to call 911 increased significantly among employees from baseline to follow-up: pain or discomfort in the jaw, neck, or back (awareness increased from 69% to 91%); feeling weak, light-headed, or faint (awareness increased from 79% to 89%); call 911 if someone is having a heart attack or stroke (awareness increased from 84% to 90%). Awareness of chest pain, pain or discomfort in the arms or shoulders, and shortness of breath were more than 90% at baseline and did not increase significantly at follow-up. At baseline, 69% of respondents correctly reported five or more of the signs and symptoms of heart attack; 89% reported correctly at follow-up.
Conclusion
This low-cost workplace intervention increased awareness of the signs and symptoms of heart attack and the need to call 911.
==== Body
Introduction
Heart disease continues to be the leading cause of death in the United States in 2003, with more than 1 million Americans experiencing a new or recurrent acute myocardial infarction (AMI) (1). Timely coronary reperfusion (e.g., angioplasty, thrombolytic therapy) and arrhythmia control can reduce morbidity and mortality in persons experiencing AMI (2). Reducing the time from the initial occurrence of symptoms to hospital arrival can increase the likelihood that these therapies are used early in the course of AMI.
In the United States, the median delay time in patients hospitalized with AMI was 2.1 hours in 1997, and 32% of patients with AMI had delay times of more than four hours (3). Persons experiencing AMI may delay seeking care because of a number of factors, including inadequate knowledge of signs and symptoms, attribution of symptoms to other non-serious conditions, or other barriers such as fear, concerns about cost, and embarrassment about calling emergency medical services (4-11). Additionally, most people in the United States who experience AMI transport themselves to medical care instead of using emergency medical services (12). However, persons who believe that their symptoms are serious are less likely to delay and more likely to use emergency medical services. In addition, women may experience an array of symptoms that differ from symptoms experienced by men (13). This difference may delay recognition and acute care for AMI among women.
Increasing early awareness of the signs and symptoms of AMI and the need to use the 911 emergency telephone system can reduce delays in seeking treatment, thus reducing morbidity and mortality. This is particularly important for rural and frontier communities, where individuals must travel long distances to tertiary care facilities.
In 2003, the Montana Department of Public Health and Human Services (MT DPHHS) conducted an intervention within the state health department to increase employee awareness of the signs and symptoms of a heart attack and the need to use 911. This report assesses the effectiveness of the intervention.
Methods
Setting
The intervention was conducted within the three MT DPHHS workplaces located in Helena, Mont (site one, site two, and site three). There were 523 employees at these three sites; 70% were female; and the mean age was 47.7 years.
Intervention
Based on the original content of the Rapid Early Action for Coronary Treatment (REACT) research program developed by the National Institutes of Health (NIH), the Act in Time campaign provides information on the warning signs of heart attack and the importance of calling 911 for emergency medical services (http://www.nhlbi.nih.gov/ actintime/) (14). Our adaptation of Act in Time included several components designed specifically for a workplace intervention. First, all employees at the three sites received a one-time distribution of Act in Time brochures and wallet cards with their pay stubs (15). Second, Act in Time posters were placed on bulletin boards, in hallways, and in all bathrooms at each work site during the six-week test period (Figure 1). Third, also for six weeks, all employees received weekly e-mail messages and contest questions addressing the signs and symptoms of heart attack. Participants who answered the questions correctly were included in a weekly drawing for prizes (e.g., pedometers). Approximately one third of all employees participated in these weekly e-mail contests (participation ranged from 29% to 36%). The total estimated cost for intervention materials and staff time was $1037 (mean cost per employee = $1.98).
Figure 1 Act in Time poster developed by the National Institutes of Health to provide information on warning signs of heart attack and importance of calling 911 for emergency medical services.
The "Act in Time" poster
Evaluation and data analysis
To evaluate this intervention, we conducted baseline and follow-up telephone surveys. The baseline survey took place over a two-week period in April 2003, and attempts were made to contact all 523 employees at each of the three sites. Respondents were asked a series of seven standardized questions about the signs and symptoms of heart attack from the Behavioral Risk Factor Surveillance System survey (Table 1) (16). The follow-up telephone survey was administered in July 2003 to the 401 employees responding to the initial baseline survey. The participants again were asked questions about the signs and symptoms of heart attack and the need to use 911. Respondents were also asked the following questions to assess their awareness of intervention activities:
"In the past two months, have you seen posters in your workplace with information on heart attack signs and symptoms?" Those who responded yes were asked, "Where did you see these posters? Was it in the: hallways, stairwells, elevators, bathrooms?"
"Did you participate in any of the weekly e-mail quiz contests on heart attack signs and symptoms?"
"In the past two months, did you receive a brochure and wallet card on heart attack signs and symptoms with your paycheck?"
Data analyses were completed using SPSS version 10.0 statistical analysis software (Chicago, Ill). Independent t-tests and Pearson chi-square tests were used to compare the characteristics of respondents to the baseline and follow-up surveys. Pearson chi-square tests were used to assess differences in the proportion of respondents correctly identifying each individual sign and symptom of heart attack and the proportion identifying five or more signs and symptoms of heart attack at baseline compared to follow-up. Pearson chi-square tests were also used to assess the differences in the proportion of respondents correctly answering the question regarding the need to use 911 at baseline compared to follow-up.
Results
Of the 523 employees, 401 (77%) completed the baseline survey. Of these 401 respondents, 337 (84%) completed the follow-up survey. There were no statistically significant differences in age between baseline and follow-up: baseline respondent mean age was 46.4 years (SD 9.3); follow-up respondent mean age was 46.6 years (SD 8.9). Nor were there statistically significantly differences in sex between baseline and follow-up: 71% of baseline respondents were women, and 72% of follow-up respondents were women (data not shown). Similarly, there were no differences in the proportion of respondents from each of the three work sites at baseline or follow-up (site one, 58% responded at baseline, 57% at follow-up; site two, 38% at baseline, 40% at follow-up; site three, 4% at baseline, 4% at follow-up).
Awareness of selected signs and symptoms increased significantly among employees from baseline to follow-up: pain or discomfort in the jaw, neck, or back (awareness increased from 69% to 91%); and feeling weak, light-headed, or faint (awareness increased from 79% to 89%) (Table 2). Awareness of chest pain, pain or discomfort in the arms or shoulders, and shortness of breath was greater than 90% at baseline and did not increase significantly at follow-up. The proportion of respondents who correctly reported that "sudden trouble seeing in one or both eyes" was not a sign or symptom of heart attack did not change significantly from baseline to follow-up. At baseline, 69% of respondents reported five or more of the signs and symptoms of heart attack correctly, and this increased to 89% at follow-up. Additionally, awareness of the need to use 911 emergency telephone services increased significantly from 84% to 90% between baseline and follow-up.
At baseline, women were more likely than men to report that pain or discomfort in the jaw, neck, or back was symptomatic for AMI (72% of women, 61% of men, P = .02). This difference in response between men and women persisted at follow-up (94% of women, 84% of men, P = .006). There were no significant differences in awareness of other AMI signs and symptoms or the need to use 911 emergency telephone services by sex at baseline or follow-up (data not shown). Employees 45 years and older were more likely to recognize pain or discomfort in the jaw, neck, or back compared with younger employees at baseline (74% of older employees, 61% of younger employees, P = .008). Younger employees were more likely to report feeling weak, light-headed, or faint as an AMI symptom compared with older employees at baseline (87% of younger employees, 75% of older employees, P = .003). There were no other statistically significant differences for AMI signs and symptom awareness or the need to use 911 between younger and older employees at baseline (data not shown). At follow-up, younger employees had a higher level of awareness of the need to use 911 services compared with older employees (96% of younger employees, 87% of older employees, P = .006). There were no other statistically significant differences in the awareness of AMI signs and symptoms between younger and older employees at follow-up (data not shown).
The intervention was equally effective in increasing overall awareness of signs and symptoms of heart attack among men (14 percentage point increase, P = .02) and women (23 percentage point increase, P < .001) as well as younger (22 percentage point increase, P < .001) and older (20 percentage point increase, P <.001) employees from baseline to follow-up (Figure 2). Awareness of the need to use 911 emergency telephone services increased significantly in women (9 percentage point increase, P = .005) and younger employees (11 percentage point increase, P = .004), but did not change significantly in men (1 percentage point decrease, P = .97) or older employees (3 percentage point increase, P = .34) from baseline to follow-up (Figure 3).
Results of survey questions designed to assess participant awareness of intervention activities are presented in Table 3.
Discussion
This low-cost workplace intervention was effective in increasing employee awareness of the signs and symptoms of a heart attack and the need to use 911 emergency telephone services. The intervention was equally effective in increasing awareness in both older and younger employees and had a slightly greater impact on women than men. Interestingly, the effect on increasing awareness of the need to use 911 services was found only in women and younger employees and not in men or older employees.
We were unable to identify other similar workplace intervention studies for comparison. At baseline, state health department employees were slightly more aware of signs and symptoms of heart attack and the need to use 911 compared to Montana adults overall. In a 2001 survey of Montana adults, only 60% were aware that pain or discomfort in the jaw, neck, or back were signs and symptoms of a heart attack, and 74% were aware that feeling weak, light-headed, or faint were signs and symptoms of a heart attack (17). More than 90% of adult Montanans in 2001 were aware of the signs and symptoms of chest pain, pain or discomfort in the arms or shoulders, and shortness of breath, and 82% knew to call 911 if someone is having a heart attack or stroke.
Large community intervention studies using mass media campaigns have had mixed effects on heart attack signs and symptoms awareness, use of emergency medical services, and reduction in patient delay in receiving services for persons experiencing AMI (14,18-24). A recent review of the literature provides a number of strategies for improving future community-based efforts to reduce patient delay times. These strategies include targeting high-risk groups; addressing emotional (e.g., denial) and social (e.g., inclusion of family members in education programs) issues; emphasizing cognitive aspects such as the physiologic consequences of delay; educating individuals on how to evaluate their symptoms; and developing messages specific to men and women (25). Integrating workplace awareness campaigns within larger community-based efforts may be an effective approach for reaching family and friends of persons at high risk for AMI. State health departments are attractive workplaces to pilot such interventions.
This study, however, has a number of limitations. First, all MT DPHHS employees in the three sites were exposed to the intervention, and a comparison group not receiving the intervention was not used. Other factors may have increased employee awareness outside of the intervention, although we believe that this is unlikely. Second, we used telephone surveys of employees to evaluate this intervention, and respondents were asked "aided" questions to indicate which of the possible symptoms described by the interviewer were symptoms of a heart attack. Previous studies using unaided, open-ended questions have found lower levels of heart attack awareness (26). Aided questions may overestimate awareness of signs and symptoms, and unaided questions may underestimate awareness. Third, the baseline telephone survey itself may have increased respondent awareness of the signs and symptoms. Fourth, the follow-up telephone survey took place during the summer months (July and August) and resulted in a smaller sample size (n = 337). The lack of response was due mostly to contact with answering machines, no answers, or no eligible respondent at telephone number (15%). Finally, we were not able to quantify the relative contributions of each of the intervention activities to increases in awareness.
Our findings show that this low-cost intervention can be easily replicated in other workplaces. The State of Montana will promote this type of intervention at work sites through the newly convened Governor's Council on Worklife Wellness. Increased awareness of the signs and symptoms of heart attack and the need to use 911 are important for individuals at high risk of AMI as well as family members and friends who are often the first people to have contact with persons potentially experiencing AMI.
This project was supported through a cooperative agreement (U50/CCU821287-02) with the Centers for Disease Control and Prevention (CDC) Cardiovascular Health Branch. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the CDC. We would like to thank and acknowledge Linda Priest and the staff members of Northwest Resource Consultants for their expertise and work on the telephone surveys. We also thank Jason Swant and Linda Schofield from the Montana Cardiovascular Health Program for their assistance with this project.
Figures and Tables
Figure 2 Awareness among Montana state health department employees of five or more heart attack signs and symptoms at baseline and follow-up, by sex and by age, 2003.
Bar chart showing awareness of 5 or more heart attack signs and symptoms among Montana state health department employees at baseline and follow-up, by sex and by age, 2003
Figure 3 Awareness among Montana state health department employees of need to use 911 emergency telephone services if someone is having a heart attack or stroke at baseline and follow-up, by sex and by age, 2003.
Bar chart showing awareness of need to use 911 emergency telephone services if someone is having a heart attack or stroke among Montana state health department employees at baseline and follow-up, by sex and by age, 2003
Table 1 Behavioral Risk Factor Surveillance System (BRFSS) Survey Questions on Signs and Symptoms of Heart Attack and Need to Use 911 Emergency Medical Telephone Services, Montana, 2003
BRFSS Survey Questionsa Response Categories (Correct Response)
Do you think pain or discomfort in the jaw, neck, or back are symptoms of a heart attack? Yes, no, do not know/not sure, refuse to answer
Do you think feeling weak, lightheaded, or faint are symptoms of a heart attack?
What about chest pain or discomfort...do you think these are symptoms of a heart attack?
Pain or discomfort in the arms or shoulder (are these symptoms of a heart attack)?
What about shortness of breath... is that a symptom of a heart attack?
Sudden trouble seeing in one or both eyes…is this a symptom of a heart attack? Yes, no, do not know/not sure, refuse to answer
If you thought someone was having a heart attack or stroke, what is the first thing you would do? Would you: Take them to the hospital, tell them to call their doctor, call 911, call their spouse or a family member, do something else, don't know/not sure, refused to answer
a Centers for Disease Control and Prevention (16).
Table 2 Numbers and Percentages of Montana State Health Department Employees Reporting Awareness of Signs and Symptoms of Heart Attack at Baseline and Follow-up, 2003
Baseline
n = 401 Follow-up
n = 337
Signs and symptoms of heart attack % n % n
Pain or discomfort in jaw, neck, or back 69 277 91a 307a
Feeling weak, light-headed, or faint 79 318 89a 301a
Chest pain or discomfort 100 399 100 337
Sudden trouble seeing in one or both eyesb 43 173 46 156
Pain or discomfort in arms or shoulder 100 399 98 330
Shortness of breath 92 370 95 319
Five or more correct responses to heart attack signs and symptomsc 69 277 89a 301a
Call 911 if someone is having a heart attack or stroke 84 337 90a 303a
a P ≤ .05.
b Correct response is "no."
c Total of six questions.
Table 3 Results of Survey Questions to State Health Department Employees, Montana, 2003
Survey Question % Responding Yes
In the past two months, have you seen posters in your workplace with information on heart attack signs and symptoms? 98
Those who responded yes to question above were asked, "Where did you see these posters?"
Hallways 67
Stairwells 16
Elevators 15
Bathrooms 96
Did you participate in any of the weekly e-mail quiz contests on heart attack signs and symptoms? 63
In the past two months, did you receive an Act in Time brochure and wallet card on heart attack signs and symptoms with your paycheck? 74
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Fogle CC, Oser CS, Blades LL, Harwell TS, Helgerson SD, Gohdes D, et al. Increasing employee awareness of the signs and symptoms of heart attack and the need to use 911 in a state health department. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/03_0029.htm.
==== Refs
1 American Heart Association (U.S.). Heart disease and stroke statistics — 2003 update American Heart Association Dallas (TX) 2002 Available from: URL:http://www.americanheart.org/downloadable/ heart/10590179711482003HDSStatsBookREV7-03.pdf
2 Ryan TJ Antman EM Brooks NH Califf RM Hillis LD Hiratzka LF ACC/AHA guidelines for the management of patients with acute myocardial infarction: 1999 update: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, Committee on Management of Acute Myocardial Infarction American College of Cardiology 2002 cited October 13, 2003 Available from: URL:http://www.acc.org/clinical/guidelines/nov96/1999/
3 Goldberg RJ Gurwitz JH Gore JM Duration of, and temporal trends (1994-1997) in, prehospital delay in patients with acute myocardial infarction: the second National Registry of Myocardial Infarction. Arch Intern Med 1999 10 159 18 2141 2147 10527291
4 Hackett TP Cassem NH Factors contributing to delay in responding to the signs and symptoms of acute myocardial infarction. Am J Cardiol 1969 11 24 5 651 658 5347938
5 Sjogren A Erhardt LR Theorell T Circumstances around the onset of a myocardial infarction. A study of factors relevant to the perception of symptoms and to the delay in arriving at a coronary care unit Acta Med Scand 1979 205 4 287 292 433667
6 Bleeker JK Lamers LM Leenders IM Kruyssen DC Simoons ML Trijsburg RW Psychological and knowledge factors related to delay of help-seeking by patients with acute myocardial infarction Psychother Psychosom 1995 63 3-4 151 158 7624459
7 Meischke H Ho MT Eisenberg MS Schaeffer SM Larsen MP Reasons patients with chest pain delay or do not call 911 Ann Emerg Med 1995 2 25 2 193 197 7832346
8 Meischke H Eisenberg MS Schaeffer SM Damon SK Larsen MP Henwood DK Utilization of emergency medical services for symptoms of acute myocardial infarction Heart Lung 1995 Jan–Feb 24 1 11 18 7706094
9 Johnson JA King KB Influence of expectations about symptoms on delay in seeking treatment during myocardial infarction Am J Crit Care 1995 1 4 1 29 35 7894552
10 McKinley S Moser DK Dracup K Treatment-seeking behavior for acute myocardial infarction symptoms in North America and Australia Heart Lung 2000 Jul–Aug 29 >4 237 247 10900060
11 Moser DK Dracup K Beyond sociodemographics: factors influencing the decision to seek treatment for symptoms of acute myocardial infarction Heart Lung 1997 Jul–Aug 26 >4 253 262 9257135
12 Cummins R Advanced cardiac life support 1997 American Heart Association Dallas (TX)
13 McSweeney JC Cody M O'Sullivan P Elberson K Moser DK Garvin BJ Women's early warning symptoms of acute myocardial infarction Circulation 2003 11 25 108 21 2619 2623 14597589
14 Act in time to heart attack signs National Institutes of Health, National Heart, Lung, and Blood Instituten cited 2003 Sep 2 Available from: URL:http://www.nhlbi.nih.gov/actintime/index.htm
15 Luepker RV Raczynski JM Osganian S Goldberg RJ Finnegan JR Jr Hedges JR Effect of a community intervention on patient delay and emergency medical service use in acute coronary heart disease: the Rapid Early Action for Coronary Treatment (REACT) trial JAMA 2000 7 5 284 1 60 67 10872014
16 Centers for Disease Control and Prevention. Behavioral Risk Factor Surveillance System Survey Questionnaire Atlanta (GA) U.S. Department of Health and Human Services, Centers for Disease Control and Prevention 2001 Available from: URL:http://www.cdc.gov/brfss/questionnaires/index.htm
17 Montana Department of Public Health and Human Services. The burden of cardiovascular disease in the state of Montana 2003 2003 MDPHHS Helena (MT)
18 Mitic WR Perkins J The effect of a media campaign on heart attack delay and decision times Can J Public Health 1984 Nov–Dec 75 6 414 418 6525584
19 Ho MT Eisenberg MS Litwin PE Schaeffer SM Damon SK Delay between onset of chest pain and seeking medical care: the effect of public education Ann Emerg Med 1989 7 18 7 727 731 2735589
20 Moses HW Engelking N Taylor GJ Prabhakar C Vallala M Colliver JA Effect of a two-year public education campaign on reducing response time of patients with symptoms of acute myocardial infarction Am J Cardiol 1991 68 2 249 251 2063789
21 Herlitz J Hartford M Blohm M Karlson BW Ekstrom L Risenfors M Effect of a media campaign on delay times and ambulance use in suspected acute myocardial infarction Am J Cardiol 1989 7 1 64 1 90 93 2741818
22 Bett N Aroney G Thompson P Impact of a national educational campaign to reduce patient delay in possible heart attack Aust N Z J Med 1993 4 23 2 157 161 8517840
23 Gaspoz JM Unger PF Urban P Chevrolet JC Rutishauser W Lovis C Impact of a public campaign on pre-hospital delay in patients reporting chest pain Heart 1996 5 76 2 150 155 8795479
24 Meischke H Dulberg EM Schaeffer SS Henwood DK Larsen CL Eisenberg MS 'Call fast, Call 911': a direct mail campaign to reduce patient delay in acute myocardial infarction Am J Public Health 1997 10 87 10 1705 1709 9357360
25 Caldwell MA Miaskowski C Mass media interventions to reduce help-seeking delay in people with symptoms of acute myocardial infarction: time for a new approach? Patient Educ Couns 2002 1 46 1 1 9 11804764
26 Goff DC Jr Sellers DE McGovern PG Meischke H Goldberg RJ Bittner V Knowledge of heart attack symptoms in a population survey in the United States: The REACT Trial. Rapid Early Action for Coronary Treatment Arch Intern Med 1998 11 23 158 21 2329 2338 9827784
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Prev Chronic Dis. 2004 Jun 15; 1(3):A07
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Prev Chronic Dis
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Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_04_0043
15670431
Tools and Techniques
VERB™ — A Social Marketing Campaign to Increase Physical Activity Among Youth
Wong Faye MPH, RD VERB Campaign, Division of Adolescent and School Health (DASH), National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP), Centers for Disease Control and Prevention
4770 Buford Hwy, NE, Mail Stop K-85, Atlanta, GA 30341-3717 [email protected]
Centers for Disease Control and Prevention 770-488-6427
Huhman Marian PhD VERB Campaign, DASH, NCCDPHP, CDC, Atlanta, Ga
Asbury Lori VERB Campaign, DASH, NCCDPHP, CDC, Atlanta, Ga
Bretthauer-Mueller Rosemary VERB Campaign, DASH, NCCDPHP, CDC, Atlanta, Ga
McCarthy Susan MPH VERB Campaign, DASH, NCCDPHP, CDC, Atlanta, Ga
Londe Paula MD, PhD VERB Campaign, DASH, NCCDPHP, CDC, Atlanta, Ga
Heitzler Carrie MPH VERB Campaign, Division of Nutrition and Physical Activity, NCCDPHP, CDC, Atlanta, Ga
7 2004
15 6 2004
1 3 A102004
The VERB campaign is a multiethnic media campaign with a goal to increase and maintain physical activity among tweens, or children aged nine to 13 years. Parents, especially mothers aged 29 to 46, and other sources of influence on tweens (e.g., teachers, youth program leaders) are the secondary audiences of the VERB initiative. VERB applies sophisticated commercial marketing techniques to address the public health problem of sedentary lifestyles of American children, using the social marketing principles of product, price, place, and promotion. In this paper, we describe how these four principles were applied to formulate the strategies and tactics of the VERB campaign, and we provide examples of the multimedia materials (e.g., posters, print advertising, television, radio spots) that were created.
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Introduction
In response to increased concern about the health of our nation’s youth, Congress appropriated $125 million in 2001 to the Centers for Disease Control and Prevention (CDC) to develop a national media campaign to change children’s health behaviors. The CDC’s response to this broad mandate was to focus on the sedentary lifestyle of young adolescents and to develop VERB™, a multiethnic campaign launched in June 2002 to increase and maintain physical activity among tweens, or children aged nine to 13 years. These children are between childhood and adolescence and are beginning to make their own lifestyle decisions. Parents, especially mothers aged 29 to 46, and other sources of influence on tweens (e.g., teachers, youth program leaders) provide the secondary audiences for the VERB initiative.
During the past 20 years, the combination of decreased physical activity and unhealthful eating has resulted in a doubling of the percentage of overweight children and adolescents (1). Recent reports indicate that five of every eight children aged nine to 13 do not participate in any organized physical activity during their non-school hours, and almost one fourth do not engage in any free-time physical activity (2). More than one in seven children aged six to 19 years are overweight (3), and type 2 diabetes, a disease traditionally restricted to adults, has been reported among adolescents (4).
A Social Marketing Framework
VERB uses a social marketing framework that applies sophisticated commercial marketing techniques to address the public health problem of sedentary lifestyles among American children. The first year of the campaign consisted of national advertising, plus extra marketing activities in nine CDC-selected communities. The CDC based its selection of the nine communities on a variety of factors, including the size of the media market, racial and ethnic diversity, geographic diversity across the United States, existing infrastructure, and population size. Six of the nine communities received even more local advertising so that the CDC could evaluate whether the added media made a measurable difference in behavioral outcomes. These six evaluated communities were called “high-dose” communities.
Nine Communities Receiving Extra Marketing Activities
A U.S. map on which the following communities are highlighted: Washington DC, Los Angeles, Green Bay (WI), Columbus (OH), Greenville (SC), Houston, Miami, Spokane (WA), Kansas City (MO). Six of the cities (Los Angeles, Green Bay, Houston, Columbus, Greenville, and Miami) are additionally highlighted as high-dose communities.
Before campaign planners launched or named the campaign, they conducted extensive research on tweens and parents to gain an understanding of their attitudes, beliefs, and behaviors related to participation in physical activity. Research included numerous in-person focus groups, interviews, and ethnographic inquiries among multiethnic groups across the country. Additional audience research was conducted separately with African American, Hispanic/Latino, American Indian, and Asian American tweens and parents to gain deeper insights into their physical activity views and practices. The understandings gained contributed to the creation of VERB, a “for tweens, by tweens” brand that embraces the characteristics valued by tweens. VERB is not an acronym but is the word verb as a part of speech, meaning an action word. The tag line is, “It’s what you do.” (Formative research reports for the VERB campaign are available from http://www.cdc.gov/youthcampaign/research/resources.htm.)
Campaign planners applied the four Ps of commercial marketing — product, price, place, and promotion (5) — along with findings from the audience research to develop VERB as a social marketing campaign. In this paper, we describe how the four Ps helped to formulate the strategies and tactics of the VERB campaign.
Product
In social marketing, product is the desired behavior for the targeted audience. The VERB campaign’s product is physical activity — a voluntary action that requires personal choice and internal motivation if it is to be performed repeatedly. The VERB “sales package” includes the intangible benefits of physical activity (e.g., enjoyment) in addition to tangible objects or services that support behavior change (6).
For tweens, choosing to be physically active means giving up something they may like doing better. In today’s world, numerous activities compete for tweens’ attention, and tweens and their parents must overcome many barriers to become physically active, including lack of transportation, safety concerns, cost, and perceived lack of time (2). Children report spending more than 4.5 hours daily watching television, playing video games, or using the computer; parents report their children’s screen time to be almost 6.5 hours daily (7). The VERB campaign’s goal is to win or gain a greater market share of time tweens spend on sedentary activities.
The campaign’s strategy is to influence participation in physical activity by associating it with the benefits that tweens value, such as spending time with friends, playing, having fun, having an opportunity to be active with parents, and gaining recognition from peers and adults. In addition, VERB offers tweens something else they value: the opportunity to explore and discover the world around them. Early VERB advertising stimulated curiosity about the brand and enticed tweens to identify and try the activities or VERBs (e.g., swim, run, jump) that most appealed to them.
Price
Price represents a balance of product benefits and costs to a consumer. When contemplating the purchase of a tangible product such as a tennis racquet, for example, a consumer balances the potential benefits of playing tennis against the price tag (5,6). When the product is behavior change, the concept still holds: what are the benefits and costs of changing behavior (8)? For physical activity, the costs can be financial (e.g., price of dance classes), psychological (e.g., the tween does not “feel good enough” to participate in physical activity or organized sports), environmental (e.g., the neighborhood does not have sidewalks), or related to time (e.g., both parents work, leaving tweens with no time for supervised physical activity). VERB messages are designed to convince tweens and their parents that physical activity has the “right price” — that benefits outweigh costs.
Audience research yielded numerous insights into how tweens and parents view the benefits of physical activity; the CDC thus had information to show how the benefits of physical activity exceed those of non-active pursuits. The VERB campaign weaves these benefits throughout its messages, strategies, and tactics to make physical activity appealing and inviting to tweens.
Place
Download the VERB Coolness Tip Sheet (PDF 313K)
The VERB coolness tip sheet
Place is where the target audience either performs the behavior or accesses programs or services; place must be readily available to enable the desired action (5,6). A VERB place is where tweens can be physically active in a safe environment. As interest or demand rises, parents and communities need to increase the supply of accessible places and opportunities for tweens to be active every day. If the supply side is inadequate or inaccessible, tweens cannot act on their interest and intent to be more active. For VERB, a place may be a backyard, youth-serving organization, community-based organization, church, park or recreation department, school, public or private sports organization, business, government agency, or any other place that can provide facilities and year-round or periodic event-based opportunities for tweens to be physically active and have fun.
Partner organizations such as parks, schools, and youth-serving organizations can reap the benefits of tweens’ affinity for the VERB brand and interest in becoming more active. Keeping VERB a “cool brand for tweens” is a critically important goal for partners as they collaborate on the campaign. (More information on keeping VERB “cool” is available from the VERB Coolness Tip Sheet.)
Promotion
Promotion is not simply the placement of advertisements — communication messages and activities are included as well, and those in charge of promotions must consider multiple ways to reach the target audience to promote the benefits of the behavior change, including its product, price, and place components (6,7). The following sections provide descriptions and examples of the VERB campaign’s messaging strategies, advertising and marketing strategies, and campaign tactics for reaching tweens and parents.
Messaging strategies
Advertising and promotions do more than merely sell the features of a product; they depict a lifestyle that consumers aspire to achieve. By association, consumers perceive the product as providing the means to a desired outcome. In commercials, for example, a soft drink is more than a drink; it is a social experience. Running shoes are more than footwear; they make a statement about an individual’s lifestyle. In the VERB campaign, commercial strategies for marketing to youth are applied to public health and used to “sell” physical activity to tweens, creating a distinct brand culture for VERB.
VERB aims to sell physical activity to consumers, but boys and girls cannot simply go to a store to buy it. Rather, tweens must develop a positive disposition to physical activity through a positive association and relationship with the VERB brand. To make the product of physical activity compelling and cool to tweens, VERB messages diverge from the “just-the-facts” delivery that is central to many public health campaigns. Rather than say, “Engage in moderate-to-vigorous physical activity for at least 60 minutes each day,” the campaign asks tweens to discover new activities they like to do.
The fact that the campaign’s brand name, VERB, immediately connotes action increases the audience’s comprehension of the brand. To inspire tweens to do their own VERBs (physical activities), the campaign does not simply tell tweens that physical activity is for all tweens, it shows them with appealing visuals. Casting for television and print ads includes children of varied racial and ethnic backgrounds, body weights, and ability levels — including children with disabilities — to convey a sense of “kids like me do this” and “I can do that.” VERB shows tweens playing backyard games in addition to participating in organized physical activities such as team sports.
If tweens are inspired by a VERB commercial and motivated to be active, parents will more likely support their participation. Had the campaign’s strategy primarily focused on asking parents to encourage their children to be active, however, tweens might not have embraced VERB as their own brand. The campaign would have taken on a parenting agenda rather than becoming something for tweens. Notably, VERB features positive “can do” messages, not negative adult-delivered or adult-enforced “must do” or “don’t do” messages.
Parents and others who influcence tweens are asked to support, recognize, and praise children for being active; to encourage them to try new activities; and to be physically active as a family or group. VERB gives tips on how to communicate to tweens and engage them in being active in creative, positive, and fun ways. At the same time, facts on the health risks of inactivity and excessive screen time and on the benefits of being physically active are messages for adults. The messages are further tailored for different audiences, especially ethnic audiences, to address the specific parenting priorities learned through audience research.
All VERB campaign messages and the way they are presented in ads (i.e., television, radio, print) are tested with tween-aged focus groups to ensure they are motivating, clearly understood, and resonating positively. The messages are tested primarily with mothers to ensure they are acceptable and not offensive or inappropriate.
Advertising and marketing strategies
VERB™ Bumper sticker ad
Poster Image: A car is covered with a humorously large number of bumper stickers, all of them describing the sports in which the owners' children participate.
Poster Text: Encourage your kids to try new things.
Getting your kids out, active, and participating in new and different activities helps them build self esteem, gain confidence, and feel better. With your help and inspiration, you, too, could see a change in your child over time (as well as your car). VERB. It's getting kids into action.
Legal Text: For more information visit VERBparents.com HHS logo, CDC Logo, VERB logo
VERB "Bumber sticker" poster
The VERB campaign strives for high brand awareness and affinity among tweens. The theory is that when tweens are positively bonded with VERB, they will be more receptive to messages about physical activity. In the first year of VERB, marketing efforts were dedicated to creating and introducing the VERB brand to tweens. As a previously nonexistent brand, VERB initially had no value to tweens. To sell VERB successfully to tweens as “their brand for having fun,” the campaign associates itself with popular kids’ brands, athletes, and celebrities, and activities and products that are cool, fun, and motivating.
Communications for parents and tweens are separated from each other to maintain tweens’ positive affinity for the VERB brand and to avoid associating physical activity with something adults say “they have to do.” Tweens’ interests and trends evolve rapidly, whether the subject is music, clothing, electronics, or the enjoyment of being active. It is all about being “cool” to their friends and doing what’s popular. The delicate balance of keeping the VERB brand cool for tweens while relying on parents and other adult influencers’ support to help them be active is an ongoing campaign challenge.
VERB is distinct from traditional public service announcement (PSA) campaigns because advertising placement is purchased. In the first year of VERB, the campaign averaged 115 weekly gross rating points1 (GRPs) in the national television media market with 50 percent more GRPs in the high-dose communities. The purchase of media enables the campaign to control when and where advertising appears and to concentrate ad placement in delivery channels that reach the most tweens; popular delivery channels include kids’ television networks (e.g., Nickelodeon, Cartoon Network) and teen magazines (e.g., Teen People, Seventeen). Furthermore, it assures that tweens are sufficiently exposed to the advertising to recognize and develop a personal relationship with the brand and to understand the campaign’s messages to become active and have fun doing it. Having the funding to purchase media placements allows VERB to compete with commercial youth marketers to capture the attention and brand loyalty of tweens.
Campaign tactics
VERB employs a broad mix of campaign tactics to reach tweens and their parents. The campaign is designed to surround tweens at home, in school, and in the community to give VERB visible presence in tweens’ everyday lives.
Campaign Materials
"Hip Hop Scotch"
Watch "Hip Hop Scotch" VERB TV spot (RM 1.9mb)
30-second Television Spot VISUALS Different shots all taking place at Mecca Park in Houston, Texas. The lead tween introduces herself as “Andrea” and explains how to play a game called “Hip-Hop Scotch.” Spot opens with a stop-motion shot of tweens dancing. Shots featured are close-ups of the kids, as well as tweens playing the game. A hip-hop beat accompanies the shots of children and the game, while the rules are explained with voice-over. Title graphics of key words and images appear throughout, such as “Hip Hop Scotch,” “Dance Moves,” and a diagram of the boxes. Additional shots include kids playing the game and dancing, and a stop-motion shot of girl dancing. The spot ends with an aerial shot of a group of kids looking up and saying the tagline, “VERB, It’s what you do,” as graphic animation depicting the logo and tagline fade in. The closing shot is a graphic with the word “VERB” being written with chalk on asphalt.
GIRL TWEEN Andrea: Hey, I’m Andrea and we play at Mecca Park in Houston, Texas.
We’ve got lots of VERBs here. Some games we even make up. Here’s one we call Hip-Hop Scotch.
(Title Graphic— “Andrea”/“Hip-Hop Scotch”)
Andrea: It’s old school meets new school. You draw boxes, but instead of numbers you write in dance moves like spin, bounce, step, or shake. (Title Graphic— “Dance Moves”)
Andrea: Wherever it lands, that’s your thing. If it lands in the question mark, do anything. (Title Graphic— “Do anything”)
Andrea: That’s “Hip-Hop Scotch” in Houston. Every day is game day.
GIRL TWEEN Girl Tween 2 & 3: So get out there and go play! (Title Graphic— “Go Play”)
"Future"
Watch "Future" VERB TV spot (RM 3.6mb)
60-second Television Spot VISUALS A father in his mid-30s returns home from work feeling very tired. When he steps into his house, he sees his two children (aged ten and seven years) playing video games in the living room. At that point, he feels very proud and happy that he has provided a comfortable environment for his children. But when he glances through a picture of himself and his parents taken during his graduation, it triggers his memory of the old days, and then we see a flash-back of him. When he was a young child, his parents always took him out to play and supported him in all kinds of physical activities. All these activities helped him to become a healthy and well–balanced person. We then we see him back in today’s life. He decides that he shouldn’t give himself any more excuses and should be like his parents in helping his children to step into a brighter future. He takes out his baseball gloves and together with his wife, takes the children out to play. The voice-over reminds the audience that physical activities can help children do better in school, boost their confidence, and help develop both their body and mind. The spot ends with a call to action telling parents and adults that they need to encourage children to have 60 minutes of physical activity every day, as the words “VERB — A Campaign To Build Healthy Children” appear on the screen.
ADULT VOICE–OVER If you want your children to step into a brighter future, encourage them to participate in more physical activities. It could help them do better in school and boost their confidence for tomorrow’s challenges!
ADULT VOICE–OVER Girl Tween 2 & 3: So get out there and go play! (Title Graphic— “Go Play”)
SIGN–OFF VERB™
A campaign to build healthy children
www.VERBparents.com
SIGN–OFF U.S. Department of Health and Human Services
Centers for Disease Control and Prevention
"Hip Hop Scotch"
Hear "Hip Hop Scotch" VERB radio spot
30-second Radio Spot VOICE-OVER Hey, check out these kids who made up their own VERB…
VOICEOVER ANDREA: Hey, I'm Andrea and we play in Houston, Texas. We've got lots of VERBs here, some games we even make up. Here's one we call "Hip-Hop Scotch." You draw boxes. But instead of numbers, you write in dance moves. Like spin . . . bounce . . . or shake. Wherever it lands, that's your thing! If it lands on the question mark, do anything. That's "Hip Hop Scotch" in Houston.
VOICE-OVER Everyday is game day, so get out there and go play.
VOICEOVER For more cool games, log on to VERB at VERBnow.com.
VERB. It's what you do.
Made Possible by the U.S. Department of Health and Human Services and the Centers for Disease Control and Prevention.
Paid media advertising
The primary vehicle for reaching into the home is paid advertising in general market and ethnic media channels. VERB commercials air on age-appropriate television and radio channels such as Cartoon Network, Nickelodeon, The WB, ABC Saturday Morning Disney (including Radio Disney), Telemundo, and BET. Print advertising is placed in youth publications such as Sports Illustrated for Kids, TIME for Kids, Teen People, and xSeventeen. Examples of parent publications include Family Circle, Parent Magazine, Ebony, and Indian Country Today. Spanish and Asian in-language advertising and advertorials appear in publications such as Korea Times, World Journal, and Los Padres. (An inventory of current VERB advertising is available from http://www.cdc.gov/youthcampaign/advertising/index.htm.)
Added-value opportunities
In addition to paid media placements, the campaign negotiates added-value opportunities from media partners. VERB’s media partners donate their talent and properties or placements to help promote VERB’s physical activity messages to tweens. For example, media partners have produced VERB PSAs using their television talent, such as the stars of The WB’s Gilmore Girls and 7th Heaven and properties such as Disney’s Kim Possible and Cartoon Network’s Courage the Cowardly Dog. The PSAs are aired in prime time during these shows. For parents, The WB produced VERB PSAs featuring Reba McEntire and CBS produced VERB PSAs featuring Deion Sanders. Other examples include VERB sponsorship of Nickelodeon’s Wild and Crazy Kids Show, Sports Illustrated for Kids’ Road Show, and Teen People’s Break for the Beach. These added-value PSAs and sponsorships increase the “cool factor” and marketing reach of the VERB campaign among tweens.
Activity promotions
Several times a year, VERB features promotions that invite community-based organizations and schools throughout the United States to participate. For example, in 2003, VERB proclaimed the day of the summer solstice (the longest day of the year) as the “Longest Day of Play” and created a promotion with Radio Disney to motivate tweens to be active all day long. During fall 2003, when the clocks were turned back one hour from daylight saving to standard time, VERB featured its “Extra Hour for Extra Action” (EHEA) promotion, which included a kit of innovative and fun VERB materials for teachers and youth-serving organizations to use in activating tweens. Participating EHEA schools and organizations were eligible to apply for a small grant to support physical activity at the end of the three-week promotion.
Download the Weekly Reader (PDF 477K)
VERB™ — A Social Marketing Campaign to Increase Physical Activity Among Youth
The VERB Weekly Reader
VERB Student Planner 2003–2004 Download the current Student Planner (PDF 16mb). This planner is also available from VERB in HTML format.
The VERB Student Planner
Schools
School is a natural venue for reaching tweens; it also gives youth a prime opportunity to discover their interests and develop skills. For example, working with youth publications like Weekly Reader and TIME for Kids, VERB distributes custom-developed materials to middle schools throughout the country. Primedia’s Channel One allows VERB advertising to reach tweens through schools. In-school vehicles include book covers, day planners, and customized lesson plans that incorporate physical activity into the classroom and encourage tweens to try many different VERBs.
Community-based events and grassroots marketing
VERB participated in existing community events, including cultural festivals such as the Harvest Moon Festival (Los Angeles, Calif), Calle Ocho (Miami, Fla), and the Gathering of Nations Pow Wow (Albuquerque, NM). At these grassroots community events, VERB hosted an “activity zone,” a dedicated space for tweens to try out different activities such as kicking a soccer ball, dancing, performing martial arts, or other activities. Another community-based tactic is the use of “street teams,” teams of five to eight college-aged men and women hired to engage tweens in being physically active at events and tween hangouts, including malls, parks, and community centers. Street teams create buzz about VERB and build affinity for the brand as tweens tell their friends and siblings about their fun experiences and show off their VERB premiums. The street teams distribute VERB-branded premiums to tweens, such as foot bags, T-shirts, temporary tattoos, and Frisbee disks.
Contests and sweepstakes
To increase the value of the product and reward tweens for being active, many media partners sponsor VERB contests and sweepstakes. For example, Channel One sponsored a pedometer-based middle-school competition, Make Every Move Count. The schools that accumulated the most steps won an "Action Pack" of physical activity equipment and materials to support their physical activity programs. In addition, YM (Your Magazine) featured the VERB Move It to Groove It contest, where tween contestants competed to win a video dance party for their entire school.
Public relations
VERB continuously communicates with the news media, stakeholders, and partner organizations to offer information on the importance of youth physical activity to parents and other influencers and to spotlight current campaign activities, such as events and promotions. The campaign maintains good relationships with key members of the tween/teen and parent news media to keep them current on the campaign and to serve as resources for information about youth physical activity, childhood overweight, and related topics. News media materials are tailored to meet specific needs, media tours are conducted in key markets, and special news media coverage is arranged when appropriate.
Community partnerships
In the first year of VERB, the campaign developed local partnerships in the nine cities that received extra marketing activities to bring the VERB brand to life and to establish a foundation of support in those locations. Now, the goal of VERB is to recruit organizations across the country to become site partners (organizations that can provide opportunities for tweens to be physically active) or outreach partners (organizations that can reach parents or influence the environment to support tweens' participation in physical activity). The campaign is actively reaching out to organizations that have a network of affiliates or chapters across the country and also is contacting regional, state, and local organizations. (More information on VERB partnerships is available from http://www.cdc.gov/youthcampaign/partners/index.htm)
VERB poster "It's What You Do. Native Style."
VERB poster: It's What You Do. Native Style. Images are of healthy American Indian kids playing and exercising.
VERB poster "Sal y Juega."
Sal y Juega poster, version 3. Images are of sports equipment and exercising kids.
Corporate partnerships
VERB also is seeking partnerships with corporations to extend the reach and appeal of the campaign to tweens. For example, VERB is successfully negotiating partnerships with professional sports leagues for its ProVERB initiative; those who have made commitments include the National Football League, National Hockey League, Major League Soccer, and Women’s Tennis Association. These sports leagues will provide content for the VERBnow.com Web site for tweens, donate athlete-signed merchandise for prizes, and provide opportunities for VERB sponsorship of their grassroots sports clinics.
Web sites
In partnership with AOL, VERB has created http://www.VERBnow.com, a Web site designed exclusively for tweens that includes the VERB Recorder, where tweens can report their participation in physical activity and become eligible to win prizes for being active. A parent site, http://www.VERBparents.com, includes in-language pages (Spanish, Chinese, Korean, and Vietnamese) in partnership with ethnic media partners. In addition, http://www.cdc.gov/VERB was created for partners and stakeholders to access information about the VERB campaign and to view advertising.
Summary
The VERB campaign is a public health and marketing partnership based on the social marketing principles of product, price, place, and promotion. It brings together a diverse array of public health, marketing, and community experts to engage tweens in being physically active every day by playing, having fun, and trying new VERBs, or new ways to be physically active. A lifestyle or behavior change such as increasing physical activity is difficult to achieve and even more difficult to sustain. One can speculate, however, that success in changing behavior among tweens is more likely to be achieved when the following conditions are met: consumers have an in-depth understanding of the product and price associated with it, they have easy access to appropriate places where they can perform the behavior in everyday life, and product promotion portrays benefits in a positive, appealing fashion and reaches audiences through channels they value.
Rigorous multiyear evaluation of the VERB campaign will determine its effectiveness in motivating tweens to be more active. To ensure objectivity, the CDC has retained an evaluation contractor. The outcome evaluation is designed as a nationally representative longitudinal study of more than 6000 tweens and their parents across the country, half of whom are from the six high-dose communities. A baseline survey was conducted before the campaign's launch in June 2002. Two follow-up surveys, one conducted in 2003 and one to be completed in 2004, will measure the effectiveness of the campaign. The evaluation methodology, a longitudinal dose-response analysis, controls for numerous baseline factors and allows evaluators to measure changes in physical activity specifically attributable to the VERB campaign.
The authors acknowledge and thank former CDC VERB campaign strategists Michael Greenwell, Suzanne Gates, and Claudia Parvanta; former campaign team members Jami Fraze, Victor Medrano, and Enbal Shacham; and current campaign staff Bill Wood, Wanda Price, Juanita Mondesire, Lula Anna Green, Heidi Melancon, Dana Robinson, Cynthia Mitchell, and Judy Berkowitz. We also thank the VERB campaign contractors Frankel (Chicago, Ill); Saatchi & Saatchi (New York, NY), Publicis Dialog (Chicago, Ill), Garcia 360 (San Antonio, Tex); G&G Advertising (Alburquerque, NM); A Partnership, Inc. (New York, NY); PFI Marketing (New York, NY); and Westat (Rockville, Md).
1 Gross rating points is a measure of estimated exposure to advertising. Reach is defined as the proportion of the target audience that has an opportunity to be exposed to the ad. Frequency is the number of times an average target audience member is estimated to have an opportunity to view the advertisement in a given time period, usually weekly or monthly. Multiplying reach times frequency produces the GRP measure. Because GRPs are the product of reach and frequency estimates, a GRP estimate reflects many different possible exposure patterns. For example, if 10% of a population could be reached five times in a week, the weekly GRPs for that advertisement would be 50 (10 x 5); if 50% of the population could be reached once in a week, the GRPs would also be 50 (50 x 1).
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Wong F, Huhman M, Heitzler C, Asbury L, Bretthauer-Mueller R, McCarthy S, et al. VERB™ — a social marketing campaign to increase physical activity among youth. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0043.htm.
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1 Ogden CL Flegal KM Carroll MD Johnson CL Prevalence and trends in overweight among US children and adolescents, 1999-2000 JAMA 2002 288 1728 1732 12365956
2 Centers for Disease Control and Prevention. Physical activity levels among children aged 9-13 years — United States, 2002 MMWR Morb Mortal Wkly Rep 2003 52 33 785 788 12931076
3 2003 National Health and Nutrition Examination Survey; NHES 11/111 (1963-70) NHANES I (1971-74), NHANES II (1976-80), NHANES III (1988-94) Hyattsville (MD) Centers for Disease Control and Prevention, National Center for Health Statistics NHANES 1999 available from: URL: http://www.cdc.gov/nchs/about/major/nhanes/ Databriefs.htm
4 Goran MI Ball GD Cruz ML Obesity and risk of Type 2 diabetes and cardiovascular disease in children and adolescents J Clin Endocrinol Metab 2003 88 1417 1427 12679416
5 Andreasen AR Marketing social change: changing behavior to promote health, social development, and the environment 1995 San Francisco (CA) Jossey-Bass 368
6 Turning Point Social Marketing Collaborative, Centers for Disease Control and Prevention, Academy for Educational Development. CDCynergy: social marketing edition, version 1.0 [CD ROM] 2003 Atlanta (GA) CDC, Office of Communication
7 Woodard EH Media in the home 2000: the fifth annual survey of parents and children 2000 Philadelphia (PA) The Annenberg Public Policy Center of the University of Pennsylvania
8 Kotler P Roberto N Lee N Social marketing: improving the quality of life 2nd 2002 Thousand Oaks (CA) Sage Publications
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==== Front
Prev Chronic Dis
Preventing Chronic Disease
1545-1151
Centers for Disease Control and Prevention
PCDv13_04_0009
Tools and Techniques
The VERB™ Campaign Logic Model: A Tool for Planning and Evaluation
Huhman Marian PhD VERB Campaign, Division of Adolescent and School Health (DASH), National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP), Centers for Disease Control and Prevention
4770 Buford Hwy, NE, Mail Stop K-85, Atlanta, GA 30341 [email protected]
Centers for Disease Control and Prevention 770-488-6437
Heitzler Carrie MPH VERB Campaign, Division of Nutrition and Physical Activity, NCCDPHP, CDC, Atlanta, Ga
Wong Faye MPH, RD VERB Campaign, DASH, NCCDPHP, CDC, Atlanta, Ga
7 2004
15 6 2004
1 3 A112004
The VERB campaign uses a logic model as a tool to share information, to facilitate program planning, and to provide direction for evaluation. Behavior change and communication theories are incorporated to help hypothesize how behavior change might occur. Evaluation of the campaign follows the process of the logic model. The elements of the logic model are described and further explanation “pops up” as the reader rolls over the graphic of the logic model.
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Introduction
A logic model is a picture of how planners think their program is going to work. It is a systematic, visual way to present the elements of a project and its desired outcomes. Also called a program’s “theory of action,” the logic model expresses not only the obvious components, such as the program’s activities, but also the underlying assumptions and theoretical framework of the program’s interventions (1). Throughout the life of VERB™ (2), the logic model has been a tool for campaign planners to communicate with stakeholders, participating creative agencies, and evaluators. As a road map that guides campaign planning, message development, and outcome evaluation, the logic model has changed as the strategy of VERB has evolved in response to research and results from VERB’s evaluation. Here we describe the elements of the VERB logic model, summarize key theories that have influenced its development, and link evaluation activities to the components of the model. The reader can scroll through the graphic of the logic model for additional information on the model’s components.
Elements of the Model
Enlarged image and descriptive text
VERB Campaign Vision: All youth leading healthy lifestyles.
VERB Campaign Mission: To increase and maintain physical activity among tweens (children aged nine to 13 years).
The Inputs of the campaign are: Consultants
Staff
Research & Evaluation
Contractors
Community Infrastructure
Partnerships
All Inputs contribute to campaign activities. Campaign activities include: Advertising
Promotions
Web
Public Relations
National & Community Outreach
All Activities lead to short-term outcomes for both tween and parent audiences. The short-term outcome for the campaign is tween and parent awareness of and buzz about the campaign brand and its messages. Awareness and buzz lead to mid-term outcomes, which include changes in: Subjective norms
Beliefs
Self-efficacy
Perceived behavioral control
The logic indicates that if these changes occur, positive buzz will be created among tweens about physical activity. Tweens will enlist support from their parents and generate intentions to take part in physical activity.
Awareness and understanding of the campaign and brand messages by parents leads to changes for parents in Knowledge, Beliefs, and Expectations.
The logic indicates that if these changes take place among parents, and if tweens are enlisting their parents' support, parents will support tweens' participation in physical activity, and Parents and influencers will mobilize and advocate for physical activity.
The mobilization of parents and influencers and advocacy for physical activity as well as national and community outreach lead to the Availability and access to organized and non-organized settings for physical activity.
Tweens' behavioral intention as well as parent support and available and accessible settings are likely to result in tweens engaging in physical activity.
The Long-Term Outcomes include tweens engaging in and maintaining physical activity, thereby reducing chronic diseases. The model also indicates a possible displacement strategy: tweens who participate in physical activity may also have a Reduction of unhealthy, risky behavior.
This is the VERB™ Logic ModelThis is the VERB™ Logic Model
When mapping out an activity, it is often best to start with the desired destination. For VERB, the destination is expressed in the model under the heading of Long-Term Outcomes by the statement of broad vision, All youth leading healthy lifestyles, and specific mission, To increase and maintain physical activity among tweens (children aged nine to 13 years). The Congressional language that appropriated funding for VERB included the words of the vision statement. Campaign planners selected physical activity and the tween age group as VERB’s focus to reach the vision. The endpoint of the logic model, the box titled Reduction in chronic diseases, reflects the desired long-term impact of VERB’s vision, an outcome that is broader than the scope of VERB and that will take years to realize. Nonetheless, the goal of tweens engaging in and maintaining physical activity is within reach of the planned five years of the VERB campaign and will affect the development of chronic diseases as children age into adulthood (3).
Inputs
The logic model reads from left to right, beginning with Inputs, or fundamental resources, to the campaign. These inputs include Consultants (experts in marketing, youth physical activity, and evaluation), organizational resources such as project Staff, and the Research & Evaluation infrastructure at the Centers for Disease Control and Prevention (CDC). Contractors represent a major input and include general-market creative and public relations agencies, creative agencies specializing in ethnic markets, and an evaluation contractor. Community Infrastructure is an input that expresses the importance of context for the child’s engagement in physical activity. Partnerships provide an essential resource for guiding, supporting, and extending the reach of the VERB campaign.
Activities
Activities are the products that result from the project’s inputs. As a media campaign, VERB focuses on both paid and donated Advertising in the form of television and radio commercials, print advertisements, and out-of-home ads (e.g., billboards, mall kiosks). Important Promotions include special events, in-school promotions, contests, and sweepstakes. VERB hosts three Web sites, one each for tweens, parents, and partners. Additionally, VERB provides and monitors information through Public Relations activities. National & Community Outreach has been actualized by engaging partners such as the YMCA and Boys & Girls Clubs and by mobilizing community-based constituents whose goals are aligned with those of VERB.
Short-term and mid-term outcomes
Tweens
Activities lead to Short-Term Outcomes and then to Mid-Term Outcomes for both the tween and parent audiences. The first boxes, sometimes called proximal outcomes, relate to tween and parent awareness of the campaign brand and its messages. An underlying assumption of the VERB model is that individuals must first attain a high degree of awareness to achieve behavior change. Awareness for VERB begins with the VERB brand, the vehicle for message delivery. If a brand is created that is “cool” to the target, tweens will talk — or generate Buzz about the campaign and brand messages — further heightening interest in the brand and the messages.
VERB’s messages appeal to tweens’ needs to have fun, to enjoy themselves while being active with friends, and to have confidence that they can be physically active. These messages are linked to desired changes in how tweens experience physical activity in their lives, depicted in the next box of the logic model. Specifically, VERB aims to change tweens’ a) Subjective norms (the belief that important referents approve or disapprove of a behavior); b) Beliefs, especially in the benefits of physical activity; c) Self-efficacy, or confidence about being physically active; and d) Perceived behavioral control, or how strongly tweens believe they can engage in physical activity even if there are barriers to overcome.
Proceeding to Mid-Term Outcomes for tweens, VERB planners believe that as tweens internalize the benefits of physical activity, they will act on these beliefs by a) generating Positive buzz for physical activity among tweens (now about physical activity, not just the VERB ads); b) enlisting support of parents to help them be physically active; c) going through a phase of intend[ing] to do physical activity; or d) going immediately to a long-term outcome of engage[ment] in physical activity. Some children, especially younger children, may skip c) (intention) and go directly to engagement in physical activity because they are not deliberate in their decision-making process, or parents may sometimes intervene prior to intention.
Parents
For parents, short- and mid-term outcomes also begin with message awareness. VERB messaging for parents is meant to work differently, however, than it does for tweens. Because VERB must be kept genuine as a cool “by kids, for kids” brand, the brand is de-emphasized for parents. Parent-directed messages aim to affect how parents prioritize physical activity in their child’s life, giving the parents specific recommendations for physical activity for their child, helping them develop skills on how to verbally and nonverbally support their child’s physical activity, urging them to expect their child to be physically active, and, finally, encouraging parents to be physically active with their child. Campaign planners hypothesize that as parents internalize changes in Knowledge, Beliefs, and Expectations, parents will support tween’s participation in physical activity, enhanced by tweens enlisting support from them. As depicted in the model, planners also expect that as parents prioritize their child’s physical activity needs, the parents, as well as other influencers of tweens (e.g., coaches, teachers), will mobilize and advocate for physical activity.
VERB views parents and other influencers as important forces in helping to ensure that options and opportunities are available for children to be active. For children to engage in organized or free-time activities, they need teams, clubs, and safe and appealing places to be active. As depicted in the model, VERB perceives Activities of National & Community Outreach as essential to improving Availability and access to organized and non-organized settings for physical activity. VERB acknowledges that places to be active are part of the critical socioecological perspective that surrounds the campaign and affects its immediate and long-term success.
Long-term outcomes
The box Tweens engage in physical activity is framed by a double line to indicate that it is the primary distal outcome of the VERB campaign. This box represents a broad conceptualization of engagement in physical activity that ranges from inactive tweens doing at least something to minimally to moderately active tweens doing more; it also includes tweens trying a new activity. VERB ads seek to motivate the minimally active tween and to keep tweens in general interested in physical activity. Promoting new activities may be an important route for some tweens to experience the benefits of physical activity. As depicted in the logic model, intention may precede engagement; parent support and availability and access to physical activity settings are important in facilitating tweens’ engagement in physical activity.
Tweens being physically active is a necessary but not sufficient outcome of the VERB campaign. Thus, the double-lined box leads to the endpoint of the VERB campaign, the critical and sustained effect of VERB on tweens: Tweens maintain physical activity. VERB planners believe that for tweens to maintain physical activity, communities must sustain efforts to provide safe and appealing settings for tweens to be physically active, depicted by the arrow connecting the box Availability and access to organized and non-organized settings for physical activities to the box Tweens maintain physical activity.
Theoretical Constructs
Theories related to social marketing and behavioral change have played an important role in developing the logic model. Research in marketing, communication, and physical activity provided a theoretical structure for advising the creative agencies on developing the VERB messages and for helping evaluators hypothesize a model of change. The logic model’s boxes and directional arrows represent the underlying assumptions and the beliefs or theories about how children will change in response to VERB advertising. Below, we describe the theoretical framework that drives the VERB logic model.
Branding theory
Branding theory (4) posits that the target audience will develop a relationship with the brand that 1) begins with association with brand attributes, 2) builds affinity to those attributes over time, and 3) results ultimately in long-term loyalty to the brand. Hypothesized steps in building the VERB brand relationship include the following:
Association with an image that is cool, fun, and socially appealing.
Production of a positive affective response, such as “I like VERB” or “I want to be VERB,” and a positive cognitive response, such as “I think I can do [what is seen in the advertisement], too.”
Integration of these associations into the child’s identity: for example, “I am a cool, fun kid because I am a VERB kid.”
Evolution of the child’s beliefs — with repeated exposure — toward, for example, “I want to do X and I will try X because I am a cool, fun VERB kid.”
Motivation to change through the VERB brand occurs because VERB supports the child’s existing motives, needs, aspirations, and values. The effect would be that the child first juxtaposes VERB values with his or her own, and then shifts toward adopting the VERB motives, needs, aspirations, and values.
Support for these processes is provided by researchers with the truth™ anti-smoking campaign, who have posited that through internalization of the “truth brand,” youth adopt self-images as a “truth teen” that lead to positive dispositions not to smoke (5). Similarly, VERB assumes that children will adopt behaviors portrayed in the ads that support a self-image of being physically active through a process known as “identification” — how much children want to be like the people they see on television. Identification is associated with important antecedents to behavior change, such as expectations of benefits, and is especially salient with younger children (6).
Message design
Theories of message design help explain the steps for building awareness of a brand like VERB. For example, if branding evokes a high level of interest and identification with the advertisement, then information processing theory, such as the Elaboration Likelihood Model (7), suggests that the child would be willing to exert more cognitive effort to pay attention to the ad, understand it, and actively process its message. Messages provoke active processing when the presentation of content is unusual, unfamiliar, or novel (8) — all key features of the VERB advertisements — and when there is a discrepancy between expectation and reality, such as with the “Paint the Town” VERB promotions in which a water tower is wrapped to look like a soccer ball.
VERB campaign planners decided early on that VERB would be a positive campaign with advertising exclusively characterized by laughter, play, fun, and enjoyment of physical activity. Message designers believe that creating positive feelings in the child will lead to immediate, but also enduring, influence over later cognitive processing (9). Positive appeals are good at attracting attention; they invoke “approach” behaviors, meaning they make it more likely that the audience will be receptive to the forthcoming message. Monahan has asserted that positive appeals are more likely to be recalled, to change attitudes, and to lead to compliance with the desired behavior (9). Moreover, media campaigns that promote commencement of a desirable, positive behavior show larger effect sizes than cessation campaigns that attempt to extinguish a risky or undesirable behavior (10).
Articulating how VERB would lead to change in physical activity behaviors was especially challenging because most health-related behavior change theories are still being tested for their applicability to children. Two theories, the theory of planned behavior (11) and social cognitive theory (12), have been used to plan or test interventions in children and were applied to VERB. The logic model incorporates elements of the two theories into boxes under Short- and Mid-Term Outcomes.
Theory of planned behavior
The theory of planned behavior proposes that one can predict people’s intention to perform a behavior from the attitudes they hold toward that behavior, from a measure of their subjective norms, and from the control they have to perform the behavior (13). The theory proposes that intentions correlate with observed actions (14). A study of Canadian youth reports support for the association of norms, attitudes, and behavioral control with intentions to be physically active (11).
Social cognitive theory
While the theory of planned behavior is a personal-level theory, social cognitive theory emphasizes the interplay of intrapersonal factors, environment, and behavior (15,16). Intrapersonal factors have cognitive, affective, and biological aspects. Environmental factors encompass the immediate social environment, such as teachers, peers, and parents, in addition to physical environments, such as the presence and appeal of a backyard, driveway, or neighborhood. Principles of social cognitive theory that are especially applicable to child physical activity behaviors include 1) perceptions of the environment, knowledge, and skill to perform an activity; 2) others’ observations of and reinforcement for activity trial; 3) beliefs about the likely outcomes of a behavior; and 4) the values that the child places on the outcome. In addition, self-efficacy (the confidence of the child to perform the behavior) is a critical part of the theory and has been shown to be an important prerequisite for the physical activity of children (17).
Information processing theory
Another theory that guided campaign strategists to manage expectations for the size of behavioral effects is McGuire’s hierarchical steps of information processing (18). McGuire’s model posits that the impact of persuasive communication is mediated by three broad stages of message processing: attention, comprehension, and acceptance. Attention depends on exposure and awareness; comprehension is predicated on understanding the message; and acceptance includes intention and, finally, behavior change. In McGuire’s model, because of the inherent variability in how people process media messages, a percentage of the audience is lost at each stage. Thus, high levels of exposure and awareness are needed to create measurable population effects.
Evaluation
The logic model has been an important tool in bringing rigor and direction to VERB’s process and outcome evaluations. In the beginning stages of the campaign, the exercise of mapping how the program would work brought clearer vision and purpose to campaign efforts, which in turn eased the development of measurable objectives, making evaluation more efficient and productive. Every box of the logic model beyond the Inputs section has been evaluated for process or outcome. For example, one of VERB’s major promotional events was evaluated with on-site intercept interviews and a follow-up survey. In addition, VERB’s creative agencies use the VERB Brand Tracking Survey primarily to assess key features of the brand’s resonance with tweens. VERB’s staff use the tracking survey continuously to track the numbers of tweens who have an awareness and understanding of VERB and its messages. They also use the survey to track multiple qualitative features like “wear-out” of commercials and to help modify the channels of VERB’s messages.
The instruments that measure campaign outcomes reflect the constructs expressed in the Short- and Mid-Term Outcomes boxes. The primary outcome tool, the Youth Media Campaign Longitudinal Survey includes items that tap tweens’ perceptions of social norms, self-efficacy, beliefs, and perceived behavior control. Survey items measure tweens’ intentions to be active and perceptions of parental support as well as parental knowledge, attitudes, and behavior. The survey also includes five measures of physical activity behavior to evaluate the long-term outcomes of the campaign. (The survey as well as other evaluation resources are available at http://www.cdc.gov/youthcampaign/research/index.htm.)
Finally, a conceptual and practical benefit of a logic model is reinforcement of the idea among VERB program staff that projects like VERB represent a continuous process where results of evaluation are used to modify Inputs and Activities. To maximize impact of the VERB campaign on the physical activity levels of American tweens, VERB planners are continuously engaged in this feedback loop, responding to audience research data, the monthly tracking study, and the outcome evaluation to refine VERB messages and their delivery.
Summary
The VERB campaign’s logic model is a learning and management tool that began long before the VERB brand existed. The logic model is a workhorse for the VERB campaign; it serves as an effective tool for sharing knowledge among stakeholders, creative agencies, and program staff, contributes to more effective programming, and provides conceptual structure and practical direction for VERB’s evaluation.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Huhman M, Heitzler C, Wong F. The VERB™ campaign logic model: a tool for planning and evaluation. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0033.htm.
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1 W.K. Kellogg Foundation (U.S.) Logic model development guide W.K. Kellogg Foundation Battle Creek (MI) 2001
2 Wong F Huhman M Heitzler C Asbury L Bretthauer-Mueller R McCarthy S VERB™ — a social marketing campaign to increase physical activity among youth Prev Chronic Dis [serial online] cited 2004 Jun 15 Available from: URL:http://www.cdc.gov/pcd/issues/2004/jul/04_0033.htm 2004 7
3 2003 National Health and Nutrition Examination Survey; NHES 11/111 (1963-70) NHANES I (1971-74), NHANES II (1976-80), NHANES III (1988-94) Hyattsville (MD) Centers for Disease Control and Prevention, National Center for Health Statistics NHANES 1999 available from: URL: http://www.cdc.gov/nchs/about/major/nhanes/ Databriefs.htm
4 Aaker D Building strong brands Free Press New York (NY) 1996
5 Evans WD Wasserman J Bertolotti E Martino S Branding behavior: the strategy behind the truth® campaign Soc Marketing Q 2002 8 3 17 29
6 Austin EW Pinkleton BE Fujioka Y The role of interpretation processes and parental discussion in the media’s effects on adolescents’ use of alcohol Pediatrics 2000 105 343 349 10654953
7 Petty RE Cacioppo JT Communication and persuasion. Central and peripheral routes to attitude change Springer-Verlag New York (NY) 1986 262
8 Louis MR Sutton RI Fujioka Y Switching cognitive gears: from habits of mind to active thinking Hum Rel 1991 44 55 76
9 Monahan JL Maibach E Parrott RL Thinking positively: using positive affect when designing health messages Designing health messages: approaches from communication theory and public health practice Sage Publications Thousand Oaks (CA) 1995 81 98
10 Snyder LB Rice R Atkin C How effective are mediated health campaigns? Public communication campaigns 3rd Sage Publications Thousand Oaks (CA) 2001 181 190
11 Mummery WK Spence JC Hudec JC Understanding physical activity intention in Canadian school children and youth: an application of the theory of planned behavior Res Q Exerc and Sport 2000 71 116 124 10925808
12 Perry CL Williams CL Forster JL Wolfson M Wagenaar AC Finnegan JR Background, conceptualization and design of a community-wide research program on adolescent alcohol use: Project Northland Health Educ Res 1993 8 1 125 136 11067180
13 Ajzen I The theory of planned behavior Organ Behav Hum Decision Processes 1991 50 179 211
14 Doll J Ajzen I Accessibility and stability of predictors in the Theory of Planned Behavior J Pers Soc Psychol 1992 63 754 65
15 Bandura A Social foundations of thought and action: a social cognitive theory Prentice Hall Englewood Cliffs (NJ) 1986 544
16 Baranowski T Perry CL Parcel GS Glanz K Lewis FM Rimer BK How individuals, environments, and health behavior interact: social learning theory Health behavior and health education: theory, research, and practice Jossey-Bass, Inc San Francisco (CA) 1997 153 78
17 Sallis JF Prochaska JJ Taylor WC A review of correlates of physical activity of children and adolescents Med Sci Sports Exerc 2000 32 963 75 10795788
18 McGuire WJ McClintock CG Attitude change: the information-processing paradigm Experimental social psychology Holt, Rinehart & Winston New York (NY) 1972
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Book Review
How Healthy Are We? A National Study of Well-Being at Midlife
Sutphen Sussan K MD Choice Care Occupational Medicine Atlanta, Ga
7 2004
15 6 2004
1 3 A12Brim Orville G. Ryff Carol D. Kessler Ronald C.
How Healthy Are We? A National Study of Well-Being at Midlife.
Chicago: The University of Chicago Press. 2004. ISBN: 0-226-07475-7 (cloth) Price: $42.00 688 pages, 74 figures, 104 tables 2004
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Every human being is the author of his own health or disease.
— Siddhartha Gautama, The Buddha, 563–483 BC
Many Americans might agree with this statement, but if asked to rate their own physical, mental, and health beliefs, what would their responses be? How would they respond at midlife? How Healthy Are We? A National Study of Well-Being at Midlife answers these questions.
In 1990, The John D. and Catherine T. MacArthur Foundation established the Research Network on Successful Midlife Development (MIDMAC), directed by one of the book’s editors, Orville Brim. The multidisciplinary network was tasked with examining adults aged 40 to 60 as part of the foundation’s series of studies on midlife development. MIDMAC identified the following goals: Establish specific indicators for a successful midlife.
Establish a factual basis for the course of midlife.
Identify factors that may influence the course of midlife development.
Identify strategies that individuals may use to deal with midlife challenges.
MIDMAC developed a national survey known as MIDUS (Midlife Development in the United States) (available at http://midmac.med.harvard.edu/research.html) to help meet these goals. In How Healthy Are We?, Brim and colleagues present a collection of articles that summarize MIDUS survey findings.
The authors organize the text into four sections. Chapter 1 provides background on MIDMAC and the development of the MIDUS survey, including information on samples, design, measures, and content. Chapters 2 through 6 discuss assessments of physical health and the impact of age, gender, and socioeconomic status. Chapters 7 through 14 explore assessments of psychological well-being and quality of life. Chapters 15 through 21 focus on contexts that affect midlife experiences, including work, family, and geography. Each chapter provides background research, information on how MIDUS was designed and implemented, results, and a comparison between MIDUS and prior studies.
The audience for this text includes a broad range of scientists, such as sociologists, epidemiologists, mental health practitioners, psychologists, and anthropologists, reflecting the range of disciplines involved in developing the survey. In addition, the audience includes physicians, public health practitioners, and policy makers.
Of particular interest from a policy perspective is Chapter 17, which centers on work, family, and social class. The authors find that the lower the income level, the more likely it is that work is juxtaposed with poor social support, a chronically ill child, or other caretaking responsibility. Low-income jobs do not provide flexibility in sick leave or work hours, resulting in a disproportionate number of children who suffer from unmet health and developmental needs — and ultimately impacting our nation’s health.
Chapter 6, a discussion of menopause and the aging process, is another well-written and accessible chapter. The author finds that, for most women, menopause is a benign event unassociated with the severe physical and psychological distress traditionally portrayed.
Other interesting findings in the book include the following: Women report worse health than men, but they devote more effort to health maintenance and have greater longevity.
The social/income gradient to ill health is greater in men than women and is independent of education.
Adults rate the development of relationships with others as the most important factor in having a good life, followed by health, then family.
While daily stresses or demands are greater in the lives of young and midlife adults compared to individuals in other life stages, midlife adults feel they have a greater level of mastery and control over stresses and demands than young adults do.
Overall, How Healthy Are We? answers the title question through research establishing midlife as a time when most adults have a positive view of physical health, have established supportive family relationships, are pleased with their financial situation, have a good quality of life, and have control and mastery of their work. Education, socioeconomic standing, race, and gender affect all of the above.
How Healthy Are We? also can serve as a basis for brainstorming community-based approaches for tackling chronic health issues and for devising new methods to encourage behavioral change. For example, an awareness of gender differences in health-maintenance behavior, noted in Chapter 1, could change approaches to promoting weight loss or smoking cessation for men and women.
Because the MIDUS survey included individuals aged 25–74, a more complete picture emerges on midlife in relation to the other life stages. How Healthy Are We? differs from other texts on midlife by presenting this expanded perspective. Some texts define midlife from a traditional psychological or biological perspective using staging or physical changes. Others take a health-management approach, focusing on specific aspects of midlife such as parenting or sexuality, or examining midlife in the context of larger societal trends such as divorce, two-income families, and longer life expectancy.
MIDMAC and MIDUS serve as excellent blueprints for an integrative approach to investigation. The book describes carefully developed pilot studies and creatively used satellite studies that provide information on a national level as well as on specific target groups. The public health profession could apply this approach to a wide range of problems.
The authors provide great insight into the physical, psychological, and social development that takes place during this important stage of life and establish a clear baseline for further exploration of midlife issues.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
Suggested citation for this article: Sutphen SK. How healthy are we? A national study of well-being at midlife [book review]. Prev Chronic Dis [serial online] 2004 Jul [date cited]. Available from: URL: http://www.cdc.gov/pcd/issues/2004/jul/04_0040.htm.
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Prev Chronic Dis
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Centers for Disease Control and Prevention
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Errata
Erratum, Vol. 1, No. 1
7 2004
15 6 2004
1 3 A132004
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The title of the article “Law as a Tool for Preventing Chronic Diseases: Expanding the Spectrum of Effective Public Health Strategies” appeared incorrectly in the article and in the suggested citation of the article as “Law as a Tool for Preventing Chronic Diseases: Expanding the Range of Effective Public Health Strategies” (Vol. 1: No. 1, January 2004). The title appeared correctly in the Table of Contents.
The information was corrected on our Web site on April 22, 2004. The corrected article appears online at http://www.cdc.gov/pcd/issues/2004/jan/03_0033.htm.
We regret any confusion this error may have caused.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
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Ann Clin Microbiol AntimicrobAnnals of Clinical Microbiology and Antimicrobials1476-0711BioMed Central London 1476-0711-4-151620212710.1186/1476-0711-4-15Case ReportInfection with Anaplasma phagocytophilum in a seronegative patient in Sicily, Italy: Case Report de la Fuente J [email protected] A [email protected] V [email protected] S [email protected] Marco V [email protected] A [email protected] M [email protected] AR [email protected] KM [email protected] Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA2 Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain3 Intituto Zooprofilattico Sperimentale della Sicilia, Via G. Marinuzzi n° 3, 90129 Palermo, Italy4 Istituto Zooprofilattico Sperimentale della Sicilia, Area Barcellona Pozzo di Gotto, Via S. Andrea n° 96, 98051 Barcellona, Italy2005 3 10 2005 4 15 15 10 8 2005 3 10 2005 Copyright © 2005 de la Fuente et al; licensee BioMed Central Ltd.2005de la Fuente 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
Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also.
Case presentation
Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog.
Conclusion
Infection with A. phagocytophilum was confirmed in a patient without a detectable antibody response against the pathogen. The results reported herein documented the first case of confirmed HGA in Sicily, Italy. These results suggested the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.
Human granulocytic anaplasmosisAnaplasma phagocytophilum16S rDNA sequence
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Background
Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe [1,2]. The disease was first described in the United States in 1994 [3,4] and in Slovenia in 1997 [5]. About 65 cases have been reported in Europe although some of them do not fulfill the criteria for confirmed or probable HGA [2]. Human infection with A. phagocytophilum could be confirmed by identification of morulae in granulocytes, positive PCR results using whole blood as a substrate, and/or isolation of A. phagocytophilum from the blood [2]. Serological tests, particularly indirect immunofluorescence assay (IFA), are commonly used but are often negative during the initial phase of the disease. However, high and stable antibody titers to A. phagocytophilum are usually found in patients with probable HGA who show clinical signs and symptoms that most likely are not the result of a recent infection with A. phagocytophilum [2].
Although the seroprevalence of HGA in central and southern Italy is high in workers at risk for tick bites [6], only two cases of HGA have been reported in Italy [7]. However, the analysis of A. phagocytophilum DNA was not performed in these cases. The objective of this study was to characterize the 16S rDNA sequence of a strain of A. phagocytophilum obtained from a seronegative patient with confirmed HGA in Sicily, Italy.
Case Presentation
Patient, materials and methods
On May 8, 2004, a 51-year-old man living in Barcellona Pozzo di Gotto, Messina, Sicily, Italy was admitted to the hospital with a 3-month fever (37.2°C), arthralgia, myalgia, malaise, pallor, stiffnes of hands and knee, nausea, low appetite and weight loss. The patient did not recall a tick bite but he visited hunting areas 15 days before the fever started and kept exotic birds (golden pheasants, partridges and guinea fowls) at his house for two years. The patient showed abnormal liver (462 mU/ml alkaline phosphatase, normal range (nr) = 98–280; 1.20 mg/dl total bilirubin, nr = 0.16–1.10; 0.35 mg/dl direct bilirubin, nr = 0.0–0.2) and renal (1.5 mg/dl creatinine, nr = 0.5–1.2) functions and low hemoglobin (9.9 g/dl; nr = 12–18). Pulmonary, cardiac and immunological involvement was absent. Cell counts and serum C-reactive protein levels were normal.
On September 13, 2004 the patient was admitted to the hospital for a second time. In addition to previous findings, the patient showed a mild splenomegaly, hemorrhoids, osteopenia, hiatal hernia, gastritis and inflammation of duodenum. Systemic inflammatory pathology and neoplasia were ruled out. Doctors suspected a depressive syndrome and the patient was treated with antidepressants.
Serum antibodies were determined for Lyme borreliosis, Rickettsia conorii, Coxiella burnetii and A. phagocytophilum (Fuller Laboratories, Fullerton, CA, USA), and Ehrlichia chaffeensis and A. phagocytophilum (Focus Technologies, Cypress, CA, USA) following manufacturer's recommendations. FITC-conjugated anti-human IgG (Sigma, St. Louis, MO, USA) and IgM (Aliphadia S.A., Wavre, Belgium) were used as secondary antibodies. The immunofluorescence tests for A. phagocytophilum use antigens derived from human HL-60 cells infected with the HGE-1 isolate and samples were considered negative when no fluoresce was detected at 1:80 (Fuller Laboratories) or 1:64 (Focus Technologies) dilution of the test serum, as recommended by the manufacturer. DNA was purified using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma) or TriReagent (Sigma) from patient's blood samples collected in September 8 and October 8, 2004. PCR analysis for R. conorii, C. burnetii, E. chaffeensis, Theileria spp., Babesia spp. and Anaplasma spp. were conducted on both DNA samples as previously described [8-11]. PCRs were conducted with 1 μl (0.1–10 ng) DNA using 10 pmol of each primer in a 50-μl volume (1.5 mM MgSO4, 0.2 mM dNTP, 1 × AMV/Tfl 5 × reaction buffer, 5u Tfl DNA polymerase) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler for 35 cycles. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb DNA Ladder, Promega). Control reactions were done without the addition of DNA to rule out contaminations during PCR.
Amplified Anaplasma 16S rDNA fragments were resin purified (Wizard, Promega) and cloned into pGEM-T vector (Promega) for sequencing both strands by double-stranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University). Two independent clones were sequenced from each PCR. Multiple sequence alignment was performed with the program AlignX (Vector NTI Suite, version 5.5; InforMax, North Bethesda, MD, USA).
Results and discussion
The analysis of the clinical symptoms and laboratory results of the patient described herein suggested the possibility of an infectious agent as the cause of the illness. Viral infections were ruled out and the investigation was directed towards the identification of tick-borne pathogens.
Serology for Lyme borreliosis, R. conorii, C. burnetii, E. chaffeensis and HGA and PCR analyses for R. conorii, C. burnetii, E. chaffeensis, Theileria spp. and Babesia spp. were negative. However, positive PCR results were obtained for Anaplasma spp. in patient's blood samples collected in September 8 and October 8, 2004. PCR reactions for Anaplasma spp. 16S rDNA were conducted with independent DNA extractions in two laboratories (in Palermo, Sicily and in Ciudad Real, Spain) to confirm the results. Control reactions ruled out PCR contaminations and the sequence of the 16S rDNA fragment obtained from the patient (Genbank accession number DQ029028) was identical in both DNA samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog (Genbank accession number AY055469; ref. [12]).
The patient described herein fulfilled the criteria for confirmed HGA, including prolonged fever, arthralgia, myalgia, malaise, nausea, abnormal liver function and positive PCR and sequence analysis for A. phagocytophilum DNA. However, the patient was seronegative for up to six months after detection of pathogen infection by PCR. These results suggested that the patient presented a chronic stage of infection with symptoms produced by a secondary illness related or not to A. phagocytophilum and/or could has been infected with a strain of the pathogen that could not be detected by existing serological tests or induces a low antibody response. Strain genetic differences have been reported in A. phagocytophilum and may be associated with variations in pathogenicity and host tropism [10,12-14], although the exact relationship between these factors is presently unknown.
Conclusion
The results reported herein demonstrated a prolonged A. phagocytophilum infection in a patient without a detectable antibody response against the pathogen. These results documented the first case of prolonged A. phagocytophilum infection in Sicily, Italy and suggest the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.
Authors' contributions
JF carried out the sequence analysis, designed the study and drafted the manuscript. AT and SC conceived the study, and participated in its design and coordination and helped to draft the manuscript. VN and AA carried out the molecular genetic studies. VDM and MR collected and analyzed the clinical data. ARM carried out the immunoassays. KMK helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This research was supported by The Ministry of Health, Italy, the Instituto de Ciencias de la Salud (ICS-JCCM), Spain (project 03052-00) and the Endowed Chair for Food Animal Research and the Oklahoma Agricultural Experiment Station Project 1669 (K.M. Kocan). V. Naranjo is funded by Junta de Comunidades de Castilla – La Mancha (JCCM), Spain. We thank S. Scimeca, R. D'Agostino and A. Corrente (Intituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy) for technical assistance.
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Bakken JS Dumler JS Human granulocytic ehrlichiosis Clin Infect Dis 2000 31 554 560 10987720 10.1086/313948
Lotric-Furlan S Petrovec M Avsic-Zupanc T Strle F Comparison of patients fulfilling criteria for confirmed and probable human granulocytic ehrlichiosis Scand J Infect Dis 2004 36 817 822 15764167
Bakken JS Dumler JS Chen S-M Eckman MR Van Etta LL Walker DH Human granulocytic ehrlichiosis in the upper Midwest United States, a new species emerging? JAMA 1994 272 212 218 8022040 10.1001/jama.272.3.212
Chen S-M Dumler JS Bakken JS Walker DH Identification of a granulocytotropic ehrlichia species as the etiologic agent of human diseases J Clin Microbiol 1994 32 589 595 8195363
Petrovec M Lotric Furlan S Zupanc TA Strle F Brouqui P Roux V Dumler JS Human disease in Europe caused by a granulocytic Ehrlichia species J Clin Microbiol 1997 35 1556 1559 9163481
Santino I Cammarata E Franco S Galdiero F Oliva B Sessa R Cipriani P Tempera G Del Piano M Multicentric study of seroprevalence of Borrelia burgdorferi and Anaplasma phagocytophila in high-risk groups in regions of central and southern Italy Int J Immunopathol Pharmacol 2004 17 219 223 15171823
Ruscio M Cinco M Human granulocytic ehrlichiosis in Italy: first report on two confirmed cases Ann NY Acad Sci 2003 990 350 352 12860650
Tzianabos T Anderson BE McDade JE Detection of Rickettsia rickettsii DNA in clinical specimens by using polymerase chain reaction technology J Clin Microbiol 1989 27 2866 2868 2512328
To H Kako N Zhang GQ Otsuka H Ogawa M Ochiai O Nguyen SV Yamaguchi T Fukushi H Nagaoka N Akiyama M Amano K Hirai K Q fever pneumonia in children in Japan J Clin Microbiol 1996 34 647 651 8904431
Stuen S Nevland S Moum T Fatal cases of Tick-borne fever (TBF) in sheep caused by several 16S rRNA gene variants of Anaplasma phagocytophilum Ann NY Acad Sci 2003 990 433 434 12860670
de la Fuente J Naranjo V Ruiz-Fons F Vicente J Estrada-Peña A Almazán C Kocan KM Martín MP Gortázar C Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus) in central Spain Eur J Wild Res 2004 50 187 196 10.1007/s10344-004-0060-1
Massung RF Lee K Mauel M Gusa A Characterization of the rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila DNA Cell Biol 2002 21 587 596 12215262 10.1089/104454902320308960
Massung RF Priestley RA Miller NJ Mather TN Levin ML Inability of a variant strain of Anaplasma phagocytophilum to infect mice J Infect Dis 2003 188 1757 1763 14639548 10.1086/379725
de la Fuente J Massung RF Wong SJ Chu FK Lutz H Meli M von Loewenich FD Grzeszczuk A Torina A Caracappa S Mangold AJ Naranjo V Stuen S Kocan KM Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains J Clin Microbiol 2005 43 1309 1317 15750101 10.1128/JCM.43.3.1309-1317.2005
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BMC Ear Nose Throat DisordBMC Ear, Nose and Throat Disorders1472-6815BioMed Central London 1472-6815-5-81616229910.1186/1472-6815-5-8Case ReportGiant cell tumor of the temporal bone – a case report Pai S Balaji [email protected] RM [email protected] Kavitha [email protected] Saraswathi G [email protected] K [email protected] Department of Neurosurgery, M.S. Ramaiah Medical College, Bangalore, India2 Department of Oromaxillofacial surgery, M.S. Ramaiah Medical College, Bangalore, India3 Department of Pathology, M.S. Ramaiah Medical College, Bangalore, India4 Department of Surgical Oncology, M.S. Ramaiah Medical College, Bangalore, India2005 15 9 2005 5 8 8 29 3 2005 15 9 2005 Copyright © 2005 Pai 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
Giant cell tumor is a benign but locally aggressive bone neoplasm which uncommonly involves the skull. The petrous portion of the temporal bone forms a rare location for this tumor.
Case presentation
The authors report a case of a large giant cell tumor involving the petrous and squamous portions of the temporal bone in a 26 year old male patient. He presented with right side severe hearing loss and facial paresis. Radical excision of the tumor was achieved but facial palsy could not be avoided.
Conclusion
Radical excision of skull base giant cell tumor may be hazardous but if achieved is the optimal treatment and may be curative.
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Background
Giant cell tumor (GCT) of bone is an uncommon primary bone neoplasm that usually occurs in the long bones. It is rarely encountered in the skull where it is preferentially seen to involve the sphenoid and the temporal bones. It is a benign neoplasm but can be locally aggressive. A tendency towards local recurrence and late malignant change with metastases especially to the lung has been reported [1,2]. Radical surgical removal is the preferred modality of treatment. We present a GCT of the temporal bone in a 26 year old male which was treated with radical surgery with a good outcome.
Case presentation
Clinical presentation
A 26 year old male was admitted with impaired hearing and tinnitus on the right side and swelling of the right temporal region which was gradually progressive for the last two years. His general physical examination was normal. Neurological examination revealed a severe right conductive hearing loss with a Grade II House-Brackman facial nerve paresis. A diffuse swelling was noted in the right temporal and preauricular region. CT scan of the brain showed a large well defined hyperdense contrast enhancing lesion originating from the right temporal bone – squamous and petrous portions with a large intracranial extension causing uncal herniation [Fig. 1 &2].
Surgical management
The patient was taken up for surgery with an intention of radical removal. Control of the right external carotid artery (ECA) was obtained in the neck [Fig. 3 inset]. Right frontotemporal scalp flap was raised. The temporalis muscle was seen to be infiltrated by the tumor and was excised seperately. The tumor was firm, reddish brown and vascular. It had destroyed the squamous temporal bone, lateral petrous portion, zygomatic arch and was seen invading the cranium pushing the temporal bone superiorly and medially along with the dura [Fig. 3]. Dura was not transgressed. Piecemeal total removal of the tumor was achieved with temporary clamping of the right ECA. The tumor was adherent to the dura but could be peeled off the dura [Fig. 4]. Biopsy was taken from surrounding bone, muscle and dura from 4 different sites. A drain was left in the large dead space created by the removal of the tumor. Cranioplasty was planned for a later date.
Postoperative period and follow up
Postoperatively the patient developed a right total LMN VII nerve palsy (Grade VI House-Brackman). Hence a right tarsorrhaphy was done to prevent exposure keratitis one week after the first operation. At the same sitting the external auditory canal was also closed to prevent communication of the dead space in the cranium with the external auditory canal after confirming the absence of any collection in the intracranial dead space. The postoperative period was otherwise uneventful. Histopathological examination revealed a neoplasm composed of numerous osteoclast like giant cells amidst a background of mononuclear plump spindle cells suggestive of a GCT [Fig. 5]. The histopathological examination of the other 4 areas of bone, dura and muscle did not reveal any tumor infiltration. Postoperative CT scan confirmed a total excision of the tumor [Fig. 6]. Since a radical excision of the tumor had been achieved it was decided to defer radiotherapy. Three months after surgery patient was normal but for the deafness and facial palsy. Follow up CT at 6 months and 12 months did not reveal any recurrence.
Discussion
Neoplasia of the skull bones are uncommon accounting for only 2.4% – 2.6% of all primary bone tumors [1]. The majority of giant cell tumors occur in the long bones usually the distal femur, proximal tibia and fibula, distal radius and ulna [3]. The skull is a rare location for GCT. In the cranium the sphenoid bone is the commonest site followed by the temporal bone [1,3-8]. This can be explained by the fact that the tumor genesis occurs in the endochondral bone instead of intramembranous bone [3,5-7]. The temporal bone has two main components – squamous and petromastoid. The squamous portion develops by intramembranous ossification, while the petreomastoid portion develops from cartilage (endochondral bone). GCTs are commonly seen to arise from the petromastoid portion as was noted in the present case. GCT is commonly seen in the 30–50 years age group with only 16% of patients below 20 years of age [6,9]. A mild female preponderance is seen but this is more pronounced in the younger age group [9]. Typically, the tumor presents as an enlarging mass associated with local pain over a period of few weeks to years [9]. GCT of the sphenoid may present with headache, visual field defects, blindness, diplopia, second through eighth cranial nerve dysfunction, endocrinopathy and change of mental status [4]. Temporal bone tumors present with pain usually behind the affected ear, deafness and facial weakness as in the present case [6]. Temporal bone GCT may invade the infratemporal fossa, paranasal sinuses and nasopharynx [6]. Intracranial extension as in our case may also be present. Dural penetration with invasion into the brain has also been seen [5,6]. Plain radiography shows radiolucent lesion of the skull and cannot be generally differentiated from other radiolucent lesions. On CT it is seen as a lytic lesion expanding the bony cortex [10]. Generally these tumors are contrast enhancing due to their vascular nature as seen in our patient.
These tumors generally tend to expand and attenuate the bony cortex rather than erode it [10]. GCT may be vascular and an external carotid angiogram may be required to demonstrate the arterial supply. In our patient the tumor was vascular but no angiogram was performed. However, temporary clamping of the external carotid artery during excision reduced the intraoperative bleeding. Grossly, these tumors are grey to yellow-brown, soft or firm and friable. Small cystic areas and grey-white necrotic foci may be seen. Microscopically, GCT consists of plump spindle shaped or ovoid cells with admixed multinucleated, cytologically benign giant cells. Variable numbers of benign multinucleated cells are seen amidst sheets of benign mononuclear spindle shaped cells with similar nuclear features. The nuclei are generally hypochromatic with inconspicuous nucleoli and mitotic figures are uncommon [4]. Histological differentiation of GCT may be challenging. The differential diagnoses consist of central giant cell granuloma (CGCG), aneurysmal bone cyst, chondroblastoma, hyperparathyroidism and fibrous dysplasia [8]. CGCG is a reactive bone lesion that occurs mainly in the jaws [11]. CGCG and GCT are histologically very similar and the main significant difference is the greater number of nuclei in the giant cells of the GCT [12,13]. CGCGs are distinguished from true GCTs by their fibrogenic, relatively acellular stroma, extensive osseous metaplasia and the clustering of giant cells around areas of hemorrhage or necrosis [5]. A key point in the differential diagnosis is that in GCT the stromal cells and giant cells resemble each other particularly with regard to their nuclei, whereas in giant cell reparative granuloma, the osteoclasts and the stromal cells of the fibroblastic type are distinctly different [14]. Jaffe has subclassified GCT into three grades but such a grading has not been found to correlate with subsequent tumor behaviour or sarcomatous transformation [1,5].
The precise ontogeny of GCT is unresolved. GCT and CGCG are histologically and pathogenetically similar [15]. Cell cycle associated proteins like MDM2, Ki-67 and PCNA have been seen to be widely expressed in CGCG and GCT. The percentage of Ki-67 and PCNA positive cells are higher in CGCG[11]. This means that CGCGs show a higher proliferative activity than GCTs. GCT cells are also seen to produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha) [16]. Studies suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. An essential cell-cell interaction in the regulation of MMP-9 expression exists in GCT [16]. Although it is the giant cell which is the most prominent feature of these lesions it is the mononuclear spindle cell which is the proliferating cell. Several pathways to induce osteoclast like giant cell formation from monocytes have been reported. The spindle cell recruits monocytes and induces them to differentiate into osteoclastic giant cells through release of cytokines [15,17]. Receptor activator of nuclear factor kappa B (RANK) ligand is also reported to play a crucial role in osteoclastic cell genesis [17-19]. It is possible that the soluble RANKL is released from the tumor derived cells and the soluble factor interacts with RANK expressed in monocytes resulting in osteoclast-like cell formation in cooperation with macrophage colony stimulating factor secreted from the cells [17]. SDF-1 has also been incriminated as one of the significant chemo-attractant factors involved in the recruitment of hematopoietic osteoclast precursor cells during tumor-induced osteoclastogenesis [20]. The histopathogenesis of GCT and CGCG thus appears to be identical but the biological and clinical behavior of GCT is more aggressive than the latter.
The treatment of choice is complete surgical excision which if achieved can be curative [1,5,9]. However the skull base location of these tumors can make total surgical excision hazardous and not possible [5,6]. The role of adjuvant radiotherapy in eliminating residual tumor tissue is controversial. Some authors claim that GCT is not radiosensitive and radiation may provoke a sarcomatous transformation in the residual tumor tissue [3,5]. However, other authors recommend a single course of moderate dose super voltage radiation in achieving a high success rate and at the same time lowering the likelihood of malignant transformation [4-6,8]. Radiotherapy remains the only option for unresectable tumors. In our patient radiotherapy was deferred for the present as radical surgical excision was achieved with no residual tumor.
Treatment of GCT and CGCG with calcitonin and osteoprotegrin has received attention. Osteoclasts express calcitonin receptors and can be inhibited by calcitonin. In GCT and CGCG, tumor giant cells and their precursors also express calcitonin receptors. Clinical studies on treatment of CGCG with calcitonin have shown positive results probably due to control of osteoclastogenesis [15]. Osteoprotegrin ligand was also selectively overexpressed in GCTs and may indicate another possible target to which antitumor therapy could be directed [21]. Osteoprotegrin influences osteoclastogenesis and may be used for the treatment of GCT and CGCG. Osteoprotegrin is a "decoy" receptor for RANKL and therefore inhibits the RANK-RANKL interaction, which is a necessary step in the osteoclastogenesis. This is the rationale for a probable therapeutic use of Osteoprotegrin. Its utility is however unproven [15].
GCT can recur especially where only a curettage is employed [6,7]. Prosser et al recommend primary curettage for intraosseous giant-cell tumors without adjuvant treatment or filling agents, but tumors with soft tissue extension or with local recurrence require more aggressive treatment [22]. Metastases occur in only 2% of cases and are usually to the lungs but spread to rare areas like lymph nodes, mediastinum, skin, scalp and pelvis has been reported [2,9]. Our patient is on regular clinical follow up every 3 months and 6 monthly CT scan. He is recurrence free at the end of 12 months.
Conclusion
Giant cell tumor involving the petrous portion of the temporal bone is rare. A radical excision if possible is the optimal treatment. Adjuvant radiotherapy is controversial and should be reserved for residual tumor and unresectable tumors. Newer modalities like calcitonin and osteoprotegrin are being investigated.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SBP performed the craniectomy and excised the tumor and drafted the manuscript.
RML and KP assisted in the surgery.
SGR did the histopathological examination and helped in drafting the manuscript.
KH helped in drafting the manuscript and references
All authors have read and approved of the manuscript.
Consent to publish
Patient's consent was obtained to publish the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Figure 1 Preoperative CT scan. Preoperative axial CT scan which shows a large hyperdense tumor arising from the petrous portion of the right temporal bone with intracranial extension and impending uncal herniation.
Figure 2 Preoperative CT scan. The figure shows the sagittal and coronal reconstruction of the tumor.
Figure 3 Tumor exposure. Right temporoparietal craniectomy and tumor exposure after right external carotid artery control (inset) and excision of the temporalis muscle. Tumor was friable, reddish brown and vascular.
Figure 4 After completion of tumor excision. After radical excision of tumor (piecemeal) the defect is covered with gelfoam.
Figure 5 Microphotograph of the tumor. Microphotograph (250×) of the specimen in H & E stains showing neoplastic stromal cells and multinucleated giant cells. The stromal cells (horizontal arrow) are round to spindle shaped with moderate amount of eosinophilic cytoplasm and a single nucleus. They are mesenchymal in origin. The multinucleated giant cells (vertical arrow) are large in size with eosinophilic cytoplasm and a large number of nuclei.
Figure 6 Postoperative CT. Postoperative CT scan of the patient showing total excision of the tumor and the normal alignment of the intracranial structures.
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Bitoh S Takimoto N Nakagawa H Namba J Sakaki S Gohma T Giant cell tumor of the skull Surg Neurol 1978 9 185 8 635765
Qureshi SS Puri A Agarwal M Desai S Jambhekar N Recurrent giant cell tumor of bone with simultaneous regional lymph node and pulmonary metastases Skeletal Radiol 2005 34 225 8 15365782 10.1007/s00256-004-0824-4
Motomochi M Handa Y Makita Y Hashi K Giant cell tumor of the skull Surg Neurol 1985 23 25 30 3964973 10.1016/0090-3019(85)90155-7
Wolfe JT Scheithauer BW Dahlin DC Giant cell tumor of the sphenoid bone J Neurosurg 1983 59 322 27 6864300
Findlay JM Chiasson D Hudson AR Chui M Giant cell tumor of the middle cranial fossa J Neurosurg 1987 66 924 28 3572521
Pradhan S Datta NR Krishnani N Ayyagiri S Tandon P Giant cell tumor of the petrous bone Indian J Cancer 1991 28 177 80 1818017
Reed L Willison CD Schochet SS JrVoelker JL Giant cell tumor of the calvaria in a child. Case report J Neurosurg 1994 80 148 51 8271002
Bertoni F Unni KK Beabout JW Ebersold MJ Giant cell tumor of the skull Cancer 1992 70 1124 32 1515987
Henderson BTH Whitwell H Giant cell tumor of the skull: Case report Neurosurg 1988 23 120 22
Lee HJ Lum C Giant-cell tumor of the skull base Neuroradiology 1999 41 305 7 10344520 10.1007/s002340050753
de Souza PE Paim JF Carvalhais JN Gomez RS Immunohistochemical expression of p53, MDM2, Ki-67 and PCNA in central giant cell granuloma and giant cell tumor J Oral Pathol Med 1999 28 54 8 9950250
Auclair PL Cuenin P Kratochvil FJ Slater LJ Ellis GL A clinical and histomorphologic comparison of the central giant cell granuloma and the giant cell tumor Oral Surg Oral Med Oral Pathol 1988 66 197 208 3174054 10.1016/0030-4220(88)90094-1
Franklin CD Craig GT Smith CJ Quantitative analysis of histological parameters in giant cell lesions of the jaws and long bones Histopathology 1979 3 511 22 511122
Kiwit JC Schober R Nicola N Schirmer M Wechsler W Osteoclastomas of the petrous bone Surg Neurol 1986 26 59 62 3715701 10.1016/0090-3019(86)90064-9
Rezegi JA Pogrel MA Comments on the pathogenesis and medical treatment of central giant cell granulomas J Oral Maxillofac Surg 2004 62 116 8 Letter to the editor
Rao VH Singh RK Delimont DC Finnell RH Bridge JA Neff JR Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha Int J Oncol 1999 14 291 300 9917505
Miyamoto N Higuchi Y Tajima M Ito M Tsurudome M Nishio M Spindle-shaped cells derived from giant-cell tumor of bone support differentiation of blood monocytes to osteoclast-like cells J Orthop Res 2000 18 647 54 11052502 10.1002/jor.1100180418
Roux S Amazit L Meduri G Guichon-Mantel A Milgrom E Mariette X RANK (receptor activator in nuclear factor kappa B) and RANK ligand are expressed in giant cell tumors of bone Am J Clin Pathol 2002 117 210 6 11863217 10.1309/BPET-F2PE-P2BD-J3P3
Morgan T Atkins GJ Trivett MK Johnson SA Kansara M Schlicht SL Molecular Profiling of Giant Cell Tumor of Bone and the Osteoclastic Localization of Ligand for Receptor Activator of Nuclear Factor {kappa}B Am J Pathol 2005 167 117 28 15972958
Liao TS Yurgelun MB Chang SS Zhang HZ Murakami K Blaine TA Recruitment of osteoclast precursors by stromal cell derived factor-1 (SDF-1) in giant cell tumor of bone J Orthop Res 2005 23 203 9 15607894 10.1016/j.orthres.2004.06.018
Skubitz KM Cheng EY Clohisy DR Thompson RC Skubitz AP Gene expression in giant-cell tumors J Lab Clin Med 2004 144 193 200 15514587 10.1016/j.lab.2004.06.005
Prosser GH Baloch KG Tillman RM Carter SR Grimer RJ Does curettage without adjuvant therapy provide low recurrence rates in giant-cell tumors of bone? Clin Orthop Relat Res 2005 435 211 8 15930941 10.1097/01.blo.0000160024.06739.ff
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-6-371615330010.1186/1471-2296-6-37Research ArticleImplementing evidence-based medicine in general practice: a focus group based study Hannes Karin [email protected] Marcus [email protected] Etienne [email protected] Bert [email protected] Frank [email protected] Anne-Marie [email protected] Belgian Centre for Evidence-Based Medicine-Belgian Branch of the Cochrane Collaboration, Kapucijnenvoer 33 blok J, 3000 Leuven, Belgium2 Department of Medical Sociology Vrije Universiteit Brussel, Laerbeeklaan 103, 1090 Jette, Belgium3 Department of General Practice, Universiteit Antwerpen, Universiteitsplein 1, 2610 Antwerpen, Belgium4 Academic Centre for General Practice, Katholieke Universiteit Leuven, Kapucijnenvoer 33 blok J, 3000 Leuven, Belgium5 Department of Public Health, Vrije Universiteit Brussel, Laerbeeklaan 103, 1090 Jette, Belgium2005 9 9 2005 6 37 37 25 3 2005 9 9 2005 Copyright © 2005 Hannes et al; licensee BioMed Central Ltd.2005Hannes 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
Over the past years concerns are rising about the use of Evidence-Based Medicine (EBM) in health care. The calls for an increase in the practice of EBM, seem to be obstructed by many barriers preventing the implementation of evidence-based thinking and acting in general practice. This study aims to explore the barriers of Flemish GPs (General Practitioners) to the implementation of EBM in routine clinical work and to identify possible strategies for integrating EBM in daily work.
Methods
We used a qualitative research strategy to gather and analyse data. We organised focus groups between September 2002 and April 2003. The focus group data were analysed using a combined strategy of 'between-case' analysis and 'grounded theory approach'. Thirty-one general practitioners participated in four focus groups. Purposeful sampling was used to recruit participants.
Results
A basic classification model documents the influencing factors and actors on a micro-, meso- as well as macro-level. Patients, colleagues, competences, logistics and time were identified on the micro-level (the GPs' individual practice), commercial and consumer organisations on the meso-level (institutions, organisations) and health care policy, media and specific characteristics of evidence on the macro-level (policy level and international scientific community). Existing barriers and possible strategies to overcome these barriers were described.
Conclusion
In order to implement EBM in routine general practice, an integrated approach on different levels needs to be developed.
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Background
Over the past years concerns are rising about the use of Evidence-Based Medicine (EBM) in health care. The calls for an increase in the practice of EBM, seem to be obstructed by many barriers preventing the implementation of evidence-based thinking and acting in general practice. There is a growing need to develop strategies that are able to address the barriers towards evidence-based thinking and acting. We searched Medline, the Cochrane Library, ACP-Journal Club, Dare, Sociological abstracts, Psychinfo and the Campbell Library to reveal studies addressing barriers towards the implementation of EBM (table 1) as well as relevant literature proposing feasible interventions for an evidence-based approach in general practice (table 2) up till May 2003 [1-22]. We used the MeSH-terms Evidence-Based Medicine, Family Physician, Attitude of Health Personal and combined them with free text words like implement$, utilization, use, barrier$, obstacle$, intervent$ and strateg$. An additional hand search was conducted for the period 2000-May 2003 in the BMJ, JAMA, Lancet, New England Journal of Medicine, Annals of Internal Medicine, British Journal of General Practice, Journal of Evidence-Based Medicine and the European Journal of Family Practice. Numerous studies have suggested that a lack of time, easy accessible EBM-resources and competences (knowledge, skills, attitudes) affect the implementation of EBM, as well as the gaps in existing scientific knowledge. Also colleagues, patients and commercial organisations have an influence on the ability to think and act Evidence-Based. Existing interventions aiming to improve the implementation of EBM are limited to logistic or educational support. The review demonstrates a lack of well-controlled empirical studies identifying clear suggestions on how to bridge barriers and improve the implementation of EBM. As a consequence it becomes very difficult to make potential actors at different levels in the health care system feel responsible for developing strategies to optimise the implementation of EBM. The exploration of barriers Flemish GPs (General Practitioners) experience when implementing EBM in daily clinical practice can lead to suggestions about an approach to break down existing barriers. This study proposes a framework, based on experiences of GPs, considering the complex networks of 'actors' and 'factors' affecting the implementation of EBM.
Table 1 Studies addressing barriers towards EBM in general practice
Study Year Olantunbosun et al, 1998 [1] Mc Coll et al 1998 [2] McAlister et al 1999 [3] Mayer et al 1999 [4] Tomlin et al 1999 [5]
Population Randomised sample of GPs and Gynaecologists in Canada Randomised sample of GPs in Wessex, United Kingdom Gps, members of the 'Canadian Society of Internal Medicine, Canada Purposeful sample of GPs of educational programs, courses, supervisors of the 'Adelaide Royal Australian college of GPs', GPs from the Darwin Urban division of GPs, Australia Purposeful sample of 8 practices of GPs in the North Tames region, members of the 'Medical Research Council General Practice Research Framework', United Kingdom
Design Quantitative: Questionnaire Quantitative: Questionnaire Cross-sectional research: Questionnaire Qualitative: Focus groups Qualitative: Semi-structured interviews
Respondents N = 154 GPs
Response rate 78% N = 452
Response rate 67% N = 294
Response rate 60% N = 27 N = 24
Barriers Factors
-Time consuming
-Decrease of the art of medicine
-Lack of evidence
-Experience not taken into account Factors
-no skills in critical appraisal
-EBM threatens GPs
-Time consuming
-No access to information
-Organisational Chaos
-No financial profits
-Gaps in evidence
-Evidence does not fit general practice
-Too much evidence
-Evidence hard to implement Factors
-Too academic
-Decrease of the art of medicine
-Movement still young
-Gaps in evidence
-not applicable to individual patient
-Decrease of importance of experience Factors
-Reduction of therapeutic freedom
-Contradictions in evidence
-Not applicable in daily practice
-Not applicable to individual patient
-Studies too quantitative Factors
-lack of time
-Lack of information sources
-Lack of knowledge and skills
-Too much pressure, less motivation
-Evidence does not count complexity of situations in practice
Actors
Patients
-erosion of autonomy Actors
Patients:
-expectations do not fit EBM
-does not except certain advice
Colleagues:
-Not evidence-based minded
Government:
-No investments
Media:
-Counterproductive messages Actors
Commercial organisations:
-have influence on evidence
Patients:
-Do not count in terms of risks Actors
Patients:
-No compliance
-Specific cultural background
-Specific values and knowledge
-Behaviour GP = avoiding conflict
-Clientism
Study Year Scott et al 2000 [6] Freeman et al 2001 [7] Young et al 2001 [8] Ely et al 2002 [9] Putnam et al 2002 [10]
Population Sample of members from the 'Internal Medicine Society', Australia and New Zealand, participants of an EBM-course program, doctors with a practice in 5 hospitals Purposeful sample of GPs out of three regions concentrated around a hospital, United Kingdom 1. GPs, participants of a research project on preventive care, selection of those willing to participate, Australia Sample of GPs in Iowa, United States Purposeful sample of GPs with a minimum of one year experience, patients with cardiovascular problems, working in the region Nova Scotia, Scotland
Design Quantitative: Questionnaire Qualitative: 3 focus groups 1. Quantitative: Questionnaire 2. Qualitative: semi-structured interviews Qualitative: observations Qualitative: 9 focus groups
Respondents N = 111
Response rate 20% N = 19 N = 60 N = 25 N = 50
Barriers Factors
-Lack of time
-No access to information
-Problems in organisation
-Lack of knowledge and skills
-GPs not motivated
-Not applicable to individual patient
-Inconsequence in evidence Factors
-Lack of logistic support
-Too many habitudes
-Decrease of importance of experience Factors
-Lack of time
-High cost of information sources
-Lack of skills
-Not applicable in daily practice
-Evidence-Based acting = less patients an hour Factors
-Lack of knowledge and skills
-Too less capacities to implement EBM in practice Factors
-Lack of time
-Lack of competences
-Evidence = dogma, confusing
-Not applicable to individual patient
-Decrease of importance of experience
Actors
Patients:
-Does not accept certain advice
-Specific characteristics
-Asks for certain treatments
-Do not always understand evidence-based message
Colleagues:
-Do not consider the patient in total
-Specialist = evidence-based mafia Actors
Patients:
-Asks for certain treatments
-Specific expectations
-Do not always understand evidence-based message Actors
Patients:
-Brings info from internet
-Not interested in EBM
-Not enough competences to understand EBM
-Creates uncertainty in the patient
Study Year Al-Ansary et al 2002 [11] Shawn et al 2003 [12]
Population All GPs out of the region Riyadh, Saudi Arabia GPs/participants of a national research program on the implementation of EBM,
Design Quantitative: cross-sectional research, questionnaire Qualitative: semi-structured interviews
Respondents N = 559
response rate 86% N = 15
Barriers Factors
-Lack of time
-No access to information sources
-Limited information sources
-No high quality training programs available Factors
-Lack of time
-Lack of information sources
-No access to information sources
-Lack of competences
-Scientific studies not attractive
-Decrease of the art of medicine
-Decrease of clinical autonomy
-Too much pressure
-Inconsequence in evidence
-Reliability and generalisation of scientific studies?
-Not applicable in general practice
-GPs actions based on intuition
Actors
Patients:
-Specific attitude Actors
Patients:
-Values and preferences of patients must be considered
Colleagues:
-Too less specialists working local
Commercial organisations:
-evidence sponsored by industry
Table 2 Studies addressing strategies to bridge barriers towards EBM in general practice
Study Year Hayward et al 1999 [13] Oswald et al 1999 [14] Brassey et al 2001 [15] Markey et al 2001 [16] Alper et al 2001 [17]
Population Stratified, randomised sample of GPs in division South of Adelaide, Australia Theoretical sample of GPs willing to participate, with patients with a non-rheumatic atrial fibrillation, 6 general practices in Cambridge, United Kingdom GPs who use the information service ATTRACT ('evidence-based summaries to clinical queries'), United Kingdom All GPs/members of the 'Monash Division of General Practice in the South-East Suburbs' of Melbourne, Australia 2 GPs 2 information-specialists, United States
Design Action research, Telephonic interviews No control group mentioned Prospective research design: 6 months follow-up No control group mentioned Quantitative: Questionnaires, No control group mentioned Quantitative: RCT Registration of answers to questions found in medical databases
Respondents N = 31 N = ? N = 42
response rate 84% N = 132
response rate 48% N = 4
Tested interventions Set-up of an online support system through which doctors can submit a form with their question(s), being answered by an information specialist Evaluation of plans of care of patients in patients records, evaluation of current type of care in the framework of criteria of a treatment protocol the doctors made themselves. Set-up of an online support system through which doctors can submit a form with their question(s), being addressed with a summary of current scientific results 'Academic detailing': introduction in EBM and exploration of knowledge and attitudes by an educative worker in the home practice of the GP Identification of qualitative databases, being able to answer questions of GPs
Conclusion GPs found the answers useful to support their clinical decisions. In four of twenty cases the answers had a positive effect on the management of the patient. Doctors noted their reasons to neglect the recommendations of the protocol very explicit. They pointed at the difficulties of applying the recommendations of the protocol on their individual patient. GPs appreciate clear summaries of scientific literature. The answers lead to a change in daily clinical practice. 'Academic detailing' leads to a significant improvement in knowledge and understanding of EBM, but does not affect the attitude towards EBM. It is not clear whether academic detailing can motivate practitioners to change their clinical practice. Existing databases are capable of answering most questions of practitioners. However, a lot of gaps in scientific knowledge should still be addressed.
Study Year Del Mar et al 2001 [18] Swinglehurst et al 2001 [19] Fritsche et al 2002 [20] Greenhalgh et al 2002 [21] Al-Ansary et al 2002 [11] Schwartz et al 2003 [22]
Population GPs with an education in information programs, Australia GPs of the region Fulham and Hammersmith, United Kingdom Participants of a course program on EBM, Berlin, Germany Selection of doctors in the field of primary care All GPs out of the region Riyadh, Saudi Arabia 3 GPs from one practice, coaching junior doctors for the university of Detroit, United States
Design Action Research, Questionnaire No control group mentioned Descriptive pilot study: Questionnaire and semi-structured interviews, No control group mentioned Quantitative: pre-post design Case studies combined with qualitative research methods Quantitative: cross-sectional research with questionnaire Prospective research design: Registration of search results, 3 months follow-up
Respondents N = 71 N = 34
response rate 34% Two Cohorts
N 1999 = 82
N 2000 = 50
N 2001 = 71 N = ? N = 559
response rate 86% N = 3
Tested or interventions Set-up of two information desks to assist practitioners in their search for medical literature (Quest and Aqua) Set-up of a clinical information system (helpdesk) to support practitioners in taking their clinical decisions Intensive 3-day course in EBM Comparison of an academic feedback system for practitioners and a practice-oriented feedback system Suggested intervention: Training programs in searching scientific literature and critical appraisal, the use of clinical guidelines and protocols Searching for evidence during the encounter with the patient
Conclusion An information desk is useful to assist practitioners with their search. However, a cost-utility analysis should be undertaken to evaluate both information desks. The helpdesks succeeds in creating a better access to 'evidence' for practitioners. GPs are satisfied with the system, but the number of users is very low. For those who used it, it actually led to a change in their clinical practice. The course led to a significant improvement of knowledge and skills towards EBM. A good information system simultaneously provides a search engine for researchers and a search engine for practitioners. Concrete actions to implement EBM in the field of health care are very necessary. Time that must be invested in a search for answers is an important barrier to use information systems during patient encounters. It can be bridged by high quality summaries of literature. Faster internet connections are necessary.
Methods
An inductive qualitative study was conducted (September 2002 – June 2003) using four focus groups with a total of 31 GPs. Purposeful sampling was used to recruit participants. Two major criteria were used to select the participants: (1) variability in interest towards EBM and (2) variability in expertise with respect to EBM. One group of 7 academics was chosen because of their status of being good informants on EBM. Group 2 consisted of 7 GPs recruited from different local peer groups in a major Flemish city. Group 3 consisted of 6 GPs recruited from an interuniversity postgraduate course in EBM. Group 4 consisted of 11 GPs from a local peer group in a small town.
A professional moderator was hired to facilitate the focus group discussion using a semi-structured interview guide. One researcher took notes on non-verbal actions of participants. Four general topics were discussed: 1. Knowledge and understanding of EBM; 2. Applicability of EBM in general practice; 3. Specific barriers to implement EBM; 4. Suggestions for bridging the barriers. At the end of the focus group (1.5–2 hours) GPs were asked to complete a short questionnaire collecting demographic data (table 3). Each focus group was recorded and transcribed verbatim. Two independent researchers identified the important concepts by coding the first two transcripts separately. No major interrater inconsistencies were found. Data collection and analysis were guided by a combination of a 'between-case' analysis [23] and an inductive strategy based on 'grounded theory' approach [24]. In the 'between-case' analysis we classified the content of all focus groups (1 group = 1 case) in a matrix, looking for themes that cut across the different cases. Concepts were classified in different levels: a micro-, a meso- and a macro level (table 4). To check the consistency of the matrix all individual ideas were then attached to a board and categorised by a group of researchers from three different disciplines (medicine, sociology, andragology). No theoretical framework was proposed to the researchers in advance. Starting from the original data, barriers and strategies were then labelled as clusters of influencing 'actors' and 'factors' (table 4) towards the ability of GPs to implement EBM. To gain a certain level of abstraction in the empirical material insights of both analyses were used to develop a classification framework (figure 1), which will be further explained in the results section.
Table 3 Demographic data of GP participants (n = 31)
()
Sex: Male (%) 22 71%
Female (%) 9 29%
Average Age (Min/Max) 45.9 25/67
Average year of graduation (sd) 1982 9.9
Province: Antwerp(%) 11 35.5%
Brabant (%) 15 51.6%
Limburg (%) 1 3.2%
Brussels (%) 4 10.7%
Practice: individual (%) 11 35.5%
group (%) 20 64.5%
Average years of practice (sd) 18 10
% present in practice: full-time (%) 26 83.9%
part-time (%) 5 16.1%
Affiliated with university/scientific organisation: Yes (%) 18 58.1%
No (%) 13 41.9%
Table 4 Explanation of the concepts used to build the classification scheme
Micro-level: problems and interventions related to the individual practice of GPs, with a direct impact on the personal acting of the GP in the consultation setting.
Meso-level: problems and interventions related to organisations and institutes, for example scientific institutes, consumer organisations, enterprises, universities, small-scale formal and informal communication circuits etc.
Macro-level: problems and interventions with an impact on the broader social environment or related to a policy level, for example government, the media etc.
Actors: all acting persons or entities in the field of health care that influence the ability of GPs to think and act according to the principles of EBM. The main characteristic of an actor is 'interaction' with the GP. Interaction can be described as a process that occurs when two or more actors interchange and have an effect upon each other.
Factors: all elements that influence the ability of general practitioners to think and act according to the principles of EBM and to which interchange and interaction cannot be attributed as a characteristic.
Figure 1 Classification scheme of influencing 'Actors' and 'Factors' on different levels.
Results
The classification scheme focuses on influencing 'actors' and 'factors' at different levels in health care that might have an impact on the ability of GPs to think and act according to the principles of EBM (figure 1). All EBM-related barriers and strategies, mentioned by the GPs, were classified as an 'actor' or a 'factor' on a micro-, meso- or macro-level. On the micro level (individual practice) we identified two 'actors' (patients and colleagues) and three 'factors' (competences, time and EBM-resources) that have an impact on the intention of GPs to handle clinical problems according to the principles of EBM. On the meso level (institutional level) two 'actors' were identified to affect implementation of EBM: commercial organisations and consumer organisations. Health care policy and the media were the influencing 'actors' on the macro level (broader social and political environment). The 'factor' 'characteristics of evidence' was also categorised on the macro level, because of its relevance to the international scientific community.
1. Problems and interventions related to the special identity of general practice as a discipline (table 5)
Table 5 Statements related to the specific identity of general practice as a discipline
Reference number* Statement
1:23.1* (EBM) is a new phenomenon... the fundamental question we have to ask ourselves is how does that part of reality, that scientific approach of health and disease fit in the totality of the GP as a person...I do have the strong impression that we are acting too fast, without taking time to reflect on our actions...
4:23.3 For me the most difficult thing is getting the diagnosis right and evidence-based. Cough, ... okay cough, but cough is a very complex item... you can't look at it with an evidence-based eye alone. I think that clinical aspects are very important indeed...
1:153.4 ... Maybe that is a task for universities to make a serious scientific-philosophic analysis of what is called EBM. A strength-weakness analysis, making the borders clear so that we can resist critics...and becoming dissidents of our own convictions.
4:36.1 I think everyone builds some decision trees based on existing knowledge and experience... EBM is another one. We should take the step to try it out at least. But it won't be easy to change a habit in no time.
*The number of the focus group (first number) does refer to the place of the interview within the hermeneutic unit of the software programme ATLAS-TI, hence it is likely that this number exceeds the number of focus groups reported due to test groups or try-out files that are used within the same hermeneutic unit.
*first nr. = focus group/second nr. = citation/third nr. = respondent
In the focus group interviews one of the important issues was: does the EBM-paradigm fit our specific way of acting and the problems that occur in our daily practice (1:23.1 – statement 1 in table 5)? Many GPs stated that their acting is built on intuition, habits and previous experience. They also indicate hat it is often impossible to handle the care for patients completely evidence-based, because the patients' symptoms are often vague and GPs are confronted with a broad spectrum of different questions and diseases (4:23.3). A continuous update on scientific information is not feasible. According to several GPs a possible strategy for improving the use of evidence will need a better demarcation of the GPs tasks and responsibilities. The rather broad spectrum of health care problems they are confronted with makes it hard to keep up to date with current knowledge. Furthermore they stated that their intellectual position must be strengthened (1:153.4). Many GPs think it is worthwhile to be stimulated and encouraged to reflect on individual decisions, patterns of prescribing and perceptions of therapeutic freedom (4:36.1). Furthermore, GPs consider very important the impact of 'evidence-based thinking and acting' on outcomes (improvement of mortality and morbidity rates, better quality of life,...) for patients to be assessed in order to keep them motivated to implement it in practice. Despite the fact that many patient outcomes can not be easily fit in existing research designs on the effectiveness of clinical practice, the participating GPs do feel the need to be aware of these outcomes in order to maintain the efforts needed for EBM.
2. Problems and interventions at the micro-level (table 6)
Table 6 Statements related to the micro-, meso- or macro-level
Reference Number Statement
MICRO 4:134.2* Look, it is like you just said: if people come to me and say:" I want that blood analyses!", I will do it...
6:30.2 Information that is brought in by patients from the internet is not evidence-based in most of the cases. It conflicts with what we know and are willing to provide. It is a tough discussion... (A:8,9)*
1:53.2 ...People who take statines for several years for example... at one point in time you have to say:" quit using them because the reason I prescribed it for you, three years ago, it does not have any value today (B: laughing). I know you took it three years, but it is okay, quit using them immediately..."
4:148.5 But take the discussion about anti-hypertensions for instance. I expect from a specialist that he knows what he is talking about, it is his job. But sometimes they are just promoting medical products, they advertise... and I think he will have had his pleasant trip organised by a company... that feeling is hard to deal with. I do not feel like they are acting evidence-based.
1:65.6 The last month I got two patients back... look, your patient does not fulfil our criteria and so he does not have to come. And I think: "Is that the kind of medicine I will be forced to do? That person comes with his complaints, whether he is fulfilling my criteria or not. But that will be the future task of the GP: helping the people who do not fit the criteria of the specialists.
1:56.1 Sometimes I have the feeling that those people who are not connected to a university or an academic hospital ... the ones that are more modal... when they take the word, when they take over, it is easier. Late adapters get convinced and start moving.
4:180.3 A GP who works alone needs a contact to people who can guide him in a certain way, because there is to less time to figure it out yourself. That step can be made very easily, because you know who can be contacted and so on...
4:181.2 Some time ago one was talking about independent educators from government for outreach visits. Now that would be interesting, for instance to visit each GP for half an hour – once a year – like commercial representatives. They can explain where the good sites are, how they are used, show all options. And then after half a year they can come back to see how it went, did you use it? And now let's see how you can use it during your consultations...That would be interesting...
1:126.4 If one would like to know something about a certain topic, one interviews professor bla bla bla. He will know what it is all about... instead of organising a social debate, in which complexity of EBM can be explained to the public. Government can play an important role in that.
MESO 6:99.9 There's too much fragmentation: evidence-based journals, scientific institutions, organisations for EBM...all trying to promote evidence-based acting. It would be a good thing for those initiatives to melt together.
1:152.5 The booming business in the US... right now they are setting up commercial structures to make products of other companies evidence-based. That's business... not developing drugs, but developing evidence and set up large studies... sell them as evidence, to impress the rest of the world.
5:73.3 I held an archive of all medical information for one month for a talk a prepared for a governmental organisation. I had such a big amount of information! It is incredible how much we are influenced by commercial institutions and it is in no way comparable with the scarce information we get from independent sources. I think government must take a more active role in providing that kind of information.
1:101.4 There is a culture rising where patients are defined as consumers in a health care system. But often messages of consumer organisations are counterproductive, because they are not methodologically sound.
MACRO 1:188.6 I had a patient in my office lately that went to a specialist who said:" I have to talk to you for five more minutes because I need to gain an average of 10 minutes for a consultation (B: laughing)*. A rule from management.
4:102.2 The system is counterproductive for EBM. Some gynaecologists and GPs make a cervical smear every half year. If I tell that in the Netherlands they will have a good laugh, because they only do it once in three years. In Flanders women become 'smeared' far to often, just because it is easy money.
4:132.6 And I think government may be firm about that. If they say one cervical smear each three years, it means that there is only one pay-back to the patient. If the doctor talks you into more than one, ... sorry, you have to pay for it yourself (I:7,5)
1:212.5 It is the good care for the patient that should guide judgements about clinical practice and should be the most important parameter,... the degree of practicing evidence-based medicine can not be the sole norm (A:all)
1:98.2 ... if you hear things like allowing drug commercials on television. Well, that's like cleaning the floor while someone is painting the ceiling, because they heard on the commercials how good this drug is... and you have to explain, based on evidence, that it is not... and tell your story over and over because they all have seen it on television.
6:27.8 Yes, but all is presented so over-simplified...it makes consultations more difficult. In the past we were God himself and said: here take clamoxyle and go home. Our scheme was simple back then. On this side science is sitting and on the other side the dependent patient.
1:149.3 Economic thinking would be using the means we have as efficient as possible, based on transparent choices. We are not there yet and that's a reason why doctors should sit around the policy table too, to negotiate. We have to prevent letting public servants and insurance companies take decisions about health care on their own, because that indeed would be dangerous.
5:56.3 ..."The publishers feel that it will be helpful for clinicians to know whether their uncertainty sustains from the gap in the evidence rather than the gap in their own knowledge." So for the most questions there will be no clear answer, not because you do not know it but because evidence simply does not exist. And than it is up to you to take decisions.
6:11.8 I often ask myself... that EBM process is so slow-moving. By the time everyone has picked up the new evidence there probably will be a second movement that will reject those findings or will look at them from a different point of view.
*first nr. = focus group/second nr. = citation/third nr. = respondent – A = agreement followed by respondent – B = behaviour (software programme ATLAS-ti)
Actor: patients
The interaction process with patients has, according to many participants, an impact on the possibility to implement EBM in practice. Many patients have clear expectations on how they wish to be treated. This forces GPs in a position adapting to the patients' preferences, rather than to behave in an evidence-based manner (4:134.2). GPs state that the emancipation process of patients has blurred the autocratic position of doctors and demands for different behavioural strategies. Despite the fact that doctors consider their patients as well-informed, they lack the knowledge of EBM (6:30.2). Many GPs refer to the difficulties of patients to understand the rapid changes and insights in medicine (1:53.2). To dissolve this problem, most of the GPs are willing to take responsibility to create a safe environment in which the evidence-based story can be taught to the patient, for example by searching for evidence together through an evidence-based internet approach during consultation. Consumers need education, in which they can be taught to behave critically towards medication and media. According to several GPs consumer organisations could play an important role here.
Actor: colleagues
Conflicts with colleagues seem to be common. GPs clearly express the opinion that specialists' thinking is too commercial (4:148.5). This implies they are promoting and using products based on a commercial rather than an evidence-based approach. There is a general feeling that specialists who do act evidence-based should be protected. GPs also feel strongly for building networks with reliable colleagues. The fact that patients still consider the advice of a specialist more valuable is also brought forward. GPs are very much concerned about the fact that their role will become reduced to treating those patients who do not fit the criteria of specialists (1:65.6). Furthermore, in educational programmes more lectures and workshops should be given by colleagues who are not explicitly connected to universities, making EBM more of a real-life business (1:56.1).
Factors: time, EBM-resources and competences
Lack of time, EBM-resources, knowledge and skills are the barriers that are most recognised in existing literature. They recur during the focus group interviews. Many GPs think it is worthwhile to create a helpdesk system or a central on-line information system that provides answers to specific questions (4:180.3). On-line sources should be easy to navigate and free of charge. Guidelines and Critical Appraised Topics would be a great help. A select team of GPs should be paid to develop them. Educational programs should focus on an integration of EBM-principles in medical school and further training courses. Specialised EBM-teams should be established. The idea of outreach visits by independent educators, paid by government, gains approval by many GPs (4:181.2). As a lot of existing EBM material does not reach the GPs, they suggest to put more efforts into getting this material under their attention. Furthermore, it will be important to change GPs attitudes towards EBM. Many GPs state that EBM remains too theoretical and authority based (1:126.4). It restricts clinical practice, limiting the choices. Resistance should be broken down, for instance by finding inspiration in change management techniques, as indicated by some participants.
3. Problems and interventions at the meso-level (table 6)
The EBM movement is relatively new in Flanders. At this stage different organisations undertook efforts to stimulate EBM, for example universities, the Belgian Centre for Evidence-Based Medicine and other occupational groups. Some GPs criticise the fragmentation of initiative, which does lead to an organisational chaos in the field. GPs do see a role for government in providing a network which tightens all different institutions together (6:99.9).
Actor: commercial organisations
Several GPs criticise the fact that educational programmes and the development of guidelines is sponsored by commercial organisations, for example pharmaceutical firms and their representatives (1:152.5). Questions do rise about the honesty of their message, in which EBM is used as a fashion term. Moreover, GPs receive a lot of information from different sources. Only a small part of the information is independent and unfortunately not easily identified in the information overload (5:73.3). Suggestions are made to label independent information, distinguishing it from less relevant information. Conflicts of interest must be clearly stated. Many participants feel that spreading a uniform EBM message by all actors in the field of health care is very important and that this specific approach should also become a necessity for the commercial organisations.
Actor: consumer organisations
GPs in the sample worry about the quality of the messages spread by consumer organisations, for example self help groups. In order to stimulate EB-practice more attention should be paid to the content and form of the messages and the methods used (1:101.4). According to several GPs consumer organisations could play a major role in distributing evidence-based messages to patients that are screened for quality and in measuring their satisfaction with evidence-based treatments.
4. Problems and interventions at the macro-level (table 6)
Actor: health care policy
Belgium has a particular way of financing the GPs. GPs are most of the time independent entrepreneurs, who are financed by the number of medical acts defined in the nomenclature. Several GPs raise their voice about 'the policy system' that works counterproductive for an evidence-based approach. The time spent on research and information seeking behaviour is not paid for. One focuses necessarily on the amount of consultations, rather then evidence-based acting, and doctors adapt to the protocols they are forced to follow (1:188.6). Consultations lead to a higher income for a doctor but, as indicated by several GPs, often also to unnecessary practices (4:102.2). According to some participants a fixed income could be a solution, as well as financial incentives for extra time that is invested in acting evidence-based. Reimbursement systems should be consequent with what is promoted regarding to EBM (4:132.6). Some GPs criticise the fact that EBM seems to develop as a reductionist model to judge results of clinical acting. Most GPs think that broader outcome-measures should be taken into account, for example quality of life (1:212.5). Some concerns are expressed about the coaching of GPs to implement evidence. Too easily, government assumes that all doctors are comfortable with the evidence-based paradigm. GPs are given the noble task of being the messengers, without having enough structured information and without being enough knowledgeable for that 'new' role. Therefore many voices are raised to improve the distribution of evidence-based information.
Actor: the media
The mass media are identified as a counterproductive force, because products or services are being promoted that are not always supported by evidence (1:98.2). Patients seek answers to their questions on the internet. The quality of this information is doubtable. This changing information seeking behaviour has an impact on the relation between GPs and patients. Some GPs state that patients become negotiating partners (6:27.8). Starting a social debate could be a first step to promote coherence between different parties of interest involved in the production, interpretation, use, dissemination and promotion of evidence. In defining a rational policy towards EBM GPs find it important to be involved in the process and to integrate late-adapters in the evidence-based movement (1:149.3). The media could play an important role in representing their voice in the economic choices made to support an evidence-based health care system, since these choices are often made by governmental institutions only.
Factor: sub-optimality of 'evidence'
The sub-optimality of the evidence provided is frequently stated as a barrier to the implementation of EBM. Contradictions between similar evidence-based studies are common. Some of the studies that are promoted as 'evidence-based' are not up-to-date. Several GPs point towards the lack of evidence for many of their problems (5:56.3). There is also a time-delay between the research process and the actual adaptation of the new insights in the field (6:11.8). No explicit suggestions to overcome these barriers are given by the participants.
Discussion
The results presented in previous sections give an overview of issues mentioned by different GPs during the focus group sessions. Compared to what already was known from previous qualitative research (table 1.2) this study adds two more 'actors' that influence the implementation of EBM in general practice: consumer organisations at the meso-level and health care policy at the macro level. Providing logistic and educational support seems to be important to participants in previous research programmes, as well as for Flemish practitioners. Recent reviews on the effects of decision support systems to support evidence-based practice conclude that these strategies improve practitioners' performance [25,26]. Practitioners start using online evidence during consultations much more than previously reported [27,28]. Educational programmes have also proven to impact on practitioners' competences [29,30]. However, they should be moved from the classroom to clinical practice [31]. The approach for implementing EBM in general practice needs to start from the specific characteristics of the profession. Despite the fact that GPs are keen to consider evidence in clinical decision making, there is a widespread belief that intuition plays a vital role in primary care [32]. GPs are not likely to change this belief until the impact of the evidence-based thinking and acting on patients' outcomes is assessed.
Apart from these insights the focus group research reveals that interventions should also focus on communication, rational policy making and the building of networks between different 'actors' to impact on an actual implementation of EBM in practice. The analytic classification proposed in this paper has the advantage to identify the complexity of problems and by consequence to identify and focus on interventions. To our knowledge no other study in the field of difficulties in the implementation of EBM in general practice has proposed a theoretical framework wherein barriers can easily be located and which naturally leads to the development of strategies to tackle them at the right level. We feel the framework is of relevance to the international community of GPs and those who promote EBM in this context. The classification model as presented seems to be a useful tool to orient change management processes.
Some specific barriers for GPs are directly linked to the specific characteristics of the Belgian health care system. These characteristics should be taken into account in other countries or regions. Considering this aspect we want to warn for generalisations even in Belgium. Although the organisation of the health care system is a federal issue, the results presented could be biased since health care traditions are culturally diverging in Flanders, the Walloon provinces and Brussels. However, these contextual issues are fundamental to address when developing policies or interventions.
The issue of generalisation brings us to some other methodological considerations. The 31 general practitioners who participated in this study formed a small sample of Flemish GPs. The sample was neither random nor representative. Given the basis on which most of the GPs joined the study we assume that their interest in EBM would be greater than that of their colleagues in the field. However, we are convinced that precisely this positive attitude and experience revealed knowledgeable results to contemplate further about interventions facilitating an evidence-based approach. The focus group methodology was considered appropriate to identify barriers and suggestions to overcome them. Methodological rigour was assured by using a combined approach of 'between-case analyses' and an inductive strategy based on 'grounded theory approach'. The first two focus groups were coded by two independent researchers and the analysis was checked and discussed with two senior researchers. However, when assessing the outcomes of the focus groups it was noticed that saturation was not reached. The last focus group interview still generated seven new ideas: five new barriers and two more interventions. Therefore we cannot guarantee that all factors, actors and suggestions were identified. More methodological research on predictive models for saturation points in qualitative research is necessary.
Another interesting methodological issue is that we found a high level of 'agreement' between participants. A possible explanation for the lack of 'extreme cases' could be that the topic under study (the implementation of EBM) was relatively new to many participants. Conversation in the focus groups were about 'recognition' and 'explanation' rather than 'debate' and 'opposing opinions' about suggestions. This probably explains why we inventoried more barriers than suggestions on interventions and strategies.
Conclusion
The classification model can be used as a frame of reference to initiate change management and policy interventions. For a successful implementation of EBM different 'actors' should be mobilised at different levels, as they are able to prevent GPs from acting evidence-based. However, potential conflicts between 'actors' can be bridged by (1) creating the necessary communication platforms between actors at different levels and within different disciplines (including patients, policy makers, researchers, consumer organisations, representatives from the media, commercial organisations, primary care practitioners, secondary care practitioners etc.), (2) communicating a consistent and uniform message on the principles of EBM and (3) modifying the current health care system towards a more evidence-based approach (for example an update of the reimbursement system, incentives that stimulate evidence-based acting, (re)opening the discussion on a fixed income vs. an income based on consultations). Future research projects should focus on the changing relations between GPs and other 'actors'. Furthermore, (4) qualitative educational programmes should be offered, ideally within working hours, with a reasonable compensation for the income lost and organised by independent organisations, (5) access to EBM-resources should be free of charge, easy accessible and whenever possible provided in the mother tongue of the practitioners and (6) gaps in knowledge need to be addressed by researchers world wide. A better understanding of the complex relationships as proposed in the conceptual framework (figure 1) and the 'factors' influencing the implementation of EBM will hopefully lead to the funding of research projects in which a set of strategic priorities can be inventoried and worked out to support and stimulate a coherent evidence-based approach within the health care system. In this important negotiation process all identified actors in the field of health care have to be involved.
Competing interests
The author(s) declare that they have no financial competing interests. However, answers to questions could have been influenced by a convenience bias, as the participating GPs were aware that the focus groups were carried out by a research team known to be in favour of EBM.
Authors' contributions
KH set up the focus groups, coded the manuscripts, analyzed the qualitative data and drafted the manuscript. BA coordinated the research process and participated in the design of the study. EV assisted in the coding process and participated in the data-analysis. ML helped developing the conceptual framework. AD and FB interpreted the results and commented on the draft version of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We wish to acknowledge the time and enthusiasm of the GPs who took part in the focus groups. We like to thank Professor R. Schepers from the department of sociology of 'Katholieke Universiteit Leuven' for her comments on the final drafts of the manuscript.
==== Refs
Olatunbosun OA Edouard L Pierson RA Physicians' attitudes toward evidence- based obstetric practice:a questionnaire survey BMJ 1998 316 365 6 9487175
McColl A Smith H White P Field J General practitioners' perceptions of the route to evidence-based medicine: a questionnaire survey BMJ 1998 316 361 65 9487174
McAlister FA Graham I Karr GW Laupacis A Evidence-based medicine and the practicing clinician J Gen lntern Med 1999 14 236 42 10.1046/j.1525-1497.1999.00323.x
Mayer J Piterman L The attitude of Australian GP's to evidence-based medicine: a focus group study Fam Pract 1999 16 627 32 10625142 10.1093/fampra/16.6.627
Tomlin Z Humprey C Rogers S General practitioners' perceptions of effective health care BMJ 1999 318 1532 5 10356011
Scott I Heyworth R Fairweather P The use of evidence-based medicine in the practice of consultant physicians Aust N Z J Med 2000 30 319 26 10914748
Freeman AC Sweeney K Why do general practitioners do not implement evidence?: qualitative study BMJ 2001 323 1100 2 11701576 10.1136/bmj.323.7321.1100
Young JM Ward JE Evidence-based medicine in general practice: beliefs and barriers among Australian GP's J Eval Clin Pract 2001 7 201 10 11489044 10.1046/j.1365-2753.2001.00294.x
Ely JW Osheroff A Ebell MH Chambliss ML Vinson DC Stervermer JJ Pifer EA Obstacles to answering doctors' questions about patient care with evidence:qualitative study BMJ 2002 324 1 7 11777781 10.1136/bmj.324.7339.710
Putnam W Twohig PL Burge FI Jackson LA Cox JL A qualitative study of evidence in primary care: What the practitioners are saying CMJA 2002 166 152 30 12074118
Al-Ansary LA Khoja TA The place of evidence-based medicine among primary health care physicians in Riyadh region, Saudi Arabia Fam Pract 2002 19 537 42 12356709 10.1093/fampra/19.5.537
Shawn T Guilherme CD Ross EGU Evidence-based medicine in primary care: a qualitative study of family physicians Fam Pract 2003 4 1 9
Hayward JA Wearne SM Middleton PF Silagy CA Weller DP Doust JA Providing evidence-based answers to clinical questions: a pilot information service for general practitioners Med J Aust 1999 171 547 50 10816708
Oswald N Bateman H Applying research evidence to individuals in primary care: a study using non-rheumatic atrial fibrillation Family Practice 1999 16 414 9 10493714 10.1093/fampra/16.4.414
Brassey J Elwyn G Price C Kinnersley P Just in time information for clinicians: a questionnaire evaluation of the ATTRACT project BMJ 2001 322 529 30 11230069 10.1136/bmj.322.7285.529
Markey P Schattner P Promoting evidence-based medicine in general practice-the impact of academic detailing Family Practice 2001 18 364 6 11477042 10.1093/fampra/18.4.364
Alper BS Stevermer JJ White DS Ewigman BG Answering family physicians' clinical questions using electronic medical databases Journal of family practice 2001 50 [online]
Del Mar CB Silagy CA Glasziou PP Weller D Spinks AB Bernath V Anderson JN Hilton DJ Sanders SL Feasability of an evidence-based literature search service for general practitioners Med J Aust 2001 175 134 7 11548078
Swinglehurst DA Pierce M Fuller JC A clinical informaticist to support primary care decision making Qual Health Care 2001 10 245 9 11743154 10.1136/qhc.0100245..
Fritsche L Greenhalgh T Falck-Ytter Y Neumayer HH Kunz R Do short courses in evidence-based medicine improve knowledge and skills? Validation of Berlin questionnaire and before and after study of courses in evidence-based medicine BMJ 2002 325 1338 41 12468485 10.1136/bmj.325.7376.1338
Greenhalgh T Hughes J Humprey C Rogers S Swinglehurst D Martin P A comparative study of two models of a clinical informaticist service BMJ 2002 324 524 11872553 10.1136/bmj.324.7336.524
Schwartz K Northrup J Israel N Crowell K Lauder N Neale AV Use of on-line Evidence-based Resources at the point of care Fam Med 2003 35 251 6 12729308
Miles M Huberman A Qualitative data analysis: an expanded sourcebook 1994 California (US): Sage Publications
Strauss A Corbin J Grounded theory in practice 1997 London (UK): Sage Publications
Kawamoto K Houlihan CA Balas EA Lobach DF Improving clinical practice using clinical decision support systems: a systematic review of trials to identify features critical to success BMJ 2005 330 765 15767266 10.1136/bmj.38398.500764.8F
Garg AX Adhikari NK McDonald H Rosas-Arellano MP Devereaux PJ Beyene J Sam J Haynes RB Effects of computerized clinical decision support systems on practitioner performance and patient outcomes: a systematic review JAMA 2005 293 1223 38 15755945 10.1001/jama.293.10.1223
Magrabi F Coiera EW Westbrook JI Gosling AS Vickland V General practitioners' use of online evidence during consultations Int J Med Inform 2005 74 1 12 15626631 10.1016/j.ijmedinf.2004.10.003
Van Duppen D Aertgeerts B Goossens F Van Linden A Neirinckx J Seuntjens L Hannes K Internet implementation of Evidence Based Medicine during patient visits (online-on-the-spot) Health Expectations
Hyde C Parkes J Deeks J Milne R Systematic Review of effectiveness of teaching critical appraisal [critical appraisal] 2000 Oxford University Institute of Health Sciences, Centre for Statistics in Medicine
Parkes J Hyde C Deeks J Milne R Teaching critical appraisal skills in health care settings The Cochrane Database of Systematic Reviews 2001
Coomarasamy A Khan KS What is the evidence that postgraduate teaching in evidence based medicine changes anything? A systematic review BMJ 2004 329 1017 15514348 10.1136/bmj.329.7473.1017
Tracy CS Dantas GC Upshur RE Evidence-based medicine in primary care: qualitative study of family physicians BMC family practice 2003 1 6 12740025 10.1186/1471-2296-4-6
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BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-5-621617152310.1186/1472-6963-5-62Research ArticleHow to promote, improve and test adherence to scientific evidence in clinical practice Caminiti Caterina [email protected] Umberto [email protected] Francesca [email protected] Rodolfo [email protected] Epidemiology Service, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, Parma, Italy2 Division of Neurology, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, Parma, Italy3 Division of Medical Oncology, Azienda Ospedaliera di Cremona, Viale Concordia, 1, Cremona, Italy2005 19 9 2005 5 62 62 9 5 2005 19 9 2005 Copyright © 2005 Caminiti et al; licensee BioMed Central Ltd.2005Caminiti 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
Negative variation in the management of patients with the same clinical condition is frequent, and affects quality of care. Recent studies indicate that single interventions are not an effective solution. We aim to demonstrate that a multifaceted strategy can favor the introduction of research into practice, and to assess its long-term effects on a set of common medical conditions exhibiting significant negative variation at our institution.
Methods
The strategy, devised and agreed upon by a multidisciplinary group, was first applied to one relevant medical condition – cerebral ischemic stroke. To test its effectiveness a quasi-experimental study was conducted, comparing an intervention group with historical controls. After validation the strategy was extended to other pathologies, and its long-term effect measured using evidence-based quality indicators. Adherence to each indicator was determined prospectively on a six-month basis for a period of at least two consecutive years. Measures are expressed as proportions with 95% confidence intervals.
Results
Validation findings demonstrated that the strategy improved compliance with scientific evidence: the percentage of patients who received a CT scan within 24 hours of hospital presentation rose from 56% to 75%, (χ2 = 7.43 p < 0.01); admissions to selected wards increased from 45% to 64%, (χ2 = 7.81 p < 0.01); the number of physical medicine visits within 24 hours of the request grew from 59% to 91% (χ2 = 14,40 p < 0.001). Over a four-year period the program was gradually applied to 14 medical conditions. Except for 3 cases, compliance with the pathway, i.e. number of eligible patients for whom data on the care process is collected, was above the minimum requirement of 75%. Indicator adherence generally exhibited a positive trend, though variability was observed both among different conditions and between different semesters for the same pathology.
Conclusion
According to our experience, incorporation of research into practice can be favored by systematically applying a shared, multifaceted strategy, involving multidisciplinary teams supported by central coordination. Institutions should device a tailor-made approach, should train personnel on implementation strategies, and create cultural acceptance of change. Just like for experimental trials, human and economic resources should be allocated within health care services to allow the achievement of this objective.
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Background
A large variance in the utilization of health technology (concerning both drugs, equipment, and medical/surgical procedures as well as the organizational and support framework for such performances) is increasingly evident, putting patients at risk of receiving unnecessary care, or of being deprived of performances already proven effective [1-3]. This awareness has made quality of care a central issue [4]. In the past few years various approaches to this phenomenon have been developed, suggesting alternative methodological solutions involving different professional groups. Quality assurance (QA), technology assessment (TA), clinical epidemiology (CE), and continuous quality improvement (CQI), are just a few of the disciplines involved in the critical and constructive evaluation of clinical practice. The main and widely accepted indications suggested by research in this field [5,6] are the following: 1) in order to ensure effectiveness, organizational, socio-cultural, and structural aspects must be taken into account; 2) in order to improve clinical practice the sources of problems must be identified, and all parties must be actively involved; 3) in order to verify and improve the quality of care, accurate local data must be readily available. Following the above-mentioned indications, we have decided to develop and implement at our hospital a multifaceted strategy, comprising the creation and application of integrated care pathways (ICPs), and the monitoring of quality indicators (QIs).
In this article we first describe this strategy and aim to demonstrate that it improves adherence to scientific evidence and consequently reduces negative deviations in patients with stroke. We then describe our experience over a four-year period with the gradual extension of the strategy to other eligible medical conditions and the testing of its long-term effect.
Methods
The study began in June 2000 and was implemented at one of the largest Italian health care facilities, with 1420 beds (of which 330 for out-patients and 90 for long-term care), and over 60,000 admissions per year.
This initiative was carried out as part of a project sponsored by the Italian Ministry of Health, and was designed in order to provide health planners with indications on factors which favor or hinder the routine use of clinical "best practice", in a natural context of a health care facility, and to suggest the rationale for new intervention studies.
Strategy's definition and validation
The strategy comprises a sequence of interventions, each designed to remove barriers which have been demonstrated by preceding studies to hinder EBM practice [2]. It was developed with the involvement of various professionals: clinicians, nurses, administrators, epidemiologist, sociologist, patients' representative; this factor has made it possible to devise a shared program, suited to our local reality. Integrated care pathways, quality indicators and relative audit were chosen as tools necessary to health care workers to define the best process in the workplace and monitor variation.
ICPs' creation and application
Integrated care pathways are structured multidisciplinary care plans which detail essential steps in the care of patients with a specific clinical problem [7]. What follows is the description of three main aspects of the process followed for the creation and application of ICPs at our institution, outlined in detail in Figure 1 along with the relative mean times.
Figure 1 Phases of ICP development and relative mean times.
1. Multidisciplinary team (MT) creation
Motivated clinicians and nurses involved with the condition join the MT voluntarily. While the MT should not be too large, it is important that all departments involved with the pathology be represented. The members will then appoint the coordinator – a key figure for the success of the pathway.
2. Agreement on objectives and actions
The MT meets regularly to identify key aspects and discuss local problems of the process of care. The team's discussion is based on the evidence gathered through a careful literature analysis. This will eventually lead to the creation of a document which contains the ICP's objectives, required interventions, the description of the pathway through time-task matrices, and the data collection sheet used for the analysis of the care process. This document will be subject to periodical reviews and modified when appropriate. The document is disseminated to all professionals involved with the pathology in question, in order to build consensus and favor its application.
3. Implementation phase
It's a sort of experimental stage, necessary to educate health care professionals on the use of the ICP; during this phase the data, gathered with an ad hoc data collection sheet, will be used to describe the care process, and MT compliance, i.e. the number of eligible patients for whom the data collection sheet is filled out, will be verified; this value is considered acceptable if greater than 75% [7].
Development of indicators and analysis through audit
Based on the analysis of the information collected with paper forms, during the implementation phase, and on detected variation, each member of the MT will suggest possible quality indicators. The MT will discuss them and assess them against criteria such as validity (strength of evidence), reliability and feasibility of data collection (they should be easy to retrieve, calculate and interpret), and sensitivity to change [8]. The indicators which best meet the criteria, about 3–6, will be chosen, and form the minimum set of QIs, gathered prospectively using an electronic record [9] and monitored to measure adherence to scientific evidence. To ensure the correct interpretation of observed variations, inclusion and exclusion criteria for each QI are explicitly defined and considered during data processing.
Multiprofessional audit is performed regularly (approximately every 3 months) in order to discuss any negative deviations, to determine the causes, and act upon them if needed [7,9-13]. This should ensure that improvement is gradually achieved and maintained.
Study design and statistical analysis for validation
The above-mentioned strategy was first applied to one relevant medical condition – cerebral ischemic stroke – in order to test its effectiveness. We opted for a quasi-experimental design, a controlled study in which an intervention is assigned without the use of randomization [14]. To determine whether the introduction of the intervention had changed behavior in terms of improved adherence to scientific evidence, we compared clinical records of all patients with cerebral ischemic stroke consecutively hospitalized during 2 homogeneous periods, preceding and following the strategy's introduction. The size of the considered sample, about 100 cases per group, was adequate to detect a difference of at least 20%, with significance level α = 0.05 and power 1-β = 0.80. The comparison between proportions was examined using the χ2 test [15]. We considered P values < 0.05 to be significant, P values < 0.01 strongly significant and P values < 0.001 extremely significant. Data were processed using the SAS System (version 8.1).
Strategy's extension and long-term effects
Following the validation phase, the strategy was gradually applied to other medical conditions chosen according to the following criteria: high admission frequency (at least 100 admissions/year), large variations in clinical practice affecting patient outcome, and high level of interest among local staff. Frequency and variation are verified through the analysis of discharge summaries, clinical records, or studies conducted at a local level for other purposes.
The activity of the MTs is coordinated by the Support Panel (SP). This group – made up of 1 epidemiologist, 1 professional nurse, 1 data manager, and 1 bibliographic researcher – verifies that the chosen medical conditions meet the selection criteria, tests compliance with the program, intervenes in the case of conflict, and provides support to the management in the collection and analysis of data and literature.
For the medical conditions which completed all the steps required by the multifaceted strategy, we recorded a minimum set of quality indicators to monitor the strategy's long-term effect. Data analysis was carried out following the methodology commonly applied in descriptive observational studies [14]. The analysis consisted in the prospective collection of specific indicators, which must be gathered for all eligible patients (following the inclusion criteria established by the multidisciplinary team) consecutively admitted to hospital in a given period (the same dynamic target population). The analysis of such data enables to assess adherence to key aspects of the care process, both in terms of constant improvement and maintenance of improvement overtime once it has been achieved. We determined adherence to each indicator on a six-month basis for a period of at least 2 consecutive years (long-term effect). Each measure is expressed as a proportion with relative 95% confidence interval.
Results
Strategy's validation
We compared two periods: the first 6 months of 2001, when the intervention was in use, and the corresponding period of the previous year, when it was not. Trained personnel examined 224 clinical records of eligible patients discharged with a primary diagnosis of cerebral ischemic stroke. The comparison employed the following 3 quality indicators, adapted from the indications established in 1999 by the American Heart Association/American College of Cardiology [16]:
- Performance of a CT scan within 24 hours of hospital presentation;
- Patient admission to dedicated wards;
- Timely involvement of the rehabilitation team
The results of the comparison between the intervention and control groups, carried out to demonstrate improvement of behavior, are the product of the following specific interventions agreed upon by the MT (table 1):
Table 1 Pre-post comparison of compliance with QIs for Cerebral Ischemic Stroke
PRE-INTERVENTION POST-INTERVENTION
INDICATOR N. patients assessed Value (95%CI) N. patients assessed Value (95%CI)
Performance of CT scan within 24 h of hospital presentation 110 56% (47–65) 114 75% ++ (67–83)
Admission to a dedicated ward 110 45% (36–54) 114 64% ++ (55–73)
Early intervention of the rehabilitation team * 57 59% (46–72) 64 91% +++ (84–98)
Level of test significance χ2
+ p < 0.05
+ + p < 0.01
+ + + p < 0.001
* Patients for whom rehabilitation was indicated.
- Cerebral CT scan is crucial to determining the type of stroke and consequently planning the correct therapy [17]. Earlier, patients with suspected stroke usually underwent CT scan in the admission ward, often experiencing important delays, while in some cases the test was not performed. It was therefore decided to provide CT scans for suspected stroke in the emergency department. After the intervention the percentage of patients who received a CT scan within 24 hours of hospital presentation rose from 56% to 75%, (χ2 = 7.43 p < 0.01).
- At our institution, stroke patients used to be hospitalized according to bed availability, which resulted in high dispersion. Considering the great importance of specialized stroke care, as demonstrated in the literature [18], the ICP requires that all subjects presenting to the ER with suspected stroke be reported by telephone to one of the referring physicians who will be in charge of the patient. Consequently, admissions of stroke patients to selected wards increased from 45% to 64%, (χ2 = 7.81 p < 0.01).
- Prior to the intervention, physical medicine evaluation and rehabilitation were not always promptly initiated, mostly due to a lack of communication between the wards and the physical medicine department. Since early intervention is central in improving disability and quality of life [18], a referring physical medicine physician was chosen who is immediately informed via fax when a new stroke patient is admitted to hospital. This will enable the rapid provision of an individualized evaluation and rehabilitation program. After the introduction of this measure the number of patients with rehabilitative indication seen by the rehabilitation team within 24 hours grew from 59% to 91% (χ2 = 14,40 p < 0.001).
Strategy's extension and long-term effects
Over a four-year period the strategy was gradually applied to 14 medical conditions, involving nearly 150 health care workers, and allowing the hospital management of over 7000 patients/year to be verified.
The 14 conditions have reached different stages, depending on the time they were included in the program, and also on factors related to the individual MTs. Table 2 provides for each pathway initiation date, MT composition, number of eligible patients/year, and MT compliance. The latter has been recorded for 13 out of 14 ICPs – in June 2004 the one on Pulmonary Thromboembolism had yet to begin implementation. Except for the ICPs on Breast Cancer, Supraventricular Tachiarrhythmias, and Pediatric Head Injury (53%, 55%, and 62% respectively), compliance was good, reaching optimal rates for Liver Cirrhosis (94%), Stroke (89%), Pediatric Pneumonia and Melanoma (88%), Child Delivery (86%), and chronic obstructive pulmonary disease (COPD) (84%).
Table 2 Integrated care pathways in use at our institution
ICP Initiation Date Eligible/Year+ MT Composition MT Compliance ++
Cerebral Ischemic Stroke June 2000 220–230 2 emergency medicine physicians, 1 geriatrist, 1 physical medicine physician, 1 neurologist, 1 neuroradiologist, 2 internists, 1 psychiatrist, 1 physiotherapist, 2 nurses. 89%
Pediatric Head Injury June 2001 100–110 1 neuropsychiatrist, 2 pediatricians, 1 radiologist, 2 nurses. 62%
Chest Pain June 2001 1800–2000 3 cardiologists, 1 cardiosurgeon, 2 emergency medicine physicians, 2 internists, 3 nurses. 79%
COPD June 2001 430–450 3 pulmonologists, 1 emergency medicine physician, 1 geriatrist, 1 physical medicine physician, 2 nurses. 84%
Child Delivery June 2001 2000–2100 3 gynecologists, 1 pediatrician, 1 obstetrician, 1 nurse. 86%
Lung Cancer June 2001* 200–220 3 pulmonologists, 2 thoracic surgeons, 1 oncologist, 1 radiologist, 1 radiotherapist, 1 physical medicine physician, 1 pathologist, 2 nurses. 77%
Breast Cancer December 2001 320–340 2 oncologists, 3 surgeons, 1 plastic surgeon, 1 radiologist, 1 radiotherapist, 1 pathologist, 1 biologist, 1 nurse. 53%
Pediatric Pneumonia December 2001 110–130 6 pediatricians, 1 neonatologist, 1 radiologist, 2 nurses. 88%
Liver Cirrhosis December 2001 470–490 2 gastroenterologists, 3 infective disease physicians, 2 nurses. 94%
Supraventricular Tachiarrhythmias March 2002 680–700 3 cardiologists, 4 internists, 2 emergency medicine physicians, 1 nurse. 55%
Hip Arthroplasty October 2002 360–380 4 orthopedists, 1 radiologist, 1 physical medicine physician, 1 anesthetist, 1 hematologist, 2 nurses. 82%
Melanoma January 2003 110–130 3 dermatologists, 2 plastic surgeons, 1 nuclear medicine physician, 2 oncologists, 1 general surgeon, 1 pathologist, 1 nurse. 88%
Non-Hodgkin Lymphomas June 2003 90–110 1 oncologist, 1 radiotherapist, 1 radiologist, 2 hematologist, 1 nuclear medicine physician, 1 surgeon, 2 internists, 1 nurse 77%
Pulmonary thromboembolism April 2004 190–210 1 emergency medicine physician, 2 pulmonologists, 1 nuclear medicine physician, 2 internists, 1 geriatrist, 1 cardiologist, 1 radiologist *
* For this pathway the implementation phase was not completed by June 2004, thus data are not available.
+ Eligible/Year: number of patients who meet the inclusion criteria set forth in the ICP document.
++ MT Compliance: number of eligible patients for whom the data collection sheet is filled out; this value is considered acceptable if greater than 75%.
By June 2004, indicators had been defined for 7 conditions. For 5 of those (Stroke, Chest Pain, COPD, Child Delivery, Lung Cancer) the effect of the strategy has been monitored for at least 2 years; for the remaining two (Liver Cirrhosis and Pediatric Pneumonia) indicators have been monitored since the second semester of 2003, and thus are not included in our long-term analysis. For three conditions (Supraventricular Tachiarrhythmias, Breast Cancer and Pediatric Head Injury) the minimum set of indicators had not been measured. Hip Arthroplasty, Melanoma and Non-Hodgkin Lymphomas had completed their implementation phase, and the MTs were working on defining the quality indicators to begin monitoring.
Table 3 shows the trend of adherence to each indicator for five semesters, the two semesters of 2002 and 2003, and the first semester of 2004, except for Lung Cancer for which only 4 semesters are shown because implementation was delayed.
Table 3 Trend of adherence to each indicator by semester
CONDITION INDICATOR 1st semester 2002 Percentage (95%CI) 2nd semester 2002 Percentage (95%CI) 1st semester 2003 Percentage (95%CI) 2nd semester 2003 Percentage (95%CI) 1st semester 2004 Percentage (95%CI)
STROKE CT scan performed within 24 hours of hospital presentation[17] 91%
(86–97) 90%
(84–96) 96%
(92–100) 96%
(93–99) 93%
(88–98)
Patients admitted to a dedicated ward[18] 63%
(56–71) 74%
(67–81) 81%
(75–87) 80%
(74–86) 82%
(75–89)
Visits by the physical medicine specialist within 24 hours of request for patients with rehabilitative indication [18] 78%
(69–87) 65%
(55–75) 94%
(89–99) 82%
(74–90) 75%
(66–84)
Patients transferred to a post-acute care facility according to their clinical indications [41] 89%
(78–100) 78%
(64–91) 80%
(72–89) 79%
(72–86) 73%
(64–82)
Psychiatric tests (GDS) administered to patients eligible for the test [42] 67%
(58–78) 57%
(46–69) 73%
(64–83) 74%
(65–83) 75%
(66–84)
Patients discharged alive who underwent 1 follow-up visit 1–3 months after the onset of stroke [16] 57%
(47–68) 67%
(57–76) 80%
(72–88) 69%
(60–78) 74%
(65–83)
CHEST PAIN Patients discharged from the ED and readmitted to hospital within 1 month [43] 3.2%
(1–6) 1.4%
(0–3) 0.5%
(0–2) 1.9%
(0–4) 0%
Hospitalized patients admitted to the appropriate ward according to pain characteristics [44] 64%
(61–67) 61%
(58–64) 81%
(78–84) 73%
(70–76) 77%
(74–80)
Patients with AMI admitted to the coronary ICU within 60 minutes of hospital presentation [45] 63%
(55–70) 65%
(58–72) 77%
(70–84) 70%
(63–77) 76%
(69–83)
Diagnoses of AMI in the discharge summaries inconsistent with clinical records [46] 8%
(5–11) 9%
(5–13) 7%
(4–10) 7%
(4–10) 5%
(2–8)
COPD Patients hospitalized in the appropriate ward according to severity [47] 64%
(57–71) 62%
(55–69) 75%
(69–81) 92%
(88–96) 86%
(81–91)
Patients eligible for spirometry who underwent spirometry [48] 56%
(49–63) 49%
(41–56) 92%
(88–96) 92%
(88–96) 96%
(93–99)
Patients with MRC dyspnea grade >= 3 on admission who improved on discharge [49] 86%
(79–92) 83%
(77–90) 91%
(86–96) 88%
(82–94) 87%
(81–93)
Patients hospitalized for the appropriate number of days according to clinical severity [50] 93%
(88–98) 98%
(95–100) 96%
(93–100) 94%
(89–99) 97%
(94–100)
CHILD DELIVERY Cesarean sections on the total number of child deliveries [51] 41%
(38–44) 41%
(39–44) 35%
(32–38) 39%
(36–42) 39%
(36–42)
Elective cesarean sections for women with previous cesarean section without contraindications to vaginal delivery [52] 66%
(57–75) 60%
(51–69) 45%
(36–54) 64%
(55–73) 56%
(47–65)
Cesarean sections performed because of the woman's psychological refusal of vaginal delivery [53] 25%
(18–32) 17%
(11–23) 18%
(12–24) 15%
(10–20) 20%
(15–25)
LUNG CANCER Patients included in the ICP within 2 weeks of the first X-ray [54] 53%
(43–62) 55%
(43–67) 61%
(51–71) 62%
(51–73)
Patients diagnosed within 4 weeks since ICP inclusion [54] 73%
(64–82) 73%
(63–83) 78%
(69–87) 62%
(51–73)
Staged patients [55] 89%
(83–95) 97%
(93–100) 93%
(88–98) 96%
(92–100)
Patients transferred to the intensive care unit for the first 24 hours after surgery [56] 93%
(87–99) 87%
(77–97) 81%
(70–92) 86%
(76–96)
Patients who underwent surgery according to eligibility criteria [55] 91%
(84–98) 97%
(93–100) 88%
(81–95) 91%
(85–97)
Borderline cases discussed by the multidisciplinary group [55] 100% 100% 100% 100%
Some indicators exhibit extremely high adherence, constant through time; for instance, percentage of borderline lung cancer cases discussed by the multidisciplinary team, performance of a CT scan in stroke patients, and appropriate length of stay (LOS) for COPD patients according to severity, have never fallen below 90% in the observed semesters.
Other indicators began with a low, or fairly low adherence and then exhibited considerable improvements; for instance, the percentage of eligible patients receiving spirometry rose from 56% to 96%. The same positive trend was observed for indicators relating to the appropriateness of admission: stroke patients admitted to a dedicated ward (63%–82%); patients with chest pain hospitalized in the appropriate ward according to pain characteristics (64%–77%); and COPD patients hospitalized in the appropriate ward according to severity (64%–86%).
Finally, for some indicators adherence rates are still low compared to the standards found in the literature; for example, the percentage of cesarean sections (CSs) (39% in the last semester) remains far above the WHO recommendation of a 15% rate. Analogously, the percentage of lung cancer patients included in the ICP within 2 weeks of the first X-ray, on which suspicion is based, remains fairly poor in the 4 analyzed semesters.
For further clarity, trends for five medical conditions are portrayed graphically in Figure 2.
Figure 2 Trend of adherence to each indicator by semester. For each condition the proportion of patients receiving key interventions after the introduction of the multifaceted strategy is displayed. See text for the full description of indicators and for bibliographic references.
Discussion
In order to improve adherence to evidence-based medicine at our hospital, we applied a planned strategy comprising a combination of interventions, each addressing specific barriers to change (from professional, socio-cultural, and organizational viewpoints); we have demonstrated through a quasi-experimental study the effectiveness of this strategy. The improvement trend we demonstrate is consistent with the findings reported by Kwan et al in a very recent before-after study on the application of a Stroke pathway, concerning the indicators on timely CT scan performance and early involvement of the rehabilitation team [19].
The execution of the program we propose is complex (it requires meetings, debates, data collection, etc.), but so is the process of incorporation of evidence into practice; in fact, despite publication of several management guidelines in virtually any medical field, the literature shows that adherence to EBM indications is often poor [2,20-22]. The biggest obstacle seems to be the perception of guidelines as tools distant from the reality in which one operates, and the large amount of information often contained in guidelines, which clinicians have no time to digest [20,23]. The picture is complicated by the gap between those who practice and those who manage health care, whereby the introduction of new technologies/tests and the implementation of change is sometimes left to the initiative of individual professionals, or, conversely, allows administrators to activate or cancel procedures without assessing the effects of their decisions at a local level with those directly involved (health care workers and users). We tried solving these problems by creating a constant debate between administrators and health care workers, from the initial stages of the improvement strategy's definition.
We have demonstrated the strategy's capability to improve adherence to scientific evidence through a quasi-experimental study with historical controls, and not a randomized trial. RCTs are the gold standard for testing effectiveness, however in our case this design would have posed a number of methodological issues. For complex interventions aiming to change behavior, like ours, randomization by individual patients is not feasible and cluster randomization, by doctor or practice, is usually advised [24]. This approach, however, would have been extremely complex to take in our study, because the strategy in question involves multiple organizational levels (Emergency Department, Internal Medicine wards, Neurology, Rehabilitation, Radiology service). Furthermore, since the intervention's effectiveness depends on the subjects' active participation and on their choices, random allocation may lessen its effect, and a clinical trial would in fact be inappropriate [25,26].
The analysis of clinical records as a means of validating the program also has some well-known limitations (exhaustivity, accuracy, etc.), however it offers the great advantage of allowing timely access to the information needed to documenting an intervention's impact using a historical control. A possible bias may exist in the interpretation of the measured effect, which may be due to greater accuracy of documentation rather than to a real change in care. However, since a pathway's success is strictly related to the quality and quantity of available information on the patients' characteristics and the tests they undergo, the appropriate management of clinical information is part of our work's objective.
Our work concerns the adoption of an improvement strategy in a large hospital involving a set of common medical conditions, and its long-term effectiveness. Studies on the subject generally focus on a single condition, or on a few easy-to-standardized pathologies [27-34]. Instead, our work concerns the use of 14 ICPs, of which 77% exhibit good compliance with the strategy. Noteworthy is also the number of patients, approximately 7000/year, whose hospital management is verified through: the analysis of data prospectively collected, the comparison with the most recent scientific indications, and the discussion of deviations by a group of experts directly involved in the management of the condition. What makes our experience different is that it focuses on the strategy's effectiveness in the long-run, while research on ICPs found in the literature generally reports on short-term findings. A very recent Australian study [35] does focus on the long-term effect of ICP use, but the number of patients included in the analysis is quite small.
Despite some variability, data generally demonstrate a positive trend, showing improvements for most indicators with fairly frequent drops in both second semesters, which most probably reflect the greater difficulties encountered during the summer holiday season, when personnel is reduced, thus affecting the provision of services. The measures we adopted to achieve change in behavior were both educational (critical literature analysis, sharing and dissemination of the ICP, systematic clinical audits, etc.), and organizational/managerial (performance of CT scans in the emergency department, rapid involvement of the rehabilitation team via fax, use of a portable spirometer for bedridden patients, etc.). As also noted by Panella et al in a recent Italian experience with the application of an ICP program [36], constant dialogue within MTs and between managers and clinicians, based on variance analysis, is essential to ensure best clinical practice.
The employment of this strategy is not easy, and some medical conditions have encountered greater difficulties than others. The ICP on Breast Cancer has been inactive since September 2002; though it was initiated in December 2001, it has not yet completed its implementation phase, and indicators have not yet been defined. This pathway's problems are mostly caused by the instability consequent to the change of the director of the Oncology Department, which led to the lack of a leading figure in the multidisciplinary group, an essential aspect for improvement of clinical practice [7]. The ICP on Pediatric Head Injury was also inactive since January 2002. Indicators have been defined but never applied. The main obstacle for this ICP seems to be the concern of many physicians of losing their professional freedom, also fearing possible ethical and legal implications in the care of children [37]. The ICP on Supraventricular tachiarrhythmias, which was in its implementation phase, shows the lowest MT compliance, 55%. The difficulty seems to lie mainly in the large amount of data to be collected, since the MT had not identified the most interesting key aspects, and in the fact that this group of patients is managed in several different wards. Finally, the ICP on Child Delivery has proven very ineffective, exhibiting very low adherence with the selected QIs. Various major obstacles have been identified for this ICP: first of all, the lack of sound scientific evidence supporting the pathway's indications, which were derived from authoritative sources (WHO) but are mostly based on expert opinion and remain controversial. Furthermore, it is believed that the high and rising cesarean section rates in most countries may be due in part to physician attitudes of defensive medicine. Legal suits are very frequent when a vaginal delivery has a bad outcome, but they are unlikely when an unnecessary cesarean section is performed [38]. Protection against legal litigation would thus be required to enhance compliance with indications aiming to reduce unnecessary cesarean section rates. Other actions which could help achieve WHO goals are the provision of epidural anesthesia free of charge (in most Italian institutions it is rather costly for patients), and the equalization of financial compensation for CS and vaginal delivery, since in Italy the former is much higher and this might encourage some gynecologists to support the performance of unnecessary CSs.
Conclusion
This research highlights the importance of multidisciplinary involvement in the creation and application of a strategy to favor EBM introduction into practice. We believe hospitals should ensure the incorporation of research into routine practice by systematically applying a shared, multifaceted strategy, as single interventions cannot be effective in all situations [2]. Our experience confirms what recently emphasized in the literature, i.e. that multiple approaches are the most effective, and that the choice of interventions should be guided by the analysis of determinants of professional behavior and by specific clinical and organizational circumstances [39]. Health care institutions willing to adopt analogous tools should first assess local characteristics carefully and develop a tailor-made approach.
The presence of central coordination, competent in scientific methodology and implementation techniques, is necessary to support the work of individual multidisciplinary teams, to favor agreement between health care professionals and managers, and to constantly remind all parties involved of the importance of the program, as motivation is likely to decrease with time. A far-reaching change in culture is essential to favor the acceptance of change in an organization. Institutions should offer their professionals adequate training on implementation strategies and on the nature and importance of clinical practice improvement [40].
The main down side of this program, as emphasized in the literature on quality improvement strategies [7] is its high costs in terms of staff time. This can be a cause of reluctance for some clinicians and managers. However, the introduction of scientific evidence into practice does necessarily require time and money. We therefore believe that, just like for experimental trials, economic and professional resources should be allocated to allow the achievement of this objective.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CC is the person responsible for the program discussed in the paper. She conceived of the study, and supervised the entire process, from the creation of the program to its application, to statistical analysis, to the drafting of the manuscript. US is the coordinator of the MT on cerebral ischemic stroke. He worked on the definition of the pathway and was responsible for the data gathering process. FD performed bibliographic research and literature analysis, participated in the coordination of the work, and helped to draft and translate the manuscript into English. RP offered expertise on the methodology to follow, and provided comments and suggestions in the preparation of the paper. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The idea of this project was first conceived by our hospital's former Managing Director, Dr. Gianni Giorgi. This work would never have been completed without his creativity, competence and enthusiasm. We thank Giacomo Annaloro, Maria Cristina Cornelli, Simona Fontechiari, Adriana Gelmini, Maria Grazia Ollari, and Maria Rosaria Pieri Nerli, for their precious contribution to the definition of the strategy. We are grateful to all the clinicians and nurses of each multidisciplinary team, who dedicated so much time and effort to this work. Our special thanks go to the following MT coordinators, for their high motivation and commitment: Diego Ardissino (Chest Pain and Supraventricular Tachiarrhythmias), Gianfranco Cervellin (Pulmonary Thromboembolism), Giuseppe De Panfilis (Melanoma), Carlo Ferrari (Liver Cirrhosis), Alessandro Grignaffini (Child Delivery), Dario Olivieri (COPD), Bruno Panno (Hip arthroplasty), Giovanna Pisi (Childhood Pneumonia), Vittorio Rizzoli (Non Hodgkin Lymphomas), Michele Rusca (Lung Cancer). We thank Davide Romano and Renata Todeschini for their precious help with editing. Finally, we are very thankful to our Managing Director, Sergio Venturi, and Medical Superintendent, Gianbattista Spagnoli, for their essential support to the initiative.
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Jencks SF Cuerdon T Burwen DR Fleming B Houck PM Kussmaul AE Nilasena DS Ordin DL Arday DR Quality of medical care delivered to Medicare beneficiaries: A profile at state and national levels JAMA 2000 284 1670 1676 11015797 10.1001/jama.284.13.1670
Grol R Grimshaw J From best evidence to best practice: effective implementation of change in patients' care Lancet 2003 362 1225 1230 14568747 10.1016/S0140-6736(03)14546-1
Shortell SM Bennett CL Byck GR Assessing the impact of continuous quality improvement on clinical practice: what it will take to accelerate progress Milbank Q 1998 76 593 624 510 9879304 10.1111/1468-0009.00107
Bodenheimer T The American health care system–the movement for improved quality in health care N Engl J Med 1999 340 488 492 9971876 10.1056/NEJM199902113400621
McDonald IG Quality assurance and technology assessment: pieces of a larger puzzle J Qual Clin Pract 2000 20 87 94 11057990 10.1046/j.1440-1762.2000.00372.x
Smith TJ Hillner BE Ensuring quality cancer care by the use of clinical practice guidelines and critical pathways J Clin Oncol 2001 19 2886 2897 11387362
Campbell H Hotchkiss R Bradshaw N Porteous M Integrated care pathways BMJ 1998 316 133 137 9462322
Campbell SM Braspenning J Hutchinson A Marshall M Research methods used in developing and applying quality indicators in primary care BMJ 2003 326 816 9 12689983 10.1136/bmj.326.7393.816
Kaltenthaler E McDonnell A Peters J Monitoring the care of lung cancer patients: linking audit and care pathways J Eval Clin Pract 2001 7 13 20 11240836 10.1046/j.1365-2753.2001.00275.x
Kitchiner D Davidson C Bundred P Integrated care pathways: effective tools for continuous evaluation of clinical practice J Eval Clin Pract 1996 2 65 69 9238576
Kitchiner D Bundred P Integrated care pathways increase use of guidelines BMJ 1998 317 147 148 9657806
Dalton P Macintosh DJ Pearson B Variance analysis in clinical pathways for total hip and knee joint arthroplasty J Qual Clin Pract 2000 20 145 149 11207952 10.1046/j.1440-1762.2000.00382.x
Sweeney AB Flora HS Chaloner EJ Buckland J Morrice C Barker SG Integrated care pathways for vascular surgery: an analysis of the first 18 months Postgrad Med J 2002 78 175 177 11884705 10.1136/pmj.78.917.175
Kleinbaum DG Kupper LL Morgenstern H Epidemiologic research: principles and quantitative methods 1982 New York: Van Nostrand Reinhold 44 5
Fleiss JL Statistical Methods for Rates and Proportions 1981 2 John Wiley & Sons. New York
Measuring and improving quality of care: a report from the American Heart Association/American College of Cardiology First Scientific Forum on Assessment of Healthcare Quality in Cardiovascular Disease and Stroke Circulation 2000 101 1483 1493 10736296
Adams HP JrAdams RJ Brott T del Zoppo GJ Furlan A Goldstein LB Grubb RL Higashida R Kidwell C Kwiatkowski TG Marler JR Hademenos GJ Stroke Council of the American Stroke Association Guidelines for the early management of patients with ischemic stroke: A scientific statement from the Stroke Council of the American Stroke Association Stroke 2003 34 1056 1083 12677087 10.1161/01.STR.0000064841.47697.22
Stroke Unit Trialists' Collaboration Organised inpatient (stroke unit) care for stroke (Cochrane Review) The Cochrane Library 2004 Chichester, UK: John Wiley & Sons, Ltd
Kwan J Hand P Dennis M Sandercock P Effects of introducing an integrated care pathway in an acute stroke unit Age Ageing 2004 33 362 7 15047573 10.1093/ageing/afh104
Gallagher EJ How well do clinical practice guidelines guide clinical practice? Ann Emerg Med 2002 40 394 398 12239494 10.1016/S0196-0644(02)00059-8
Roberts CM Ryland I Lowe D Kelly Y Bucknall CE Pearson MG Audit Sub-committee of the Standards of Care Committee British Thoracic Society Clinical Effectiveness and Evaluation Unit, Royal College of Physicians: Audit of acute admissions of COPD: standards of care and management in the hospital setting Eur Respir J 2001 17 343 349 11405509 10.1183/09031936.01.17303430
Barnard J Siriwardena AK Variations in implementation of current national guidelines for the treatment of acute pancreatitis: implications for acute surgical service provision Ann R Coll Surg Engl 2002 84 79 81 11995768
Shaneyfelt TM Building bridges to quality JAMA 2001 286 2600 2601 11722277 10.1001/jama.286.20.2600
Medical Research Council A framework for development and evaluation of RCTs for complex interventions to improve health 2001
Grimshaw J Freemantle N Wallace S Russell I Hurwitz B Watt I Long A Sheldon T Developing and implementing clinical practice guidelines Qual Health Care 1995 4 55 64 10142039
Black N Why we need observational studies to evaluate the effectiveness of health care BMJ 1996 312 1215 1218 8634569
Johnson KB Blaisdell CJ Walker A Eggleston P Effectiveness of a clinical pathway for inpatient asthma management Pediatrics 2000 106 1006 1012 11061767 10.1542/peds.106.5.1006
Dowsey MM Kilgour ML Santamaria NM Choong PF Clinical pathways in hip and knee arthroplasty: a prospective randomised controlled study Med J Aust 1999 170 59 62 10026684
Pitt HA Murray KP Bowman HM Coleman J Gordon TA Yeo CJ Lillemoe KD Cameron JL Clinical pathway implementation improves outcomes for complex biliary surgery Surgery 1999 126 751 756 10520925 10.1067/msy.2099.100672
Miller PR Fabian TC Croce MA Magnotti LJ Elizabeth Pritchard F Minard G Stewart RM Improving outcomes following penetrating colon wounds: application of a clinical pathway Ann Surg 2002 235 775 781 12035033 10.1097/00000658-200206000-00004
Chang SS Smith JA JrGirasole C Baumgartner RG Roth BJ Cookson MS Beneficial impact of a clinical care pathway in patients with testicular cancer undergoing retroperitoneal lymph node dissection J Urol 2002 168 87 92 12050498 10.1097/00005392-200207000-00021
Marrie TJ Lau CY Wheeler SL Wong CJ Vandervoort MK Feagan BG A controlled trial of a critical pathway for treatment of community-acquired pneumonia JAMA 2000 283 749 755 10683053 10.1001/jama.283.6.749
Choong PF Langford AK Dowsey MM Santamaria NM Clinical pathway for fractured neck of femur: a prospective, controlled study Med J Aust 2000 172 423 426 10870534
Feagan BG A controlled trial of a critical pathway for treating community-acquired pneumonia: the CAPITAL study. Community-Acquired Pneumonia Intervention Trial Assessing Levofloxacin Pharmacotherapy 2001 21 89S 94S 11446524 10.1592/phco.21.10.89S.34535
Wolff AM Taylor SA McCabe JF Using checklists and reminders in clinical pathways to improve hospital inpatient care Med J Aust 2004 181 428 31 15487958
Panella M Marchisio S Di Stanislao F Reducing clinical variations with clinical pathways: do pathways work? Int J Qual Health Care 2003 15 509 21 14660534 10.1093/intqhc/mzg057
Every NR Hochman J Becker R Kopecky S Cannon CP Critical pathways: a review Circulation 2000 101 461 5 10653841
Wagner M Choosing caesarean section Lancet 2000 356 1677 80 11089840 10.1016/S0140-6736(00)03169-X
Grilli R Ballini L Il Pensiero Sceintifico Editore Evidence-based medicine e miglioramento della pratica clinica Etica, conoscenza e sanità 2005 Roma: Liberati A 229 257
Horne MK Monash CSSP Consortium The Monash University Consortium: factors involved in the local implementation of clinical evidence into practice Med J Aust 2004 180 S89 91 15139844
Chiu L Shyu WC Liu YH Comparisons of the cost-effectiveness among hospital chronic care, nursing home placement, home nursing care and family care for severe stroke patients J Adv Nurs 2001 33 380 386 11251725 10.1046/j.1365-2648.2001.01703.x
Johnson G Burvill PW Anderson CS Jamrozik K Stewart-Wynne EG Chakera TM Screening instruments for depression and anxiety following stroke: experience in the Perth community stroke study Acta Psychiatr Scand 1995 91 252 257 7625207
Pope JH Aufderheide TP Ruthazer R Woolard RH Feldman JA Beshansky JR Griffith JL Selker HP Missed diagnoses of acute cardiac ischemia in the emergency department N Engl J Med 2000 342 1163 1170 10770981 10.1056/NEJM200004203421603
Lee TH Goldman L Evaluation of the patient with acute chest pain N Engl J Med 2000 342 1187 1195 10770985 10.1056/NEJM200004203421607
Van de Werf F Ardissino D Betriu A Cokkinos DV Falk E Fox KA Julian D Lengyel M Neumann FJ Ruzyllo W Thygesen C Underwood SR Vahanian A Verheugt FW Wijns W Task Force on the Management of Acute Myocardial Infarction of the European Society of Cardiology Management of acute myocardial infarction in patients presenting with ST-segment elevation. The Task Force on the Management of Acute Myocardial Infarction of the European Society of Cardiology Eur Heart J 2003 24 28 66 12559937 10.1016/S0195-668X(02)00618-8
Cannon CP Battler A Brindis RG Cox JL Ellis SG Every NR Flaherty JT Harrington RA Krumholz HM Simoons ML Van De Werf FJ Weintraub WS Mitchell KR Morrisson SL Brindis RG Anderson HV Cannom DS Chitwood WR Cigarroa JE Collins-Nakai RL Ellis SG Gibbons RJ Grover FL Heidenreich PA Khandheria BK Knoebel SB Krumholz HL Malenka DJ Mark DB Mckay CR Passamani ER Radford MJ Riner RN Schwartz JB Shaw RE Shemin RJ Van Fossen DB Verrier ED Watkins MW Phoubandith DR Furnelli T American College of Cardiology key data elements and definitions for measuring the clinical management and outcomes of patients with acute coronary syndromes. A report of the American College of Cardiology Task Force on Clinical Data Standards (Acute Coronary Syndromes Writing Committee) J Am Coll Cardiol 2001 38 2114 2130 11738323 10.1016/S0735-1097(01)01702-8
Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD). Workshop Summary Am J Respir Crit Care Med 2001 163 1256 1276 11316667
MacNee W Calverley PM Chronic obstructive pulmonary disease. 7: Management of COPD Thorax 2003 58 261 265 12612309 10.1136/thorax.58.3.261
Bestall JC Paul EA Garrod R Garnham R Jones PW Wedzicha JA Usefulness of the Medical Research Council (MRC) dyspnoea scale as a measure of disability in patients with chronic obstructive pulmonary disease Thorax 1999 54 581 586 10377201
Cotton MM Bucknall CE Dagg KD Johnson MK MacGregor G Stewart C Stevenson RD Early discharge for patients with exacerbations of chronic obstructive pulmonary disease: a randomized controlled trial Thorax 2000 55 902 906 11050257 10.1136/thorax.55.11.902
World Health Organization Appropriate technology for birth Lancet 1985 2 436 437 2863457
Brill Y Windrim R Vaginal birth after Caesarean section: review of antenatal predictors of success J Obstet Gynaecol Can 2003 25 275 286 12679819
Saisto T Halmesmaki E Fear of childbirth: a neglected dilemma Acta Obstet Gynecol Scand 2003 82 201 208 12694113 10.1034/j.1600-0412.2003.00114.x
"Programma Nazionale linee guida" PNLG
British Thoracic Society Society of Cardiothoracic Surgeons of Great Britain and Ireland Working Party BTS guidelines: guidelines on the selection of patients with lung cancer for surgery Thorax 2001 56 89 108 11209097 10.1136/thorax.56.2.89
The EACTS/ESTS Working Group on Structures in Thoracic Surgery Structure of General Thoracic surgery in Europe
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BMC Med GenetBMC Medical Genetics1471-2350BioMed Central London 1471-2350-6-351620212310.1186/1471-2350-6-35Research ArticleAge at onset of Huntington disease is not modulated by the R72P variation in TP53 and the R196K variation in the gene coding for the human caspase activated DNase (hCAD) Arning Larissa [email protected] Peter H [email protected] Carsten [email protected] Jürgen [email protected] Jörg T [email protected] Department of Human Genetics, Ruhr-University, 44780 Bochum, Germany2 Department of Neurology, St. Josef-Hospital, Ruhr-University, 44791 Bochum, Germany2005 3 10 2005 6 35 35 10 2 2005 3 10 2005 Copyright © 2005 Arning 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
TP53 is an attractive candidate for modifying age of onset (AO) in Huntington disease (HD): The amino-terminus of the mutated huntingtin (htt) exon 1 translation product has functional properties which may affect critically the TP53 pathway in HD neurons. The pathogenic domain of mutant htt interacts with nuclear transcription factors, and it potentially modulates TP53-induced transcriptional events. A single nucleotide polymorphism (SNP) resulting in the R72P exchange in TP53 protein might modulate the variation in AO. In addition, also the R196K replacement in human caspase activated DNase (hCAD) may theoretically affect the AO.
Methods
We have genotyped the polymorphisms R72P and R196K in a well established cohort of 167 unrelated HD patients.
Results
The expanded CAG repeat explained 30.8% of the variance in AO. Adding the genotypes of the SNPs investigated did not affect the variance of the AO variance explained.
Conclusion
In this replication study, no association was found explaining a significant amount of the variability in AO of HD thus contradicting a recent report.
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Background
Huntington disease (HD) is an autosomal dominantly transmitted, neurodegenerative disorder characterized by motor abnormalities, cognitive dysfunction and psychiatric symptoms [1]. HD is caused by an expansion of a polyglutamine tract in the amino-terminal portion of the protein huntingtin (htt), which apparently acquires a deleterious gain of function [2]. The length of the polyglutamine tract is the most important factor in determining AO of HD, although substantial variability remains after controlling for repeat length, particularly in cases where CAG repeat numbers range in the high 30 s or low 40 s [3]. Therefore, defining independent AO modifying factors is of great importance, since they may provide further clues pertaining to the pathology arising from the expanded repeats. In mammals, TP53 is the most important tumor suppressor gene. A variety of intracellular stress signals activate TP53 to induce either transient cell cycle arrest with stimulation of repair activities, senescence or apoptosis [4]. It has been proposed that the amino-terminal portion of mutant htt encoded by exon 1 exertsfunctional properties with significant consequences for the TP53 pathway in HD neurons. The pathogenic domain of mutant htt interacts with critical transcription factors and potentially modulates TP53-induced transcriptional events [5]. It has been shown that the two variations of the common codon 72 polymorphism (Arg /Pro) in the TP53 gene differ with respect to the ability to suppress cellular transformation [6] and induce apoptosis [7]. Therefore, the R72P polymorphism represents a good candidate for modulating the AO of HD. Recently this polymorphism was described to explain a significant part of the variance in the AO in HD [8]. In the same study also the SNP R196K was investigated in the human caspase activated DNase protein (hCAD), since DNase is responsible for DNA degradation during apoptosis [9]. Variations in the latter SNP were likewise found to exert an additionally significant impact on the variation of AO. We reinvestigated here whether TP53 and hCAD variations modulate the AO of HD in our established cohort.
Methods
Our study population consists of 167 unrelated patients with the clinical diagnosis of HD [10], recruited from the Huntington Center (HZ) NRW, Bochum (Germany). Clinical assessment and determination of the motor AO was performed exclusively by two experienced neurologists/psychiatrists of the HZ NRW. Controls (healthy blood donors from the German population of Essen) were also genotyped for comparison. Informed consent was obtained from all patients and controls. We purposely selected a cohort with limited extent of CAG expansions (41–45), since within this defined range the expansions show linear correlation with AO. Within this range ~75% of all elongated CAG alleles are comprised, and they are distributed normally. CAG repeat sizes were determined after PCR amplification of genomic DNA from peripheral white blood cells. CAG repeats were amplified by established methods [11]. The polymorphism R72P in the TP53 gene was demonstrated after PCR amplification using the following primers: 5'-GAGGACCTGGTCCTCTGACT-3' and 5'-GTAGGTTTTCTGGGAAGGGA-3'. PCR was carried out in a final volume of 10 μl with 50 ng of DNA, 200 μM dNTP and 1 U Taq Polymerase. Thermal cycling was performed with an initial denaturing step for 5 minutes at 94°C.; 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were digested with the restriction enzyme Bsh1236I I at 37°C overnight and visualised on 2% agarose gels stained with ethidium bromide. This digestion yielded altogether 3 bands of different sizes: a 254 bp fragment (restriction site absent) corresponding to the C allele as well as a set of 160 and 94 bp fragments corresponding to the G allele (restriction site present). The polymorphism R196K in the hCAD gene was demonstrated using the following primers: 5'-CCTCTGACCACAGGACTGG-3' and 5'-TCTGTCGAAGTACGTGCCAT-3' under the same conditions mentioned above. PCR products were digested with the restriction enzyme Alu I at 37°C overnight. Digested DNA was electrophoresed on 3% agarose gels. The restriction fragments of the G allele were 129, 99 and 13 bp in length. The A allele harbors an additional restriction site so that the 99 bp fragment is digested into 73 and 26 bp. The variability in AO attributable to the CAG repeat number was calculated by linear regression. For the regression analysis, we used the AO as the dependent variable, the respective genotypes as independent variables.
The CAG repeat number was considered as numerical variables, the other putative modifying genotypes were considered as nominal variables. SPSS Ver.11.0 for Windows (SPSS Inc.) was used for all statistical analyses.
Results and discussion
As detailed in an earlier study concerning our clinically thoroughly characterised HD cohort, the expanded CAG repeat explained 30.8% of the variance in AO [10]. Adding the genotypes of the SNPs investigated here did not affect the variance of the AO variance explained (Table 1). Chattopadhyay et al. further conducted a case-control study for these SNPs and found genotype GG in the TP53 gene to be a significant risk factor for HD. In order to retest also this assumption, we performed a case-control study as well. As expected, there was no difference between the case and control groups. All frequencies observed were in Hardy-Weinberg equilibrium and correspond to those reported for the general Caucasian population [12].
Conclusion
Our study failed to replicate the association between the genotypes at the R72P polymorphism in TP53 and R196K polymorphism in hCAD genes with the AO of HD. Since we employed a larger cohort of exceptionally well-characterized HD patients the initial association may have been due to chance or to bias as introduced by population stratification. It is, however, possible that a true association exists in Indians. The effect size may also be quite small, requiring an even larger sample size than ours for Caucasians.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LA initiated the study, carried out the molecular genetic studies and drafted the manuscript. PHK participated in the data analysis. JA and CS had ascertained the clinical status of the patients, and JTE participated in the study design, the coordination and finalized the analyses as well as the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Table 1 Genotype frequencies and linear regression analysis of the SNPs in TP53 and hCAD genes
Gene Genotypes Number (%) Gene (polymorphism) R2 ΔR2 Unexplained variance (%) p value
HD patients Controls
TP53 GG 89 (53) 86 (51) HD CAG .308 -
CG 69 (41) 69 (41)
CC 9 (5) 13 (8) HD CAG +
TP53 (R72P) .314 .006 0.9 .130
hCAD GG 141 (84)
AG 23 (14) HD CAG +
AA 3 (2) hCAD (R196K) .305 .003 0.4 .590
==== Refs
The Huntington's Disease Collaborative Research Group Cell 1993 72 971 983 8458085 10.1016/0092-8674(93)90585-E
DiFiglia M Sapp E Chase K Schwarz C Meloni A Yound C Martin E Vonsattel JP Carraway R Reeves SA Boyce FM Aronin N Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons Neuron 1995 14 1075 1081 7748555 10.1016/0896-6273(95)90346-1
Kehoe P Krawczak M Harper PS Owen MJ Jones AL Age of onset in Huntington disease: sex specific influence of apolipoprotein E genotype and normal CAG repeat length J Med Genet 1999 36 108 111 10051007
Vogelstein B Lane D Levine AJ Surfing the p53 network Nature 2000 6810 307 310 10.1038/35042675
Steffan JS Kazantsev A Spasic-Boskovic O Greenwald M Zhu YZ Gohler H Wanker EE Bates GP Housman DE Thompson LM The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription Proc Natl Acad Sci U S A 2000 97 6763 6768 10823891 10.1073/pnas.100110097
Thomas M Kalita A Labrecque S Pim D Banks L Matlashewski G Two polymorphic variants of wild-type p53 differ biochemically and biologically Mol Cell Biol 1999 19 1092 1100 9891044
Bonafe M Salvioli S Barbi C Trapassi C Tocco F Storci G Invidia L Vannini I Rossi M Marzi E Mishto M Capri M Olivieri F Antonicelli R Memo M Uberti D Nacmias B Sorbi S Monti D Franceschi C The different apoptotic potential of the p53 codon 72 alleles increases with age and modulates in vivo ischaemia-induced cell death Cell Death Differ 2004 11 962 973 15131588 10.1038/sj.cdd.4401415
Chattopadhyay B Baksi K Mukhopadhyay S Bhattacharyya NP Modulation of age at onset of Huntington disease patients by variations in TP53 and human caspase activated DNase (hCAD) genes Neurosci Lett 2005 374 81 6 15644269 10.1016/j.neulet.2004.10.018
Enari M Sakahira H Yokoyama H Okawa K Iwamatsu A Nagata S A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD Nature 1998 6662 43 50
Arning L Kraus PH Valentin S Saft C Epplen JT NR2A and NR2B receptor gene variations modify age at onset in Huntington disease Neurogenetics 2005 6 25 8 15742215 10.1007/s10048-004-0198-8
Warner JP Barron LH Brock DJ A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes Mol Cell Probes 1993 7 235 239 8366869 10.1006/mcpr.1993.1034
Donehower LA p53: guardian AND suppressor of longevity? Exp Gerontol 2005 40 7 9 15664727 10.1016/j.exger.2004.10.007
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BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-5-331616229810.1186/1472-6920-5-33Research ArticleLessons learned in developing family medicine residency training programs in Japan Murai Mitsuya [email protected] Kazuya [email protected] Michael D [email protected] Kashima Hospital, Kashima City, Japan2 Department of General Medicine, Nagoya University Hospital, Nagoya, Japan3 University of Michigan Medical Center, Department of Family Medicine, Ann Arbor, USA2005 15 9 2005 5 33 33 6 5 2005 15 9 2005 Copyright © 2005 Murai et al; licensee BioMed Central Ltd.2005Murai 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 family medicine is not well established as a discipline in Japan, a growing number of Japanese medical schools and training hospitals have recently started sougoushinryoubu (general medicine departments). Some of these departments are incorporating a family medicine approach to residency training. We sought to learn from family medicine pioneers of these programs lessons for developing residency training.
Methods
This qualitative project utilized a long interview research design. Questions focused on four topics: 1) circumstances when becoming chair/faculty member; 2) approach to starting the program; 3) how Western ideas of family medicine were incorporated; and 4) future directions. We analyzed the data using immersion/crystallization to identify recurring themes. From the transcribed data, we selected representative quotations to illustrate them. We verified the findings by emailing the participants and obtaining feedback.
Results
Participants included: five chairpersons, two program directors, and three faculty members. We identified five lessons: 1) few people understand the basic concepts of family medicine; 2) developing a core curriculum is difficult; 3) start with undergraduates; 4) emphasize clinical skills; and 5) train in the community.
Conclusion
While organizational change is difficult, the identified lessons suggest issues that merit consideration when developing a family medicine training program. Lessons from complexity science could inform application of these insights in other countries and settings newly developing residency training.
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Background
Like many countries in the world, the discipline of family medicine is not well established in Japan. This is surprising, given an effective national health insurance system [1,2], and large number of ambulatory care physicians in Japan. In 2002, about 34.4% of physicians reported working as a solo practitioner or as an employee in a "clinic" (defined as a physician's office without beds or with fewer than 20 beds) [3]. The majority of Japanese physicians receive specialty training in university hospitals or large hospitals for five to ten years, then about one third of them become private practitioners in a clinic [4]. Japanese physicians are not restricted by regulations based on their training or board certification and can label themselves by the kind of practice they wish to have [5]. By default, these sub-specialty-trained doctors become Japan's self-taught primary care doctors, but they are neither systematically trained for this field nor do they have much opportunity to gain knowledge and skills necessary for primary care through continuing medical education.
Previous authors have synthesized information on the need for family medicine in Japan and provided status reports on the development of family medicine [4-7]. While the family medicine movement in Japan began in the 1970's, the first department of general medicine was not established until 1981 at Kawasaki Medical School. The most important academic organization supporting family physicians is the Japanese Academy of Family Medicine (JAFM). Established in 1986, it has taken a leadership role in establishing family medicine in Japan. Like most medical societies in Japan, the JAFM has membership based on interest, not criteria such as board certification or completion of specific residency training. As family medicine is still a young discipline in Japan, most members have trained in non-family medicine programs. The physician members include a diverse group: those trained in a family medicine training program in Japan or abroad (mostly the US), those trained in another specialty or multiple specialties and became a general practitioner after entering practice, and those who trained in a general internal medicine program in Japan or abroad.
A barrier to family medicine's development continues to be inconsistent support from the Japanese government. At times enthusiastic, at times apathetic about family medicine's establishment, the government has faced stiff opposition from the Japan Medical Association and previously has backed down from its own initiatives to support family medicine. Interestingly, the Japanese Ministry of Health, Labour, and Welfare (MHLW) recently approved creation of sougoushinryoubu (translation: general medicine departments)[8]. Of the 80 medical schools in Japan, 30 have established sougoushinryoubu, and 96 training hospitals have established, or are preparing, a new training program [8]. Unfortunately, the MHLW did not provide clear direction about the content and purpose of these departments, and most have developed according to one of three patterns. About ten of the medical school sougoushinryoubu decided to use family medicine as a model for their development. An even smaller number appear to be pursuing a general internal medicine model, and the remainder has not made a commitment to a discipline. These often function as basic triage departments to funnel patients to hospital-based sub-specialists who do not want to manage undifferentiated problems.
Given our interest in the development of family medicine in Japan, and the lack of literature describing the issues involved in starting a department of family medicine training program, we were interested in the experiences and opinions of the pioneers who are embracing development of family medicine. The objectives of this research were to investigate their experiences with developing family medicine residency training in Japan, and to draw lessons for others.
Methods
This qualitative project utilized a long interview research design [9] since the intent was to elicit, in detail, the family medicine pioneers' experiences with, and their opinions about, the development of family medicine training programs in Japan. During a one-month research elective from his US residency training program, one of us (MM) traveled to Japan in November 2000 to conduct interviews with as many Japanese faculty "pioneers" as could be arranged. As the authors are members of the JAFM, and actively participate, we are familiar with the small number of individuals taking a leadership role in family medicine activities in the JAFM, and directly contacted potential candidates. The eligibility requirements of recruited sites included: having at least one faculty member graduated from a family medicine program in Canada or the U.S., or who had had several months of learning experiences in undergraduate/family medicine in the U.S.; and/or having at least one faculty member with broad and long experience in community-based general practice.
One of us (MM) conducted in-depth, open-ended 40 to 75 minutes duration interviews. He conducted each interview in an office, or other private setting selected by the participant. We developed the interview guide in the format proposed by Crabtree and Miller for conducting "Long Interviews." [9]. The interviews began with a "grand tour" style question: "Think back and describe the circumstances that lead up to your becoming the chair/faculty member of the program." This was followed with a series of probe questions (see Appendix 1). Interview questions focused on four topics: 1) circumstances when becoming chair/faculty member; 2) approach to starting the program; 3) how Western ideas of family medicine/general medicine were incorporated; and 4) future directions. We asked participants about unanticipated issues that they had encountered. Each interview was audiotaped and field notes were kept in a study journal. The interviews were transcribed by the native Japanese-speaking researchers and a research assistant, who were instructed to record verbatim the conversation including pauses, repetitions, etc. [10].
For the content analysis, we utilized the techniques of immersion/crystallization [11,12]. The primary analysis team (MM and KK) conducted multiple readings of the transcripts and independently identified the major themes from the transcribed text. Both analysts found the participants discussed lessons they had learned, and barriers and facilitators, to development of family medicine. Within these areas, they found five overarching lessons to organize the primary findings. To ascertain these were grounded in the interviews, they searched the transcribed text to identify examples corroborating the findings.
After organization into a narrative format, we sent these results to the participants by email, and requested their comments on our interpretations, and specifically on the issue of economics of starting a training program as this area had been discussed little during the interviews. There were no substantive changes based on the feedback.
Results
The participants were ten Japanese faculty leaders of family medicine programs: two program directors, five chairpersons, and three faculty members. The facilities where they work include two private medical colleges, two private community hospitals, and three public universities. The participants included nine male, and one female, physicians.
Based on our analysis of the text and field notes, we identified five themes, two highlighting the barriers and three signifying facilitators for developing family medicine training. We organized them as five lessons: 1) "They just don't get it;" 2) developing a core curriculum is difficult; 3) start with undergraduates; 4) emphasize clinical skills; and 5) train in the community.
Lesson 1: They just don't get it
In the environments where these faculty members work, few of their colleagues understand the basic concepts and values of family medicine. In their experience, high-level administrators, CEOs, deans, and hospital directors, are the most supportive of new departments. For example, the latter group supports dispatching eligible faculty members to foreign countries to learn family medicine and to recruit board-certified family physicians, or experienced community-based general practitioners, before starting a new department. These administrators have helped resolve conflicts between the fledgling department and other specialty departments.
However, such administrators have not supported the opening of community-based family medicine teaching clinics, as they do not appreciate the need. The interviewed faculty also feel that the high-level administrators do not advocate the distinguishing features and the objectives of family medicine to faculty. These pioneers continue to work in a hostile environment alongside faculty from other departments that lack an understanding of family medicine. One participant stated, "Current Japanese specialists make no sense, saying [they practice] primary care cardiology etc." Another noted, "Faculty members of other departments with five to ten years of experience verbally harass family medicine applicants/residents with comments, such as, 'Family Medicine? Training in ambulatory care? Nonsense! Out of the question! Quit that department!' "
Even some residents in family medicine-oriented departments "don't get it" as they may resist rotations that faculty view as core, such as pediatrics, OB/GYN, or behavioral science. Their reluctance highlights a gap in the residents' intellectual understanding of family medicine and their inability to grasp the value of rotating in other departments to see patients and problems needed to acquire appropriate knowledge and skills to be a family physician. This also hinders designing a core curriculum.
Lesson 2: Developing a core curriculum is difficult
These leaders find developing a core curriculum to be difficult for three reasons: 1) residents are laborers first, learners second; 2) disinterest of residents in certain core rotations; and 3) lack of a critical mass of residents to ensure coverage for other services.
Hospital-based specialties need a work force for clinical care in their departments. The lack of work hour restrictions in Japan emphasizes the view of residents as laborers. Residents commonly "rotate" among various clinical services that are hospital-based and demand a 24-hour per day commitment to patient care by the team. Even senior family medicine residents cannot leave other rotations and return for a half-day of clinic. One participant stated, "I can't ask other departments to teach knowledge and skills relevant to primary care because rotating residents are their work-force." Residents feel pressured not to leave their rotations in other departments saying, "I feel uncomfortable leaving the rotating department to come back for a half-day clinic."
As in Lesson 1, participating faculty note that residents are sometimes opposed to certain "core" family medicine rotations. Rather, residents request elective rotations that they think will be interesting. Faculty must take these requests seriously, since the number of residents in any given year can fluctuate widely depending on graduating medical student interest and the departments are competing for these graduates. This variation in interest makes negotiation of demands about content and return to clinic while on other specialty rotations services challenging. The family medicine programs need a critical mass of residents for core clinical services, but ideally could supply a consistent number of family medicine residents for other departments.
These conflicting demands make it difficult for most programs to have a standardized residency system. One participant stated, "We don't have a national matching system like in America. It is unpredictable how many students [will] enter the program next year. Maybe five, maybe zero. In such circumstances, we are unable to provide resident availability either in [our] home or in other departments. Therefore, it is next to impossible to establish a curriculum with core rotations and elective rotations. We can't help but approve elective rotations that are simply interesting to residents. We can't stop drop-out from the program."
Lesson 3: Start with undergraduates
Five participants indicated that it is too late to start exposure to family medicine at the resident level. Residents have pre-conceived notions about the content of clinical practice. One participant stated, "Some residents declare that they are going to practice internal medicine, so they do not need pediatrics, OB/GYN, or behavioral science etc. After they graduate, they send me a letter [saying] they now realize they lack training in these areas, ha ha ha." Another criticized, "If I could teach [students] the basic concepts and the value of family medicine in 10–15 hours at the end of medical school education, [after] four years of brain-washing by "specialists," family medicine could have an impact on medical students." Many of these family medicine leaders need and want to be more involved in undergraduate curriculum reform.
Lesson 4: Emphasize clinical skills
Currently, the medical school curriculum is not structured to effectively teach clinical skills. One participant stated, "Many Japanese students are unable even to measure blood pressure, to take a medical history, and to perform a physical examination at [the time of] graduation. They aren't competent because they were not taught. Our department proved it." Medical students are hungry for basic clinical skills, such as medical interviewing and performing the physical examination; and family physicians excel at these skills. The feedback from students in programs where these skills are emphasized is uniformly positive.
As individuals, faculty members of medical school and training hospitals sometimes lack effective teaching skills. One participant stated, "They don't know the principles of adult education, nor how to teach clinical medicine. Their belief is that knowledge and skills are 'stolen' from senior residents or attending physicians, not taught." Another lamented, "There are few faculty development curricula in Japan." It is an area needing development in Japanese family medicine.
Lesson 5: Train in the community
Collectively, these faculty felt training in community-based outpatient sites has been most effective. One participant stated, "Our department started in the early 1980's. The first seven years were chaotic. There were a lot of conflicts and confusion within the department. After I took over and oriented [the department] towards family medicine in '89 most things went well, especially after opening a model office in the community." Another participant stated, "I trained residents in the community since the beginning."
University hospital-based sites have been less effective due to the poor relationship with other departments and the difficulty of being a "role model" in the tertiary care center. Specific reasons for the latter include selection bias of the patients and poor continuity of care. A faculty member states, "There is no way to solve the problem of continuity other than going to the community."
Discussion
These Japanese leaders' experiences echo many of the stories heard in the history halls of family medicine departments and residency programs in other countries with more established family medicine training. Institutional structure, politics, and national policy have a significant impact on the establishment and growth of family medicine in the culture of academic medicine in Japan. While previous literature identifies resident perspectives,[13] these lessons from faculty provide insights into issues that will likely be encountered in starting family medicine programs in, and outside, of Japan as well. While their relative importance may be different, it is likely that the issues identified here will have some universality in terms of developing a family medicine program anywhere.
Efforts to educate others about the values and content of family medicine are strongly needed. Other departments may fail to understand family medicine since it is a horizontal specialty which cuts across the lines of existing specialties [14]. Moreover, it is defined more by a value system rather than a unique body of knowledge [15]. Harvey pointed out in 1985, "There are deep philosophical differences between the traditional clinical departments and divisions... They do not understand the emphasis on the family, the educational principles, and many, many other matters of importance to family medicine programs." [16]. This may help explain the superficial support of high-level administrators.
Fledgling departments of Family Medicine around the world must make their residency training goals clear to partnering departments and their residents. Residents are a work force. Too often, training is focused on inpatient care and taught by "specialists" in tertiary care centers or large training hospitals without an understanding of the purpose of the rotation for the resident. The faculty members and senior residents in other specialty departments do not know "what" and "how" to teach family medicine residents [17,18]. As indicated by Doherty, the timing, and duration of exposure to specialists merits careful attention [19]. He states, "...most family medicine experiences in outpatient and inpatient care tend to be postponed until after internship, thereby complicating the "imprinting" experiences of young physicians in training. Thus the delicate socialization of the family physician toward a role that is neither organ-system nor technology-specific is largely in the hands of non-family physicians, who by definition cannot model the unique identity of the family physician. Next generations of family practice residents suffer from the same identity confusion as its elders (p. xiv)."[19]
A balance between hospital-based and ambulatory-based training is needed. University or tertiary care center training is important for nurturing logical approaches to post-graduate education, research, and unsolved problems [17]. On the other hand, busy practitioners have broad practical knowledge and experience in treating patients. Both offer benefits to family medicine students and residents. Training programs need to, and can develop bridges with community-based physicians [18].
Critically important to family medicine teaching is a model family medicine office as a clinical classroom for training the resident and fostering her/his identity as a family physician [20]. Japanese pioneers face significant financial and political barriers including opposition from the Japan Medical Association and ambiguous support from the government [20]. While it is possible to open a resident teaching site at the beginning of the residency program, [19] a university-affiliated clinic may be perceived as a threat to practitioners and face stiff opposition from the local medical society. Suffice it to say that the specific political barriers are likely to differ from country to country and locale to locale, but pioneers of new programs will most assuredly encounter political barriers.
Based on the experiences of these participants, future family medicine program developers can anticipate benefits from early exposure of family medicine to medical students in the undergraduate curriculum, and emphasizing teaching skills that their students, as aspiring physicians, are driven to learn. Strongly tied to this is the need to focus on faculty development.
A growing literature illustrates the relevance of complexity science to understand family practice offices as complex adaptive systems [21,22]. As would be predicted by complexity science, organizational change will be difficult given the interactions and interdependencies that exist and must evolve for new departments to grow [23-26]. For example, newly developing departments of family medicine must be imbedded within the overall hospital and university systems and will require their "buy-in" [23]. The "five lessons" here might be considered "minimum specifications"[24] for guiding development of new family medicine departments with an expectation that there would be "wide space for innovation and shared action" in each new site. Research by Anderson and colleagues provide just one illustration of how the application of complexity science can enhance care quality [27]. We believe pioneers of family medicine departments would be wise to apply the conceptual aspects of complexity science to their specific situations as a framework for organizing development efforts, and perhaps even into their teaching approaches [28].
The potential limitations of this research are selection bias and a small sample size. However, the sample is close to the population of eligible family medicine pioneers. Only one woman participated, although this level of representation is close to the population of female physicians (about 14 %) in Japan [3]. Our data collection procedure was primarily limited to individual interviews. In future research, using multiple data collection techniques[22,29,30] could facilitate through triangulation a more detailed examination of successful programs, and provide further insight into developing family medicine residency training programs. In addition, a case comparison between Japan and Korea, an Asian country that has embraced family medicine, could further illustrate the importance of social, cultural, economic, and political differences that influence development of family medicine as a discipline. Finally, additional reports about experiences from other countries and contexts could extend the dialogue about issues salient to developing family medicine training programs.
Conclusion
While organizational change is difficult, the identified lessons suggest issues that merit consideration when developing a family medicine training program. We conclude that developing family medicine departments should: make residency training goals clear to their residents and partnering departments; balance hospital-based and community-based training; develop a model family medicine office as a clinical classroom; and actively participate in medical student education. Lessons from complexity science could inform application of these insights in other countries and settings newly developing residency training.
Abbreviations
JAFM-Japanese Academy of Family Medicine
MHLW-Ministry of Health, Labour, and Welfare
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MM conceived the study, participated in the research design, conducting the interviews and transcribing them, the primary qualitative analysis, and manuscript writing. KK participated in the primary qualitative analysis and manuscript writing. MF participated in the design, coordination, confirmatory qualitative analysis, writing and revision of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to thank all the participants who graciously spent time describing their experiences. We would also like to thank Ken Yokosawa and Heather Kirkpatrick for advice on the project. Kumiko Murai assisted with transcription. We appreciate the insightful suggestions of the reviewers.
==== Refs
Ikegami N Campbell JC (eds) Containing Health Care Costs in Japan 1996 Ann Arbor, MI: University of Michigan
Ikegami N Campbell JC Medical care in Japan N Engl J Med 1995 333 1295 1299 7566019 10.1056/NEJM199511093331922
Health and Welfare Statistics Association Health care professionals Journal of Health and Welfare Statistics 2000 Tokyo, Japan: Health and Welfare Statistics Association in Japan 175 188
Ishibashi Y Why is family medicine needed in Japan J Fam Pract 1987 25 83 86 3598483
Rodnick JE Werdegar D Impressions of health care and medical education in Japan Fam Med 1985 17 166 169 3870709
Smith BW Demers R Garcia-Shelton L Family medicine in Japan Arch Fam Med 1997 6 59 62 9003172 10.1001/archfami.6.1.59
Otaki J Considering primary care in Japan Acad Med 1998 73 662 668 9653405
Kamegai M Waga kuni no primary care ni hatasu "sougoushinryo" no yakuwari (The role of general medicine in primary care in Japan) Shinryo Kenkyu 2000 357 5 15
Crabtree BF Miller WL A qualitative approach to primary care research: The long interview Fam Med 1991 23 145 151 2037216
Creswell JW Benson AC Analyzing and Interpreting Qualitative Data Educational Research: Planning, Conducting, and Evaluating Quantitative and Qualitative Research 2005 2 Upper Saddle River, NJ: Pearson Merrill Prentice Hall 256 283
Crabtree BF Miller WL Doing Qualitative Research 1999 2 Newbury Park, CA: Sage Publications, Inc
Miller WL Crabtree BF Denzin NK, Lincoln YS Clinical Research Handbook of Qualitative Research 2005 3 Thousand Oaks, CA: Sage Publications, Inc
Fetters MD Shiraishi Y Nichibei tougi no kansou (Impressions of the Japan-United States resident training forum of the 15th annual primary care conference) Jpn J Prim Care 1992 15 46 48
Stephens G Doherty WJ, Christianson CE, Sussman MB Developmental assessment of family practice: An insider's view Family Medicine: The Maturing of a Discipline 1987 New York, NY: Haworth Press 1 21
Harries DL Jarvis JQ Doherty WJ, Christianson CE, Sussman MB Family medicine and the predoctoral medical curriculum Family Medicine: The Maturing of a Discipline 1987 New York, NY: Haworth Press 91 107
Colwill JM Doherty WJ, Christianson CE, Sussman MB Family medicine in the medical school Family Medicine: The Maturing of a Discipline 1987 New York, NY: Haworth Press 71 90
Fetters MD Kitamura K Eto K Yamashita K Fujioka T Kaigyoui to daigaku sougou shinryoubu to kakehashi - Ikanishite kyouryoku kankei wo kizuiteikuka (Building bridges between general practitioners and university department of general medicine) Jpn J Prim Care 2001 24 285 291
Kassai R Exploring the value of the first family medicine residency in Hokkaido, Japan Jpn J Prim Care 2000 23 224 233
Doherty WJ Christianson CE Sussman MB Doherty WJ, Christianson CE, Sussman MB Family Medicine: The Maturing of a Discipline 1987 New York, NY: Haworth Press xi xviii
Fetters MD Jimbo M Waku kara hazureta kangae no susume: Naze dokuritsushita katei-iryou-gata shinryoujo ga hitsuyouka? (Think outside the box: Why do we need an independent Family Practice Center?) Japanese Journal of Family Practice 2005
Miller WL McDaniel RR JrCrabtree BF Stange KC Practice jazz: Understanding variation in family practices using complexity science J Fam Pract 2001 50 872 878 11674890
Crabtree BF Miller WL Stange KC Understanding practice from the ground up J Fam Pract 2001 50 881 887 11674891
Plsek PE Greenhalgh T Complexity science: The challenge of complexity in health care Br Med J 2001 323 625 628 11557716
Plsek PE Wilson T Complexity, leadership, and management in healthcare organisations BMJ 2001 324 171 172
McDaniel RR JrJordan ME Fleeman BF Surprise, Surprise, Surprise! A complexity science view of the unexpected Health Care Manage Rev 2003 28 266 278 12940348
Anderson RA Crabtree BF Steele DJ McDaniel RR Jr Case study research: the view from complexity science Qual Health Res 2005 15 669 685 15802542 10.1177/1049732305275208
Anderson RA Corazzini KN McDaniel RR Jr Complexity science and the dynamics of climate and communication: reducing nursing home turnover Gerontologist 2004 44 378 388 15197292
Fraser SW Greenhalgh T Coping with complexity: educating for capability BMJ 2001 323 799 803 11588088 10.1136/bmj.323.7316.799
Creswell JW Fetters MD Ivankova NV Designing a mixed methods study in primary care Ann Fam Med 2004 2 7 12 15053277 10.1370/afm.104
Creswell JW Plano Clark VL Gutmann ML Hanson WE Tashakkori A, Teddlie C Advanced Mixed Methods Research Designs Handbook of Mixed Methods in Social and Behavioral Research 2002 Thousand Oaks, CA: Sage Publications 209 240
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-521618149210.1186/1471-2180-5-52Research ArticleA Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results Rampersad Joanne [email protected] David [email protected] Department of Life Sciences, The University of the West Indies, St. Augustine, Trinidad, Trinidad and Tobago2 The School of Veterinary Medicine, The University of the West Indies, Mt. Hope, Trinidad, Trinidad and Tobago2005 24 9 2005 5 52 52 20 6 2005 24 9 2005 Copyright © 2005 Rampersad and Ammons; 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
Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy.
Results
Based on SDS-PAGE patterns and the presence of cry genes or a crystal, 79 candidate, non-clonal isolates of B. thuringiensis were identified from 84 samples and over 10,000 colonies. Although only 16/79 (20%) of the isolates showed DNA homology by Probe Hybridization or PCR to common cry genes, initial characterization revealed a surprisingly rich library that included a putative nematocidal gene, a novel filamentous structure associated with a crystal, a spore with spikes originating from a very small parasporal body and isolates with unusually small crystals. When compared to reports of other screens, this screen was also atypical in that only 3/79 isolates (3.8%) produced a bipyramidal crystal and 24/79 (30%) of the isolates' spores possessed an attached, dark-staining body.
Conclusion
Results suggest that the screening methodology adopted in this study might deliver a vastly richer and potentially more useful library of B. thuringiensis isolates as compared to that obtained with commonly reported methodologies, and that by extension, methodologies fundamentally different from current methods should also be explored.
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Background
Bacillus thuringiensis is a bacterium known for producing protein crystals with pesticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. Although any particular toxin has a desirably restricted host range, there is a large number of different toxins, each showing toxicity to one of many diverse pests. For these reasons there is currently great interest in isolating novel strains of B. thuringiensis with either unique host specificity or elevated toxicity. There are numerous reports on attempts to isolate novel B. thuringiensis strains from the environment [1,2]. In many of these, some form of enrichment step is used in conjunction with Phase Contrast Microscopy as the backbone of the screening strategy [3,4]. The results of these screens have a curiously similar result where a large proportion of the isolates (33.5% to 98%) contain a cry1 gene or a bipyramidal shaped crystal (known for toxicity against lepidopteran pests), [1,5,6] and relatively few, if any, novel or uniquely useful isolates are found. Herein we report on a screen for environmental isolates of B. thuringiensis on the Caribbean island of Trinidad, where a novel stain was used as an alternative to Phase Contrast Microscopy in a high-throughput method coupled with reduced selection.
Results
Isolation of native strains
The use of a gridded-slide and a stain allowed a quick and high throughput assessment of 10,349 colonies from 84 samples. The preparation method was relatively quick, requiring approximately 15 min to grid a slide of 60 colonies and 3–5 s to evaluate each stained specimen on the grid. Evaluation could be done in a continuous motion without stopping to view each specimen. The stain also offered improved resolution over Phase Contrast, allowing the visualization of very small parasporal bodies [7]. A few of the crystals did not take up the stain well, however the contrast between spores and crystals was sufficient to easily differentiate the two, even when crystal morphology mimicked that of spores (Figure 1). The spores of 24/79 (30%) isolates were characterized as containing a dark-staining body which appeared as a "cap" on the spore (Figure 2) and which persisted after sporulation. Some of the caps may have contained crystals since oval or amorphous, phase dark objects with a light center, characteristic of crystals, were observed in 16/24 capped isolates by Phase Contrast Microscopy, and SDS-PAGE analysis showed protein bands for most of the capped isolates (data not shown).
Classifying isolates as clonal (siblings) if they were from the same sample and had the same crystal or cap morphology, 79 non-clonal isolates were collected comprising mostly round or amorphous crystals with 6 rectangular and 3 bipyramidal morphologies. Some samples were found to have multiple candidates of B. thuringiensis, either of the same or different parasporal body morphology.
Two isolates (Bt1-88 and Bt2-56), showed appendages emanating from parasporal bodies. Bt1-88 was characterized generally as having multiple, perhaps three, long spikes emanating from a small spore-associated parasporal body (Figure 3). SDS-PAGE analysis of Bt1-88 revealed two pronounced low molecular weight proteins of approximately 22 and 23 kDa (data not shown). Isolate Bt2-56 was found by electron microscopy to contain a multi-fiber filament emanating from a parasporal body located within a sack-like structure presumed to be the exosporium (Figure 4). A preliminary report on this isolate has been presented elsewhere, [8].
Three samples produced isolates with relatively small parasporal bodies, Bt1-26, Bt1-40 and Bt1-90 (Figure 5). Bt1-26 was isolated from sand taken a few inches above a high tide mark on a beach, Bt1-90 from a fresh manure pile (calf, sheep and goat), and Bt1-40 from composting manure collected from a barn housing sheep, goats, cattle, horses and pigs. SDS-PAGE analysis showed major protein bands of a relatively low molecular weight, between 11–19 kDa for each of these isolates (data not shown).
Two isolates (Bt1-33 and Bt1-35) showed sequence homology to a nematocidal gene (See "cry gene profile" below). Bt1-33 showed no crystals and, among hundreds of spores within the microscopic field of vision, only a few possessed a blue-staining cap on one end of the spore. After repeated culture on media, the relative number of caps appeared to increase slightly (Figure 6A), which most probably would not have been detected by Phase Contrast Microscopy (Figure 6B). Crystals were not initially observed with either Phase Contrast Microscopy or stained specimens. However when specimens were heated strongly on the microscope slide, some specimens prepared in this manner showed oval crystals lying just lateral to the terminal end of the spore (Figure 6C). Interestingly, both Bt1-33 and Bt1-35 came from the same sample, an area of decomposing animal manure. Bt1-35 demonstrated a very small cap, however major protein bands were not observed on SDS gel analysis.
cry-cyt gene profile
Probe and PCR analysis indicated that of the 79 non-clonal isolates, only 16 showed homology to any one of the major groups of cry genes tested (Table 1). cry and cyt genes were not detected in the capped isolates by either PCR or Probe Hybridization, except for Bt2-14, which was positive by PCR for a cry4 and cyt2 gene (Table 1). PCR amplification of Bt1-33 with primers showing homology to known nematocidal genes produced an amplicon of the expected size which, when sequenced, showed strong homology to a known nematocidal gene (GenBank accession number U13955, cry14Aa1) (Figure 7). However, Bt1-33 contains a mutation causing a frame shift, creating a stop codon. The amplified fragment from Bt1-33 was also used to probe the other B. thuringiensis candidates, only one of which, Bt1-35, also gave a weak but unquestionable hybridization signal (data not shown).
Bioassay (quick screen)
A "quick screen" using relatively large amounts of sporulated colonies was used to ascertain if any of the isolates demonstrated appreciable activity against the 5th instar larvae of agricultural pest, Spodoptera frugiperda. For most isolates, leaf pieces were completely eaten and given a score of 1.0 (non-toxic). Only one isolate and three control strains achieved a score greater than 2 and were considered toxic, (Bt2-46, with bipyramidal morphology and a score of 2.7; BGSC4A3, B. thuringiensis serovar thuringiensis HD2, Score 2.0; BGSC4D1, B. thuringiensis serovar kurstaki HD1, Score 2.0 and BGSC4J3, B. thuringiensis serovar aizawai HD133, Score 2.7).
Discussion
With few exceptions [9,10] the crystal and spore of B. thuringiensis are released separately during sporulation. It was thus surprising that 24/79 non-clonal, candidate B. thuringiensis isolates possessed spores with a "capped" appearance. Some of these caps appeared to contain crystals since phase dark objects with a light center could be seen in many of the caps, including Bt2-56, which was subsequently confirmed by Electron Microscopy to possess a parasporal body within the exosporium (Figure 4). The functional significance of spore-associated crystals is not totally understood however benefits such as protection against UV degradation and better access to the target organism have been attributed to crystals attached within an exosporium [11]. If this is true, and the caps are shown to contain useful toxins, then those isolates showing the capped morphology may eventually prove useful in field applications. Clearly, more attention needs to be given to this phenotype and its mechanism of expression. For example, if the phenotype is plasmid encoded, could strains that undesirably liberate crystals be transformed to enclose their crystals within a protective cap?
Examples of filamentous appendages associated with bacterial spores are relatively rare where only a few isolates of Clostridium and Bacillus are reported to possess spores with appendages [12,13]. Even rarer is the association of an appendage with a parasporal body, as was observed with both Bt1-88 and Bt2-56. Very little is presently known about Bt1-88 and studies are underway to characterize both the strain and the ultra structure of the appendage-associated complex. From preliminary studies undertaken with Bt2-56, this isolate clearly demonstrates an intimate relationship between the filament and the parasporal body where the parasporal body appears to help anchor the filament to the spore (Figure 4 and [8]). Both these isolates raise a number of questions fundamental to our basic understanding of bacteria and the role of spore associated appendages. Among these is the obvious question of a possible relationship between the appendage and B. thuringiensis crystals, i.e., did some crystals evolve to act as an anchoring structure for spore associated filaments and if so, do they still retain any toxin activity? Similarly, what is the role of the filament and does it aid in pathogenicity?
Sequence data indicated that a gene in Bt1-33 is highly homologous to the cry14Aa1 gene, [GenBank: U13955] which has demonstrated activity against nematodes [14]. It was surprising that this relatively small study would produce an isolate containing a putative nematocidal gene when a much larger study utilizing the same primers did not [15]. However the gene in Bt1-33 is probably non-functional due to a frame-shift mutation, raising the question of why would it be maintained by the bacterium? One explanation could be that the gene is clustered in the genome with other genes necessary for pathogenicity as part of a pathogenicity island, and is being maintained in the genome indirectly through selection for the other genes in the group. Interestingly, PS80JJ1 (the strain gene U13955 is found in), has been shown to contain at least two other δ-endotoxin proteins Cry34Aa1 and Cry35Aa1, which together form a binary toxin shown to be active against Diabrotica virgifera (western corn rootworm). The corn root worm, like the nematode, is a soil-dwelling organism that B. thuringiensis are rarely active against. It is perhaps significant that U13955 is associated with other toxins that target a soil-dwelling organism, raising the question of whether PS80JJ1 is adapted against soil-dwelling organisms. If so, then perhaps like PS80JJ1, Bt1-33 may also possess other similarly rare toxin genes that target soil-dwelling organisms.
Using the PCR amplified fragment from the cry14Aa1-like gene in Bt1-33 as a probe, another isolate, Bt1-35, showed a weaker but definite hybridization signal. Perhaps significant is that it was isolated from the same sample as Bt1-33. The sequence of this gene is presently unknown however the reduced hybridization signal shown suggests that it may be an evolutionary relative or even share limited sequence, such as a functional domain.
The isolation method used in this study diverged from many of the reported methods for B. thuringiensis isolation by utilizing a stain instead of Phase Contrast Microscopy (which is commonly used when screening for B. thuringiensis), for the identification of crystals [1,16,17]. Although Phase Contrast Microscopy was useful for observing inside spore caps, the stain uniquely allowed a fast, high throughput evaluation of bacterial colonies for the presence of crystals. Similarly, the high contrast of the stain allowed it to identify relatively small crystals or low numbers of caps in a specimen. It is doubtful that without these benefits of the stain that Bt1-88, Bt1-33/35 and the relatively small crystal producers would have been isolated. This study also sought to reduce the chance of excluding some isolates by not incorporating overt selection during the isolation of candidate bacteria (e.g., not using antibiotics in the culture medium). The importance of reducing selective pressures for the successful isolation of each strain was not determined. However, at least one of the important isolates, Bt1-33, was isolated using Method G, a selective method. Thus it is possible that selective enrichment coupled with the stain and high throughput screening could have resulted in the isolation of higher numbers of B. thuringiensis candidates than were obtained in this study. Similarly, the isolation of capped isolates, including Bt2-56, could not be directly attributed to the use of the stain or even the general isolation methodology used. For example, although not as evident as with the use of the stain, Phase Contrast Microscopy could be used to visualize the capped spores. Thus it is possible that the high level of capped spores observed is best attributable to their high or unique presence in the Trinidadian environment. For example, it is difficult to imagine how the cap-associated filament of Bt2-56 has not previously been identified elsewhere, when in this screen it was isolated from several specimens collected throughout Trinidad. However, the filament was not easily observed under Phase Contrast Microscopy without some special attention being given to finding it. For example, the filament was not observed immediately with Phase Contrast Microscopy and it was only by chance that, as a capped spore, Bt2-56 was chosen for more intense study and its filament found. Similarly, the spiked structures associated with Bt1-88 were not observed on primary isolation or initial examination with Phase Contrast Microscopy, and it was only through a re-screening of all capped spores for filaments similar to Bt2-56 that it was observed. These data would suggest that some capped spores contained in existing B. thuringiensis libraries might also possess appendages and should be re-examined specifically for their presence.
Perhaps most striking about this screen was the low percentage of isolates producing bipyramidal crystals. In literally all reported screens for environmental isolates of B. thuringiensis, the percentage of bipyramidal crystals is usually above 40% [18,1], however only 3 isolates or 3.8 % of the non-clonal isolates where shown to have a bipyramidal morphology indicating a screen that deviated significantly from the norm.
Conclusion
Results suggest that there are advantages to using a stain over the commonly used Phase Contrast Microscopy, particularly in the detection of small crystals, low numbers of crystals/capped spores and for its applicability for use in the fast, high throughput detection of B. thuringiensis candidates from the environment. There is a continuing need to explore the benefits of new methodologies for the detection of B. thuringiensis from the environment.
Methods
Sample collection and preparation
Samples were randomly collected from environmentally diverse sources (including beach sand, forest soil, aquatic and intertidal sediments, and soils from urban, rural and agricultural areas), placed in a plastic bag and held at room temperature until processing. Seven different methods (A-G) were initially used for sample preparation (Table 2), where Method F was used predominately (Table 3).
B. thuringiensis isolation
Samples (0.25 g) were prepared using one or more of Methods A-G (Table 2), then 200 μl and 20 μl were cultured on Sporulation plates (Nutrient Agar with CCY salts [19] to which antibiotics were added as indicated in Table 2) at 30°C for 2 days to allow sporulation. Using a dissecting microscope and straight wire, an individual colony that conformed to the 'Bacillus cereus group' (i.e. white to off-white with a matt appearance and irregular edges) was spotted onto a microscope slide that was pre-gridded with a wax pencil into 60 rectangles, and then stored by stabbing a grid on a nutrient agar plate. The agar plate was placed at 4°C to minimize germination of the spores. Where more than one isolate from the same sample was observed possessing the same crystal morphology, they were considered clonal and not all such isolates were kept. The slide was heat fixed and stained with Coomassie Brilliant Blue [7] and viewed under Brightfield Microscopy using a 100X immersion oil objective. Cell morphology was noted for all candidates of B. thuringiensis, which was defined as a large spore-forming rod whose ellipsoidal spore did not swell the mother cell and which produced a parasporal body upon sporulation [20,21].
SDS protein electrophoresis
Two loopfuls of bacterial growth were removed from a Sporulation plate to 50 μl of ice cold Protein Sample Buffer [22], boiled for 2 min then frozen until use. Denaturing SDS gel electrophoresis was performed on protein samples according to the method of Lamelli [23]. The relative mobility for each band was calculated by applying the equation RH = A-C/A-B where A was the position of the top of the gel, B was the position of the top of the dye front and C was the position of the top of each band of interest. Relative mobility vs the molecular weights of known proteins (116 kDa, 92 kDa, 66 kDa, 45 kDa, 21 kDa) was plotted and linear regression performed to determine the equation for the line of best fit, which was used to determine the Molecular weights (KDa) of unknown bands.
PCR of toxin genes
DNA template was prepared by lysis precipitation wherein approximately half a loopfull of cells from a sporulated plate was placed into 2 mls of L- Broth, incubated at 60°C for 10 min, then at 30°C with shaking at 250 rpm for approximately 5 h. Harvested cells were washed in water, resuspended in 200 μl of fresh lysing solution (Sodium duodecyl sulphate 3.3%; Tris 0.05 M, pH 12.5), incubated for 30 mins at 60°C and vortexed for 10 s to shear the DNA. DNA was collected by ethanol precipitation [24] and resuspended in 60 μl of 0.1 × TE buffer pH8.0. One microliter was used as template in PCR reactions. Cry and Cyt groups targeted, protocols and control strains utilized are given in Table 4. The ability of template preparations to be amplified in a PCR reaction was assessed using Bacillus 16S ribosomal RNA primers (Table 4), at 94°C for 15 sec; 55°C for 30 sec and 72°C for 2 min for 28 cycles with 1.5 mM MgCl.
Hybridization of cry genes
Probe hybridization was performed using the DIG High Prime DNA Labeling and Detection Starter Kit (Roche Molecular Biochemicals, Cat# 1 745 832). cry and cyt gene probes were made by PCR (as described above) using the following bacterial strains from the Bacillus Genetic Stock Center (BGSC) as templates: cry1- BGSC4J3; cry2- BGSC4J3; cry3- BGSC4AA1; cry4- BGSC4Q1; cry5- Bt1-33; cyt1- BGSC4Q1; cyt2- BGSC4Q1; 16S- BGSC4J3
DNA sequencing
An ABI Cycle Sequencing Big Dye Ready Reaction kit (Catalogue # 4303500) was used according to the manufacturer's instructions and reactions resolved on an ABI 377 DNA Sequencing Machine. Primers gral-nem(d) and gral-nem(r) [15] were used to amplify and sequence the putative nematocidal gene in Bt1-33.
Bioassay (quick screen)
Larvae of Spodoptera frugiperda were collected from a corn field and used to generate up to 5 generations of larvae. The width of the head capsule was used to select 5th instar larvae for use in the bioassay [25]. Larvae were starved for approximately 1 hour immediately prior to being challenged with one loopful of a bacterial isolate collected from a sporulated colony and smeared onto a 1 cm2 leaf piece (Sorghum halipense) in triplicate. Larvae were left for approximately 10 h then presented with a fresh piece of uninoculated leaf then left for a further 10–12 h. Scoring was: not eaten a score of 3; partially eaten a score of 2; completely eaten a score of 1. The toxicity value of a B. thuringiensis candidate was the average of the scores for the three trials, which were arbitrarily classified as: 1.0- not toxic; > 1.0 but < 2.0 – uncertain toxicity; = 2.0- toxic. All isolates in the library were tested, irrespective of whether they were considered clonal strains, as well as the five control strains obtained from the Bacillus Genetic Stock Center.
Authors' contributions
JR was principally responsible for the design and execution of the project and data analysis. DA contributed to project design and data analysis. Both authors read and approved the final manuscript.
Acknowledgements
We would like to thank the Caribbean Development Bank and the University of the West Indies Campus and Publication Committee for their financial support of this project. We would like to thank Dan Ziegler and the Bacillus Genetic Stock Center for control strains.
Figures and Tables
Figure 1 Poorly staining crystals. Stained (a) and Phase Contrast (b) micrographs of Bt1-32 showing spores (double arrow) and relatively lightly staining crystals (single arrow).
Figure 2 Examples of the "capped" spore morphology. Dark blue "caps" attached to lighter staining spores. A) Bt2-2, B) Bt1-48, C) Bt2-56, D) Bt1-46 and E) Bt2-14.
Figure 3 Phase contrast micrograph showing spiked appendages emanating from the spores of Bt1-88. Long straight spikes (a) emanating from a small, spore-associated parasporal body (b).
Figure 4 Electron Micrograph of Bt2-56. A Transmission Electron Micrograph of negatively stained spores from Bt2-56 containing a filament (a), and a sac-like structure containing a spore (b) and parasporal body (c).
Figure 5 Relatively small parasporal bodies. Stained Brightfield micrographs (100X) of Bt1-22 (a), Bt1-40 (b) and Bt1-90 (c).
Figure 6 Phase Contrast Microscopy of sporulated cells of Bt1-33. A) stained preparation of Bt1-33 viewed under Brightfield microscopy, 100x oil immersion, B) Bt1-33 viewed under Phase Contrast microscopy, 100x oil immersion, C) Heat-treated preparation of Bt1-33 showing phase-dark crystals lying slightly lateral and terminal to the spore, Phase Contrast microscopy, 100x oil immersion.
Figure 7 DNA sequence of PCR amplicon from Bt1-33. Comparison of the DNA sequence from Bt1-33 produced with the cry5 group primers and that of a known nematocidal gene, cry14Aa1 (GenBank accession number U13955). A "." is placed at those positions in Bt1-33 where the nucleotide is the same as that in U13955, and a letter denoting the nucleotide if it is not. Dashes "-" represent insertions required to align the sequences.
Table 1 Isolates positive for cry and cyt genes as determined by Probe Hybridization and PCR.
Sample number Isolate #
cry1
cry1 C cry1 D
cry2
cry 3,7,8
cry4
cry5/Nem
cyt1
cyt2
22 Bt1-33 H-P
22 Bt1-35 H
72 Bt2-46 H-P H-P
81 Bt2-48 P
N/A Bt2-50 H-P P P H-P
N/A Bt2-51 H H-P H
N/A Bt2-52 H-P P P H-P
N/A Bt2-53 H-P ***
N/A Bt2-54 H-P H-P H
N/A Bt2-55 H-P ***
40 Bt1-65 P H-P
58 Bt1-74 H
63 Bt1-83 H
11 Bt1-1 P
11 Bt1-4 P
11 Bt1-5 P
15 Bt1-6 P
41 Bt2-14 P P
41 Bt2-15 H-P
71 Bt2-21 H
77 Bt2-24 P
82 Bt2-35 P
(H) denotes probe hybridization and (P) PCR. Samples Bt2-(50–55) were control strains where bold type denotes genes that strains are reported to contain, and (***) designates genes that are reported to be present but were not detected.
Table 2 Summary of preparation methods A-G
Method Key points
A Sample heated for 15 min in Nutrient Broth at 65°C, then plated on Sporulation plates with 20 IU/ml penicillin G
B 4 hour Pre-incubation of sample in L-broth (30°C shaking at 250 rpm) then heated for 15 min at 65°C then plated on Sporulation plates
C Same as Method B but plated on Sporulation plates with 20 IU/ml penicillin G
D 4 hour Pre-incubation of sample in L-broth/acetate [26] (30°C shaking at 250 rpm) then heated for 15 min at 65°C then plated on Sporulation plates
E Same as Method D above except plated on Sporulation plates with 20 IU/ml penicillin G
F Sample heated for 15 min in L-broth at 65°C then plated on Sporulation plates
G Same as Method F above except plated on Sporulation plates with 20 IU/ml penicillin G
Table 3 Preparation methods and their use.
Preparation Method Number of samples Screened Number of Colonies Screened Number of Samples with B. thuringiensis Isolates
A 13 1,560 1
B 2 120 0
C 2 54 1
D 2 80 0
E 2 34 1
F 70 7,172 43
G 13 1,329 4
Total: 104 Total: 10,349 Total: 50
Table 4 Primers and probes utilized.
Target Gene Reference Control Strains
cry1 Family
Juarez-Perez et al., [27] BGSC 4J3
cry1C
Juarez-Perez et al., [27] BGSC4J3
cry1D
Juarez-Perez et al., [27] BGSC 4J3
cry2 Family
Masson et al., [28] BGSC 4J3
cry3, 7, 8
Family Ceron et al., [29] BGSC4AA1
cry4
Ben Dov et al., [6] BGSC4Q1
cry5, 12, 14, 21
Family Bravo et al., [15] none available
cyt1
Bravo et al., [15] BGSC 4Q1
Bacillus 16S rDNA Siefert et al., [30] Not Applicable
(H) denotes probe hybridization and (P) PCR. Samples Bt2-(50–55) were control strains where bold type denotes genes that strains are reported to contain, and (***) designates genes that are reported to be present but were not detected.
==== Refs
Bernhard K Jarrett P Meadows M Butt J Ellis DJ Roberts GM Pauli S Rodgers P Burges HG Natural isolates of Bacillus thuringiensis: World wide Distribution, Characterization, and Activity against Insect Pests J Invertebr Pathol 1997 70 59 68 10.1006/jipa.1997.4669
Vilas-Boas GT Lemos MVF Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil Can J Microbiol 2004 50 605 613 15467786 10.1139/w04-052
Martin PAW Travers RS Worldwide abundance and distribution of Bacillus thuringiensis isolates Appl Environ Microbiol 1989 55 2437 2442 16348022
Johnson C Bishop AH A technique for the effective enrichment and isolation of Bacillus thuringiensis FEMS Microbiology Letters 1996 142 173 177 10.1016/0378-1097(96)00261-3
Chak KF Chao DC Tseng MY Kao SS Tuan SJ Feng TY Determination and Distribution of cry-Type genes of Bacillus thuringiensis isolates from Taiwan Appl Environ Microbiol 1994 60 2415 2420 16349324
Ben Dov E Zaritsky A Dahan E Barak Z Sinai R Manasherob R Khamrev A Troitskaya E Dubitsky A Berezina N Margalith Y Extended Screening by PCR for seven cry group genes from field-collected strains of Bacillus thuringiensis Appl Environ Microbiol 1997 63 4883 4890 9406409
Rampersad J Khan A Ammons D Usefulness of Staining Parasporal Bodies when Screening for Bacillus thuringiensis J Invertebr Pathol 2002 79 203 4 12133711 10.1016/S0022-2011(02)00018-6
Rampersad J Khan A Ammons D A Bacillus thuringiensis isolate possessing a spore-associated filament Curr Microbiol 2003 47 355 357 14629020 10.1007/s00284-003-4103-8
Ohba M Aizawa K Serological identification of Bacillus thuringiensis and Related Bacteria isolated in Japan J Invertebr Pathol 1978 32 303 309 10.1016/0022-2011(78)90193-3
Hastowo S Lay BW Ohba M Naturally occurring Bacillus thuringiensis in Indonesia J Appl Bacterio 1992 73 108 113
Aronson A Sporulation and delta-endotoxin synthesis in Bacillus thuringiensis Cell Mol Life Sci 2002 59 417 425 11964120 10.1007/s00018-002-8434-6
Hodgkiss W Barker AN, Gould GW, Wolf J Filamentous appendages on the spores and exosporium of certain Bacillus species Spore Research 1971 Academic Press, London and New York 211 218
Mizuki E Ohba M Ichimatsu T Hwang SH Higuchi K Saitoh H Akao T Unique apppendages associated with spores of Bacillus cereus isolates J Basic Microbiol 1998 38 33 39 9542106 10.1002/(SICI)1521-4028(199803)38:1<33::AID-JOBM33>3.0.CO;2-X
Wei JZ Hale K Carta L Platzer E Wong C Fang SC Aroian RV Bacillus thuringiensis crystal proteins that target nematodes Proc Natl Acad Sci USA 2003 100 2760 5 12598644 10.1073/pnas.0538072100
Bravo A Sarabia S Lopez L Ontiveros H Abarca C Ortiz A Ortiz M Lina L Villalobos FJ Pena G Nunez-Valdez M Soberon M Quintero R Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection Appl Environ Microbiol 1998 64 4965 4972 9835590
Ohba M Aizawa K Distribution of Bacillus thuringiensis in Soils of Japan J Invertebr Pathol 1986 47 277 282 10.1016/0022-2011(86)90097-2
Bel Y Granero F Alberola TM Martinez-Sebastian MJ Ferre J Distribution, frequency and diversity of Bacillus thuringiensis in olive tree environments in Spain System Appl Microbiol 1997 20 652 658
Meadows MP Ellis DJ Butt J Jarrett P Burges HD Distribution, Frequency and diversity of Bacillus thuringiensis in an Animal Feed Mill Appl Environ Microbiol 1992 58 1344 1350 16348699
Stewart GSAB Johnstone K Hagelberg E Ellar DJ Commitment of bacterial spores to germinate Biochem J 1981 198 101 106 6798972
DeLucca AJ Simonson JG Larson AD Bacillus thuringiensis distribution in soils of the United States Can J Microbiol 1981 27 865 870 7306875
Smith RA Couche GA The phylloplane as a source of Bacillus thuringiensis variants Appl Environ Microbiol 1991 57 311 315 16348400
Kaelin P Morel P Gadani F Isolation of Bacillus thuringiensis from stored tobacco and Lasioderma serricorne (F) Appl Environ Microbiol 1994 60 19 25 16349149
Lamelli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970 227 680 685 5432063 10.1038/227680a0
Sambrook J Fitsch EF Maniatis T Molecular Cloning: A Laboratory Manual 1989 Cold Spring Harbor, Cold Spring Harbor Press
Dyar HG The number of moults of lepidopterous larvae Psyche 1890 5 420 422
Travers RS Martin PAW Reichelderfer CF Selective Process for Efficient Isolation of Soil Bacillus spp Appl Environ Microbiol 1987 56 1263 1266
Juarez-Perez VM Ferrandis MD Frutos R PCR Based Approach for the Detection of novel Bacillus thuringiensis cry genes Appl Environ Microbiol 1997 63 2977 3002 9251185
Masson L Erlandson M Puzstai-Carey M Brousseau R Juarez-Perez V Frutos R A Holistic Approach for determining the Entomopathogenic potential of Bacillus thuringiensis strains Appl Environ Microbiol 1998 64 4782 4788 9835562
Ceron J Ortiz A Quintero R Guereca L Bravo A Specific PCR primers directed to identify cry I and cry III genes within a Bacillus thuringiensis strain collection Appl Environ Microbiol 1995 61 3826 3831 8526493
Siefert JL Larios-Sanz M Nakamura LK Slepecky RA Paul JH Moore ERB Fox GE Jurtshuk P Jr Phylogeny of Marine Bacillus Isolates from the Gulf of Mexico Curr Microbiol 2000 41 84 88 10856371 10.1007/s002840010098
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BMC NephrolBMC Nephrology1471-2369BioMed Central London 1471-2369-6-101617658710.1186/1471-2369-6-10Case ReportCocaine-induced renal infarction: report of a case and review of the literature Bemanian Shahrooz [email protected] Mazda [email protected] Saeid M [email protected] Chief Resident, Department of Medicine, University of Southern California, Keck School of Medicine, LAC-USC Medical Center, Los Angeles, USA2 Clinical Fellow, Department of Medicine, Division of Cardiovascular Medicine, University of Southern California, Keck School of Medicine, LAC-USC Medical Center, Los Angeles, USA3 Associate Professor of Clinical Medicine, Department of Medicine, Division of Nephrology, University of Southern California, Keck School of Medicine, LAC-USC Medical Center, Los Angeles, USA2005 22 9 2005 6 10 10 27 4 2005 22 9 2005 Copyright © 2005 Bemanian 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
Cocaine abuse has been known to have detrimental effects on the cardiovascular system. Its toxicity has been associated with myocardial ischemia, cerebrovascular accidents and mesenteric ischemia. The pathophysiology of cocaine-related renal injury is multifactorial and involves renal hemodynamic changes, alterations in glomerular matrix synthesis, degradation and oxidative stress, and possibly induction of renal atherogenesis. Renal infarction as a result of cocaine exposure, however, is rarely reported in the literature.
Case presentation
A 48 year-old male presented with a four-day history of severe right flank pain following cocaine use. On presentation, he was tachycardic, febrile and had severe right costovertebral angle tenderness. He had significant proteinuria, leukocytosis and elevated serum creatinine and lactate dehydrogenase. Radiographic imaging studies as well as other screening tests for thromboembolic events, hypercoagulability states, collagen vascular diseases and lipid disorders were suggestive of Cocaine-Induced Renal Infarction (CIRI) by exclusion.
Conclusion
In a patient with a history of cocaine abuse presenting with fevers and flank pain suggestive of urinary tract infection or nephrolithiasis, cocaine-induced renal infarction must be considered in the differential diagnosis. In this article, we discuss the prior reported cases of CIRI and thoroughly review the literature available on this disorder. This is important for several reasons. First, it will allow us to discuss and elaborate on the mechanism of renal injury caused by cocaine. In addition, this review will demonstrate the importance of considering the diagnosis of CIRI in a patient with documented cocaine use and an atypical presentation of acute renal injury. Finally, we will emphasize the need for a consensus on optimal treatment of this disease, for which therapy is not yet standardized.
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Background
Cocaine abuse has been known to have detrimental effects on the cardiovascular system. Its toxicity has been associated with myocardial ischemia, cerebrovascular accidents and mesenteric ischemia. The pathophysiology of cocaine-related renal injury is multifactorial and involves renal hemodynamic changes, alterations in glomerular matrix synthesis, degradation and oxidative stress, and possibly induction of renal atherogenesis. Renal infarction as a result of cocaine exposure, however, is rarely reported in the literature.
Case presentation
A 48 year-old African American male presented to our hospital with a four-day history of severe right flank pain starting several hours after smoking cocaine. His pain was associated with subjective fevers, nausea and vomiting. There was no gross hematuria or dysuria. His blood pressure was 140/78 mmHg, heart rate 130 beats per minute, temperature 39.5 °C, respiratory rate 18 breaths per minute and his oxygen saturation was 98% on room air. Pertinent exam findings included severe right costovertebral angle tenderness, diffuse right-sided abdominal pain and a positive psoas sign on the right. Laboratory data showed a white blood cell count of 14 × 109/L (76% neutrophils), serum creatinine 1.4 mg/dL (124 μmol/L), lactate dehydrogenase (LDH) 704 U/L and serum creatinine kinase (CPK) 259 U/L. A urinalysis showed 3+ proteinuria, 1 to 3 red blood cells per high power field and no white blood cells or casts. Urine toxicology confirmed the presence of cocaine. A Computerized Tomography (CT) scan with contrast revealed a sharply-demarcated heterogenous area of focal decreased enhancement in the anterior right kidney consistent with renal infarct, but pyelonephritis could not be completely ruled out (figure 1). Volume-rendered Single Photon Emission-Computed Tomography (SPECT) image of a gallium scan showed no evidence of tracer localization in the right kidney to suggest an infection. It did reveal absence of tracer localization in the upper pole of the right kidney suggesting absence of perfusion to that area (figure 2). Blood and urine cultures were negative. A trans-thoracic and a subsequent trans-esophageal echocardiogram showed no evidence of intra-cardiac thrombus or valvular vegetations. Further screening tests for hypercoagulability (factor V Leiden, prothrombin gene, protein C and protein S, anti-thrombin III, anti-phospholipid antibody and homocysteine); collagen vascular disease (rheumatoid factor and antinuclear antibody); and lipid disorders were within normal limits. Our patient was then diagnosed with CIRI by exclusion.
Discussion
Cocaine abuse and addiction continues to be a problem that plagues the entire world. Cocaine (benzoyl methylecgonine) is available in two forms: cocaine hydrochloride and the alkaloid cocaine (freebase/crack), which is made by alkanizing the salt, followed by extraction with non-polar solvents. Both forms are well absorbed through most mucous membranes [1]. The plasma half-life is approximately 45 to 90 minutes. The elimination of cocaine is predominantly controlled by its biotransformation since its renal clearance is only 27 mL/min [2]. The pathways for the biotransformation involve plasma and liver cholinesterases that produce benzoyl and ethyl methylecgonine which are water-soluble metabolites excreted in urine [3].
The pathophysiologic effects of cocaine-induced renal injury involve several mechanisms. First, cocaine affects vascular reactivity and renal hemodynamics. This is thought to be due to its ability to inhibit uptake of catecholamines in the synapse, inhibit re-uptake of norepinephrine in sympathetically innervated tissues and release norepinephrine and epinephrine from the adrenal medulla [4,5]. There's also evidence that cocaine can directly increase calcium influx in vascular smooth muscle [6,7]. This effect has been documented in post-sympathectomy arteries as well as in umbilical arteries, which lack sympathetic innervation [6,7]. Second, cocaine has been shown to affect matrix synthesis degradation and oxidative stress in kidney [8]. Third, cocaine has been shown to accelerate renal atherogenesis [9,10].
Although major toxic effects of cocaine such as myocardial ischemia, cerebrovascular accidents, mesenteric ischemia and placental infarcts have been well- documented in the literature, renal infarction as a complication of cocaine toxicity is rare. In this article, we will attempt to raise awareness of this under-reported entity by discussing its pathophysiology, clinical presentation and the current treatment modalities.
Common risk factors for renal infarction include valvular heart disease and atheroembolic events. Hypercoagulopathy, systemic vasculitis, blunt trauma, collagen vascular disorders, vascular intervention procedures and endocarditis are other less common risk factors associated with renal infarct.
Cocaine-induced renal damage is well-documented in the literature; however, renal infarction due to cocaine is not a well-known entity. Throughout our literature search, we identified seven other such reports (table 1) [11-17]. In one patient, a renal infarct was thought to be due to unilateral right renal artery thrombosis and embolization associated with intravenous cocaine injection [11]. Kramer et al. described a patient with a right renal thrombosis and infarction associated with protein C deficiency [12]. In another report, Antonovych et al. noted unusual renal involvement in two cocaine addicts [13]. One patient was a 26 year-old male who presented with malignant hypertension, who was found to have renal infarction. From this report, it is uncertain whether cocaine-induced malignant hypertension led to renal infarction or if cocaine-induced renal infarction presented as a case of malignant hypertension. The other patient was a 39 year-old male with recent cocaine use who had an emergent surgical nephrectomy for unclear reasons, and was found to have arterial and venous thrombosis with massive infarction. Two other cases describe right renal infarctions in patients with recent cocaine use in whom no other abnormalities were found [14,15]. Edmondson et al. described a 40-year-old male with renal artery dissection and thrombosis presumed to be due to cocaine which led to renal infarction [16]. Mochizuki et al. reported a 52-year-old female with recent intranasal use of cocaine who presented with acute aortic thrombosis associated with renal infarct [17].
It is not known whether there is a gender predilection for this disorder. Interestingly, all but one of the reported cases of CIRI has occurred in males. This occurrence may be due to an increased prevalence of cocaine use in males. It is also possible that males may be more genetically prone to this disorder. However, given the small number of reported cases, one cannot make any certain conclusions about genetic susceptibility.
In our review, we also noted that all but one of the reported cases of CIRI have involved the right kidney. It is generally accepted that the right renal artery is longer than the left due to its anatomical position in relation to the great vessels. Merklin et al. investigated the varied blood supply of the kidneys by dissecting 185 kidneys obtained from adult cadavers. The length of the right renal artery from the aortic origin to its division point was as long as 8.0 cm and that of the left varied from 0.5 to 6.0 cm [18]. The calibers of these arteries were similar in diameter, and their average diameter was 5.5 mm with variations from 4 to 7 mm [18]. One must keep in mind that neither p-values nor confidence intervals were described in this study.
Steady flow models for blood flow analysis in human circulation are very complex. The simplest model for blood flow through any vessel assumes a flow of a fluid with a constant viscosity through a non-tortuous and straight cylindrical tube of circular cross-section [19]. Such fluid flows are well-described by Poiseuille Equations in the honor of J. L. M. Poiseuille who performed many experiments relating pressure gradient, flow, tube geometry and length [19].
Poiseuille formulated the factors that affect flow of fluid in a tube as:
Q = (P1-P2) π r4/8ήL
(Q = flow rate; P1- P2 = pressure difference across a circuit; r = radius; ή = viscosity of fluid; L = length of vessel).
Given the inverse relationship of flow to resistance:
Resistance = flow -1
We can then conclude that resistance in a vessel is directly proportional to its length, and indirectly proportional to the radius to the power of four:
Resistance α Length/radius4
Since the radius of the right and left renal arteries are similar in size, we postulate that the right kidney is more prone to ischemia due to the increased resistance that it encounters by the longer length of its artery. Whether this is true or not, one must take into account the limited number of reported cases along with the lack of advanced studies to investigate the exact anatomy of the renal arteries in these particular patients.
The pathophysiology of cocaine-related renal injury is multifactorial and involves renal hemodynamic changes, alterations in glomerular matrix synthesis, degradation and oxidative stress, and possible induction of renal atherogenesis [8-10,20]. The exact mechanism of how cocaine causes renal infarction, however, is unclear, and several possibilities have been proposed. Cocaine is known to enhance platelet aggregation and increase thromboxane synthesis [21]. It is also known to inhibit synaptosomal uptake of catecholamines, block the re-uptake of norepinephrine in sympathetically innervated tissues and release norepinephrine and epinephrine from the adrenal medulla [4,5,21]. Other vasoconstrictive factors such as endothelin have also been postulated to play a role in the vascular catastrophes caused by cocaine intoxication [22].
There is evidence that cocaine may enhance the renal cortical messenger RNA expression of tissue inhibitors of metalloproteinase-2 [8]. This would have the net effect of increasing matrix accumulation. Use of cocaine also increases the oxidative stress in the kidney. In experimental cultured kidney cells exposed to cocaine, there has been a lower level of intracellular glutathione, which is the most abundant cell thiol with antioxidant functions [23]. There is also supporting evidence that systemic atherosclerosis may be an inflammatory disorder. Both experimental and autopsy findings confirm that cocaine is an accelerator of atherogenesis [9,10].
Renal infarction due to cocaine abuse usually presents with severe persistent flank and/or abdominal pain associated with nausea or vomiting with or without elevated temperature. The onset of pain is usually 2–3 hours after cocaine use, but it can be delayed for up to 4 days. All routes of cocaine administration including intravenous, insufflations, and intranasal and free-basing crack cocaine have been associated with renal infarction. Leukocytosis, microscopic hematuria and elevated levels of serum LDH are common findings. However, these findings are neither sensitive nor specific for CIRI.
Various imaging techniques including CT scan, Magnetic Resonant Imaging (MRI), angiography, Ultrasound and Nuclear Scintigraphy Scans have been used to make the diagnosis. However, MRI, angiography and nuclear scintigraphy are expensive, system-specific and time-consuming, and they may not be readily available at all institutions. Ultrasound lacks sensitivity and specificity for renal infarction. Therefore, CT scan has been recommended for the diagnosis of this challenging disease. CT scan, however, becomes less reliable when the infarct is global and there is loss of viable cortical rim, or when the infarct simulates a tumefactive process.
There is no consensus on the treatment of renal infarction due to cocaine use. Prior treatment modalities in the literature range from no treatment to anticoagulation, thrombolytic use, aspirin therapy and surgical nephrectomy. Given that no evidence of underlying coagulopathy or thromboembolic events were identified in our patient, we opted to conservatively manage him with supportive therapy and pain management. He was successfully discharged on hospital day six with a serum creatinine of 1.2 mg/dL (106.1 umol/L).
Conclusion
Cocaine intoxication is associated with multiple cases of vascular injury. Renal infarction as a result of cocaine use, however, is uncommon. In a patient with a history of cocaine abuse presenting with fevers and flank pain suggestive of urinary tract infection or nephrolithiasis, cocaine-induced renal infarction must be considered in the differential diagnosis. The vasoconstrictive and thrombotic effects of cocaine are most likely the dominant factors in cocaine-induced renal infarction. The possible atherogenic effect of cocaine and the relationship of this effect to renal infarct is likely to be a long term, rather than an acute factor. The diagnosis of CIRI is based on exclusion of other entities that cause renal infarct (e.g. hypercoagulable states due to factor deficiency, autoimmune diseases and thromboembolic events), in conjunction with documented cocaine use and appropriate radiographic studies. We highly recommend non-invasive evaluation of the renal arterial supply in patients with CIRI in order to broaden our knowledge of this disorder, and conceivably better understand its selectivity for one kidney. Thus far, there is no agreement on the management of patients with this disease. In fact, it is difficult to suggest a uniform therapeutic approach to this disorder because one cannot predict whether the infarct will be due to vasospasm, thrombosis or both. In addition, the extent of the renal infarct will likely influence clinical management and outcome. Although anticoagulation, antithrombotic, conservative managements as well as surgical approaches have been attempted with variable success, perhaps the most plausible treatment would be to prevent this misfortune by patient education.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SB performed the main background search and drafted the manuscript. MM participated in the study design and helped to draft the manuscript. SMN participated in the study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The Authors would like to thank Dr. Arash D. Tehranzadeh for his interpretation of the radiological images. Written consent was obtained from the patient for publication of the study.
Figures and Tables
Figure 1 Computerized Tomography showing areas of focal decreased enhancement of the anterior lower poles (white arrow) of the right kidney suggesting renal infarction versus pyelonephritis.
Figure 2 Volume-rendered Single Photon Emission-Computed Tomography image of a gallium scan demonstrating absence of tracer localization in the upper pole of the right kidney (white arrow) compatible with infarction.
Table 1 Demographic data of previously-reported cases of cocaine-induced renal infarction.
Characteristics Wohlman [11] Antonovych [13] Kramer [12] Goodman [14] Saleem [15] Edmondson [16] Mochizuki [17]
Age (s) 32 39, 26 37 37 25 40 52
Sex Male Males Male Male Male Male Female
Route IV NR NR IN IN NR IN
Time of Onset 2 hours NR 2–3 hours 2 hours 4 days 1 day NR
Kidney Affected Right NR Right Right Right Right Left
WBC (× 103/μl) 14 NR 23 (8% bands) 16 (90% PMN) 12.3 (76% PMN) NR 19.3
Serum LDH (U/L) 368 to 1860 NR 2103 NR 351 NR 2100
Serum Cr (mg/dL) 1.2 NR 1.7 0.9 NR 1.3 0.9
Urinanalysis 5–10 RBC NR trace protein
3+ ketones
2+ blood
3–4 WBC/hpf
1–2 RBC/hpf 30 protein
80 ketones
1–5 WBC/hpf
5–10 RBC/hpf 2+ blood
2 WBC/hpf
5–10 RBC/hpf 0 RBC/hpf trace protein
large blood
3 WBC/hpf
1 RBC/hpf
Treatment heparin & coumadin nephrectomy urokinase, heparin & coumadin none aspirin heparin & coumadin heparin, antibiotics & coumadin
Co-existing Conditions NR NR DM, Protein C deficiency NR NR Renal artery dissection Aortic thrombosis
WBC (White Blood Cell; cells × 103/ul = cells × 109/ul); RBC (Red Blood Cell); hpf (High Power Field); LDH (Lactate Dehydrogenase); PMN (Polymorphonuclear Cells); N/R (Not Reported); IV (Intravenous); IN (Intra-nasal); DM (Diabetes Mellitus); Serum Cr (Serum Creatinine; 1 mg/dL = 88.4 umol/L)
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Pitts WR Lange RA Cigarroa JE Hillis DL Cocaine-induced myocardial ischemia and infarction: Pathophysiology, recognition and management Prog Cardiovasc Dis 1997 40 65 76 9247556 10.1016/S0033-0620(97)80023-0
Inaba T Cocaine: Pharmacokinetics and biotransformation in man Can J Physiol Pharmacol 1989 67 1154 1157 2688863
Chow MJ Amdre JJ Ruo TI Atkinson AJ Bowsher DJ Pischman MW Kinetics of cocaine distribution, elimination and chronotropic effects Clin Pharmacol Ther 1985 38 318 324 4028628
Chiueh CC Kopin IJ Centrally mediated release by cocaine of endogenous epinephrine and norepinephrine from the sympathoadrenal medullary system of unanesthetized rats J Pharmacol Exp Ther 1978 205 148 154 633079
Karch SB Serum catecholamines in cocaine-intoxicated patients with cardiac symptoms Ann Emerg Med 1987 16 481 482
Rogione AJ Steg PG Gal D Isner JM Cocaine causes endothelium-independent vasoconstriction of vascular smooth muscle [abstract] Circulation 1988 78 5436A
Isner J Chokshi SK Cardiovascular complications of cocaine Curr Probl Cardiol 1991 16 89 123 2015775 10.1016/0146-2806(91)90013-Z
Kapasi AJ Mattana J Wagner J Morphine amplifies cocaine-induced renal cortical expression of tissue inhibitors of metalloproteinase (TIMP)-2 [abstract] J Am Soc Nephrol 1997 6 528A
Kolodgie FD Wilson PS Corhill JF Herderick EE Mergner WJ Virman R Increased prevalence of aortic fatty streaks in cholesterol-fed rabbits administered intravenous cocaine: The role of vascular endothelium Toxicol Pathol 1993 21 425 434 8115819
Dressler FA Malekzadeh SJ Roberts WC Quantitative analysis of amounts of coronary artery narrowing in cocaine addicts Am J Cardiol 1990 65 303 308 2301258 10.1016/0002-9149(90)90292-9
Wohlman RA Renal artery thrombosis and embolization associated with intravenous cocaine injection South Med J 1987 80 928 930 3603118
Kramer RK Turner RC Renal infarction associated with cocaine use and latent protein c deficiency South Med J 1993 86 1436 1438 8272932
Antonovych TT Sabnis SG Finkelstein A Yadla RK Unusual renal involvement in two cocaine addicts [abstract] J Am Soc Nephrol 1990 1 326A
Goodman PE Rennie WP Renal infarction secondary to nasal insufflation of cocaine Am J Emerg Med 1995 13 421 423 7605528 10.1016/0735-6757(95)90129-9
Saleem TM Singh M Murtaza M Singh A Kasubhai M Gnanasekaran I Renal infarction: a rare complication of cocaine abuse Am J Emerg Med 2001 19 528 529 11593482 10.1053/ajem.2001.25778
Edmondson DA Towne JB Foley DW Abu-Hajir M Kochar MS Cocaine-induced renal artery dissection and thrombosis leading to renal infarction WMJ 2004 103 66 69 15696837
Mochizuki Y Zhang M Golestaneh L Thananart S Coco M Acute aortic thrombosis and renal infarction in acute cocaine intoxication: a case report and review of literature Clin Nephrol 2003 60 130 133 12940616
Merklin RJ Michels NA The variant renal and suprarenal blood supply with data on the interior phrenic, ureteral and gonadal arteries: a statistical analysis based on 185 dissections and a review of the literature J Int Coll Surg 1958 29 41 76 13502578
Ross J Jr West JB Dynamics of the Peripheral Circulation Best and Taylor's Physiological Basis of Medical Practice 1990 12 Maryland: Williams & Wilkins 138 158
Nzerue CM Hewan-Lowe K Riley LJ Jr Cocaine and the kidney: a synthesis of pathophysiologic and clinical perspectives Am J Kidney Dis 2000 35 783 785 10793010
Lange RA Hills LD Cardiovascular complications of cocaine use N Eng J Med 2001 345 351 358 10.1056/NEJM200108023450507
Kohan DE Endothelins in normal and diseased kidney Am J Kidney Dis 1997 29 2 25 9002526
Palamara AT DiFrancesco P Ciriolo MR Bue C Lafaria E Rotilo G Garaci E Cocaine increases Sendai virus replication in cultured epithelial cells: Critical role of intracellular redox status Biochem Biophys Res Commun 1996 228 579 585 8920954 10.1006/bbrc.1996.1701
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BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-5-331617430310.1186/1471-244X-5-33Research ArticlePublic beliefs about causes and risk factors for mental disorders: a comparison of Japan and Australia Nakane Yoshibumi [email protected] Anthony F [email protected] Kumiko [email protected] Helen [email protected] Hideyuki [email protected] Kathleen M [email protected] Department of Social Work, The Faculty of Human Sociology, Nagasaki International University, 2825-7 Huis Ten Bosch-cho, Sasebo-shi, Nagasaki, 859-3298, Japan2 ORYGEN Research Centre, Department of Psychiatry, University of Melbourne, Locked Bag 10, Parkville, Victoria 3052, Australia3 Centre for Mental Health Research, Australian National University, Canberra, ACT 0200, Australia4 Division of Neuropsychiatry, Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan2005 21 9 2005 5 33 33 17 7 2005 21 9 2005 Copyright © 2005 Nakane et al; licensee BioMed Central Ltd.2005Nakane 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
Surveys of the public in a range of Western countries have shown a predominant belief in social stressors as causes of mental disorders. However, there has been little direct cross-cultural comparison. Here we report a comparison of public beliefs about the causes of mental disorders in Japan and Australia.
Methods
Surveys of the public were carried out in each country using as similar a methodology as feasible. In both countries, household interviews were carried out concerning beliefs about causes and risk factors in relation to one of four case vignettes, describing either depression, depression with suicidal thoughts, early schizophrenia or chronic schizophrenia. In Japan, the survey involved 2000 adults aged between 20 and 69 from 25 regional sites spread across the country. In Australia, the survey involved a national sample of 3998 adults aged 18 years or over.
Results
In both countries, both social and personal vulnerability causes were commonly endorsed across all vignettes. The major differences in causal beliefs were that Australians were more likely to believe in infection, allergy and genetics, while Japanese were more likely to endorse "nervous person" and "weakness of character". For risk factors, Australians tended to believe that women, the young and the poor were more at risk of depression, but these were not seen as higher risk groups by Japanese.
Conclusion
In both Japan and Australia, the public has a predominant belief in social causes and risk factors, with personal vulnerability factors also seen as important. However, there are also some major differences between the countries. The belief in weakness of character as a cause, which was stronger in Japan, is of particular concern because it may reduce the likelihood of seeking professional help and support from others.
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Background
Mental health researchers view mental disorders as having complex causes involving an interplay of biological, psychological and social factors. However, the public's beliefs about causes are generally less sophisticated. Surveys of the public in a range of Western countries have shown a predominant belief in social stressors as causes of mental disorders. Studies from Australia, Ireland, Germany, Switzerland, UK and USA have found that social factors were most often seen as the causes of depression [1-6], whereas genetic factors were much less frequently endorsed [1-4,6]. Social factors are also seen by the public of Western countries as an important cause of schizophrenia [3,6-8]. While genetic factors are more often seen as a cause for schizophrenia than depression, they are still endorsed much less frequently than social factors [3,6]. Social factors covered in these surveys included stressful life events, traumatic experiences, family problems, and social disadvantage.
Of greater concern is the stigmatizing belief that mental disorders are caused by personal weakness or a character flaw. While this is not a predominant belief, it is fairly common. In the USA, around one-third saw "own bad character" as a cause for both schizophrenia and depression [6], implying a moral judgment of mental disorders. In Australia, around half the population believed "weakness of character" is a cause of both depression and schizophrenia [1], while in Turkey over 60% believed that this is a cause of schizophrenia [8].
While there has been considerable research on public beliefs in Western countries, there has been little research in other parts of the world and little cross-cultural comparison. The beliefs that predominate in Western countries cannot be assumed to apply elsewhere. A comparison of teachers' beliefs about schizophrenia in Japan and Taiwan found that "stress from personal relations" was commonly seen as a cause in both countries, which is similar to the belief in social factors in Western countries [9]. However, the Taiwanese were more likely than the Japanese to believe in "weakness of character", "heredity" and "stress from a disaster" as a cause. A comparison of mainly young adults from Hong Kong and England found that the Hong Kong Chinese were more likely to believe in social factors as the cause of schizophrenia, while the English were more likely to endorse genetic factors [10]. This difference was attributed to the more collectivist nature of Chinese culture. There has also been a comparison of public beliefs in Germany, Russia and Mongolia [11]. In all three countries, psychosocial factors such as stressful life events were predominantly seen as the cause of both depression and schizophrenia, whereas biological causes such as heredity and brain disease were less frequently endorsed. Taken together, these cross-cultural comparisons indicate that a belief in social causes is common in East Asian countries as well as in Western countries.
Here we report a further cross-cultural comparison involving Japan and Australia. This comparison involved surveys in both countries using the same questions about causes and risk factors for four case vignettes: depression, depression with suicidal thoughts, early schizophrenia, and chronic schizophrenia. On the basis of previous research, it might be expected that a belief in social causes would predominate in both countries. However, there are a number of cultural and health system differences between the two countries that might influence responses. Japan places a greater emphasis on hospital care compared to the emphasis on community care in Australia. The Japanese mental health care system has been described as based on the values of minimizing state financial involvement, retaining family responsibility for family members, and social control of individuals who might contribute to social disorder [12]. By contrast, Western systems are more influenced by the values of individual rights, social reintegration and government responsibility [12]. While these differences exist, it is difficult to predict what effect they might have on causal beliefs, so the study was essentially exploratory.
Methods
Survey interview
Interview questionnaires comprised a common core of questions that would allow comparisons between countries, and a country-specific component to allow investigation of issues particular to each country [13]. Copies of the Japanese questionnaire are available from YN and of the Australian questionnaire from AFJ. The interview was based on a vignette of a person with a mental disorder. On a random basis, respondents were shown one of four vignettes: a person with major depression, one with major depression together with suicidal thoughts, a person with early schizophrenia, and one with chronic schizophrenia. All vignettes were written to satisfy the diagnostic criteria for either major depression or schizophrenia according to DSM-IV and ICD-10. The vignette with depression and the one with early schizophrenia were written to satisfy at a minimal level these diagnostic criteria, so that we could ascertain the public's reaction to cases of developing disorder that had reached the point where intervention was needed. The vignette of the person with depression together with suicidal thoughts was identical to the depression vignette in all respects except the suicidal thoughts and was designed to assess how this symptom affected the public's response. The chronic schizophrenia vignette was designed to assess the response to someone with a severe long-standing disorder, where acceptance seemed less likely. Respondents were also randomly assigned to receive either male ("John") or female ("Mary") versions of the vignette. The vignettes have been given in an earlier publication [13]. After being presented with the vignette, respondents were questioned about what was wrong with the person, how they could be helped, the likely helpfulness of a range of interventions, the likelihood of recovery, knowledge of causes and risk factors, beliefs associated with stigma and discrimination, contact with people like those in the vignette, and the health of the respondent.
The only questions of relevance here are those concerned with causes and risk factors [1]. These questions were: "There are many people in the community who suffer from problems like John's. The next few questions are about possible causes of this sort of problem developing in anybody. How likely do you think each of the following is to be a reason for such problems? Could a virus or other infection, be a reason for these sorts of problems? How likely is an allergy or reaction to be the cause? Day-to-day problems such as stress, family arguments, difficulties at work or financial difficulties? The recent death of a close friend or relative? Some recent traumatic event such as bushfires threatening your home, a severe traffic accident or being mugged? Problems from childhood such as being badly treated or abused, losing one or both parents when young or coming from a broken home? How likely is it that these sorts of problems are inherited or genetic? Is being a nervous person likely to be a reason? Having weakness of character?" Response options to these questions were: very likely, likely, not likely, depends, don't know.
Then followed questions about risk factors: "The next few questions seek your opinion about whether there are some people in the community who are more likely to have these problems and others who are perhaps less likely. Do you think that women would be more likely or less likely than men to suffer these sorts of problems? Would young people, under 25 years of age, be more likely or less likely? Would older people, those aged over 65, be more likely or less likely? Would poor people be more likely or less likely to suffer these sorts of problems? Unemployed people? Divorced or separated people? Would single people, who have never been married or in a long-term relationship be more likely or less likely?". The response options were: More likely, less likely, no difference, depends, don't know.
The Japanese survey
A survey manual supplied from Australia was translated into Japanese and entrusted to Yamate Information Processing Center Ltd. for use with the target population aged 20–69 years, as a rule using the same procedures as Australia. The survey questionnaire, which was developed by the Australian researchers (AFJ, HC, KMG), was tentatively translated into Japanese. Then a native English translator, who had not seen the original English text, translated the Japanese version back into English. By comparing the two English versions, it was possible to confirm the accuracy of the original translation. There were no significant differences between the original text and the reverse translation. Finally, a Japanese version of the questionnaire was produced, which involved formatting the text into Japanese style and making slight wording adjustments. The names of the characters in the vignettes were translated into the Japanese style, viz. "A-o"(putting an o sound at the end is often used for a man's name) or "B-ko" (putting ko at the end is often used for a woman's name), instead of "John" or "Mary" which were used in the English text.
As well as the questions taken from the Australian survey, the Japanese survey asked questions concerning such issues as psychiatric health and welfare policy, the bodies implementing related services, the existence of action groups, and the change in the Japanese name for schizophrenia by the Japanese Society of Psychiatry and Neurology. These additions were made to clarify the current Japanese situation and issues in related fields. Further, an original Japanese manual was also created and adopted for use concerning points of interest in the implementation of home visits.
The survey method used was home visit interviews. It was not feasible to do a national survey of randomly selected households in Japan because of constraints of human resources, funding and time. It was therefore decided to sample a range of areas that differed in whether they were large or small cities, whether the area had many psychiatric patients or not, and whether the area had a high suicide rate or not. Using this approach, Japan was divided into 5 areas and 5 research sites were selected in each of these areas, giving a total of 25 geographic sites. As the survey was conducted during the winter, and because it was difficult to ensure that there would be enough survey interviewers, implementation in Hokkaido and Shikoku prefectures proved troublesome. Additional reasons for selection of the 25 regional sites were that they were places of comparatively high population within the relevant regions, the survey interviewers could use public transport, and the urban areas involved no particular inconveniences for the researchers to visit within a certain range using public transportation. 80 households were selected from each site, giving a total of 2000. At each site there were 4 interviewers who took responsibility for 20 households each. The survey was conducted over the period from 19 November to 12 December 2003. Each of the four vignettes was received by 250 people. Half received a male version of a vignette and half the female version.
At the start of the survey, an explanatory meeting was held for the survey interviewers in each region. As many members of the research team as possible attended these explanatory meetings. Eighty-five survey interviewers were recruited for this research with an average age of 50 and an average of 17 years' experience of survey interviewing in various types of surveys. The areas for the survey interviewers to canvass were allocated on the basis of where they lived. The question of where the individual survey interviewers should go was determined mutually among the survey interviewers themselves, and by the head survey interviewer (supervisor). As a rule, one survey interviewer conducted 20 interviews, but this was considerably flexible, given the number of years of individual experience and what the individual survey interviewer could handle. The interviews were conducted according to the following procedure: visit the target's home and present the written greetings and request (a draft had been prepared by certain survey bodies, which was put into final form after checks by the research team members), then explain the details of the survey using the documents, ask the target for their participation in the research, start the interview and follow through to completion, check that nothing had been omitted from the survey responses, and hand over the remuneration (1000 yen cash voucher). Data were not collected on the refusal rate for this survey because the emphasis was on achieving the quotas of respondents to fit the required age and gender distribution.
The Australian survey
A household survey was carried out on Australian adults aged 18 years or over by the company AC Nielsen. Households were sampled from 250 census districts covering all states and territories and metropolitan and rural areas. Up to 5 call backs were made to metropolitan selections and 3 to non-metropolitan selections. Interviewers attempted to interview the person in each household with the most recent birthday. To achieve a target sample of 4,000 interviews with adults aged 18 years or over, visits were made to 28,947 households. The outcome of these visits was: no contact after repeated visits 14,630; vacant house or lot 306; refused 7,815; person sampled within household temporarily unavailable 1,132; no suitable respondent in household 287; did not speak English 383; incapable of responding 213; and unavailable for the duration of the survey 181. The achieved sample was 3998 persons, with 1001 receiving the depression vignette, 999 the depression with suicidal thoughts vignette, 997 the early schizophrenia vignette, and 1001 the chronic schizophrenia vignette. The interviews were carried out between November 2003 and February 2004.
In addition to the common core component, the Australian survey interview had questions about awareness of depression in the media and about Australia's national depression initiative.
Ethics approval was given by the Human Research Ethics Committee of the Australian National University.
Statistical analysis
Data were pooled across male and female versions of each vignette and percent frequencies calculated. For the Japanese survey, percentage frequencies and 95% CIs were calculated using unweighted data with SPSS 12.0. For the Australian survey, percentages were calculated applying survey weights to give better population estimates. Ninety-five percent CIs were estimated using the Complex Samples procedure in SPSS 12.0. This procedure takes account of sampling weights and geographic clustering in the sample.
Because of the very different cultures of Japan and Australia, it is possible that any differences in question endorsement rates might be due to subtleties of language or to the social rules applying to the interview situation, as well as to genuine differences in beliefs about treatment and outcome. For this reason, we have not relied on statistical significance of percentage differences between countries, but rather on the broad patterns of responses, particularly where percent endorsement was ordered very differently across questions.
Results
Characteristics of the samples
Table 1 shows the age and gender distributions of the Japanese and Australian samples. Comparing the Japanese sample to the national population in the same age groups (2003 data), there was an under-representation of 50–59 year olds (20% vs 22.4%) and an over-representation of 60–69 year old males (10% vs 8.9%). Other age-gender groups showed less than 1% discrepancy.
Table 1 Age and gender distribution of the Japanese and Australian samples
Age group Japanese males % Japanese females % Japanese total % Australian males % Australian females % Australian total %
18–19 - - - 1.5 1.6 3.0
20–29 10.0 10.0 20.0 5.6 8.1 13.8
30–39 10.0 10.0 20.0 7.7 11.8 19.4
40–49 10.0 10.0 20.0 8.6 11.0 19.7
50–59 10.0 10.0 20.0 6.9 9.5 16.3
60–69 10.0 10.0 20.0 4.8 8.0 12.7
70+ - - - 6.2 8.9 15.1
Total 50.0 50.0 100.0 41.2 58.8 100.0
Comparing the Australian sample to the national population, there was an under-representation of males and of younger adults, but the sample was close to the population in marital status, country of birth and education. For the Australian sample, weights were used to correct for these biases.
Beliefs about causes and risk factors
Table 2 shows the results on beliefs about causes. In this table, the percentages pertain to each question asked separately, so that respondents could endorse any number of factors as likely causes. In both countries there was a common belief in social causes, such as day-to-day problems, death of someone close, traumatic event, and problems from childhood. This belief was common across all vignettes. The major differences were that the Australians were more likely to believe in virus or infection, allergy, and inherited or genetic, while the Japanese were more likely to endorse nervous person and weakness of character.
Table 2 Percentage (and 95% CI) of Japanese and Australian respondents giving each cause as "very likely" or "likely" for the person described in the vignette
Cause Depression Vignette Depression/Suicidal Vignette Early Schizophrenia Vignette Chronic Schizophrenia Vignette
Virus or infection
Japanese 6.2 (4.1–8.3) 6.6 (4.4–8.8) 7.2 (4.9–9.5) 7.2 (4.9–9.5)
Australian 50.5 (47.1–54.0) 41.4 (38.2–44.7) 32.1 (29.0–35.4) 33.6 (30.7–36.6)
Allergy
Japanese 10.2 (7.5–12.9) 11.4 (8.6–14.2) 12.6 (9.7–15.5) 9.4 (6.8–12.0)
Australian 44.9 (41.4–48.5) 37.6 (34.3–41.0) 31.5 (28.4–34.7) 28.4 (25.5–31.5)
Day-to-day problems
Japanese 93.6 (91.4–95.8) 91.8 (89.4–94.2) 92.0 (89.6–94.4) 91.2 (88.7–93.7)
Australian 96.8 (95.2–97.9) 95.7 (94.2–96.9) 89.6 (87.6–91.2) 86.6 (84.3–88.7)
Death of someone close
Japanese 79.8 (76.3–83.3) 81.4 (78.0–84.8) 73.4 (69.5–77.3) 74.0 (70.1–77.9)
Australian 96.3 (94.6–97.5) 94.8 (93.1–96.0) 87.4 (85.2–89.4) 83.3 (80.7–85.6)
Traumatic event
Japanese 82.6 (79.3–85.9) 79.6 (76.1–83.1) 78.2 (74.6–81.8) 80.8 (77.3–84.3)
Australian 93.9 (91.8–95.4) 92.7 (90.7–94.2) 86.5 (84.1–88.6) 82.8 (80.2–85.1)
Problems from childhood
Japanese 81.0 (77.5–84.5) 82.0 (78.6–85.4) 88.2 (85.4–91.0) 89.0 (86.2–91.8)
Australian 91.3 (89.2–93.1) 95.0 (93.2–96.3) 90.8 (88.8–92.5) 91.4 (89.4–93.0)
Inherited or genetic
Japanese 34.6 (30.4–38.8) 34.0 (29.8–38.2) 34.2 (30.0–38.4) 43.8 (39.4–48.2)
Australian 68.0 (64.8–71.0) 68.4 (65.3–71.3) 70.0 (66.8–73.0) 73.7 (70.6–76.6)
Nervous person
Japanese 81.4 (78.0–84.8) 77.4 (73.7–81.1) 74.0 (70.1–77.9) 81.8 (78.4–85.2)
Australian 67.9 (64.6–70.9) 65.6 (62.3–68.7) 58.1 (54.4–61.7) 56.9 (53.6–60.2)
Weakness of character
Japanese 73.6 (69.7–77.5) 69.2 (65.1–73.3) 73.4 (69.5–77.3) 82.0 (78.6–85.4)
Australian 43.0 (39.7–46.3) 46.1 (42.8–49.3) 39.7 (36.5–42.9) 35.1 (31.6–38.8)
Tables 3 and 4 show the data on beliefs about risk factors. Table 3 gives the percentages believing a group is more at risk and Table 4 the percentages believing a group is less at risk. As in Table 2, each group was asked about separately, so that respondents could endorse any number as more likely or less likely to be at risk. In both countries, the risk factors most strongly believed in across vignettes were being unemployed and divorced/separated, although these beliefs were more common in Australia. There were a number of cross-national differences. The Australians tended to believe that the young and the poor were more at risk of depression, but these were not seen as higher risk groups by the Japanese. In fact, the Japanese public tended to see the poor as having lower risk of depression. For schizophrenia, the Australians saw the young as having higher risk for early schizophrenia, and the poor as having higher risk for both early and chronic schizophrenia. Likewise the Japanese saw the young as having higher risk for early schizophrenia, but they again tended to see the poor as having lower risk.
Table 3 Percentage (and 95% CI) of Japanese and Australian respondents rating each group in the population as "more likely" to experience the problem described in the vignette
Group More Likely at Risk Depression Vignette Depression/Suicidal Vignette Early Schizophrenia Vignette Chronic Schizophrenia Vignette
Women
Japanese 29.4 (25.4–33.4) 24.2 (20.4–28.0) 21.4 (17.8–25.0) 23.0 (19.3–26.7)
Australian 26.8 (23.9–30.0) 27.2 (24.4–30.2) 21.1 (18.5–23.9) 14.7 (12.6–17.1)
Young
Japanese 24.2 (20.4–28.0) 24.4 (20.6–28.2) 40.4 (36.1–44.7) 26.2 (22.3–30.1)
Australian 42.5 (39.1–46.0) 48.2 (45.0–51.4) 55.3 (52.0–58.6) 28.4 (25.6–31.4)
Old
Japanese 23.4 (19.7–27.1) 21.2 (17.6–24.8) 15.0 (11.9–18.1) 29.2 (25.2–33.2)
Australian 28.6 (25.8–31.5) 29.1 (26.3–32.1) 22.0 (19.4–24.9) 38.3 (35.0–41.7)
Poor
Japanese 14.8 (11.7–17.9) 13.2 (10.2–16.2) 7.4 (5.1–9.7) 19.6 (16.1–23.1)
Australian 52.6 (49.2–56.0) 52.1 (48.6–55.5) 38.9 (35.7–42.3) 37.8 (34.4–41.2)
Unemployed
Japanese 58.4 (54.1–62.7) 50.8 (46.4–55.2) 41.0 (36.7–45.3) 50.6 (46.2–55.0)
Australian 76.3 (73.0–79.3) 76.7 (73.6–79.4) 62.7 (59.4–65.9) 54.9 (51.4–58.3)
Divorced/separated
Japanese 48.6 (44.2–53.0) 43.6 (39.2–48.0) 37.2 (32.9–41.5) 39.6 (35.3–43.9)
Australian 69.6 (66.2–72.8) 64.3 (60.9–67.6) 53.6 (50.3–57.0) 44.3 (40.9–47.8)
Single
Japanese 18.2 (14.8–21.6) 22.8 (19.1–26.5) 22.8 (19.1–26.5) 24.6 (20.8–28.4)
Australian 23.9 (21.0–27.1) 27.1 (24.2–30.1) 22.0 (19.2–25.0) 25.1 (22.2–28.2)
Table 4 Percentage (and 95% CI) of Japanese and Australian respondents rating each group in the population as "less likely" to experience the problem described in the vignette
Group Less Likely at Risk Depression Vignette Depression/Suicidal Vignette Early Schizophrenia Vignette Chronic Schizophrenia Vignette
Women
Japanese 21.6 (18.0–25.2) 22.8 (19.1–26.5) 24.0 (20.2–27.8) 24.8 (21.0–28.6)
Australian 12.0 (9.7–14.7) 13.6 (11.5–16.0) 12.8 (10.8–15.1) 18.7 (16.4–21.3)
Young
Japanese 24.0 (20.2–27.8) 20.8 (17.2–24.4) 14.6 (11.5–17.7) 27.6 (23.7–31.5)
Australian 19.5 (16.8–22.4) 18.8 (16.4–21.4) 10.9 (8.9–13.2) 30.6 (27.5–33.9)
Old
Japanese 30.2 (26.2–34.2) 25.0 (21.2–28.8) 38.6 (34.3–42.9) 27.4 (23.5–31.3)
Australian 34.8 (31.5–38.2) 37.2 (34.2–40.3) 43.2 (39.8–46.7) 25.2 (22.5–28.1)
Poor
Japanese 23.4 (19.7–27.1) 19.6 (16.1–23.1) 26.4 (22.5–30.3) 23.4 (19.7–27.1)
Australian 8.5 (6.6–10.8) 7.4 (6.0–9.2) 9.1 (7.3–11.3) 7.2 (5.7–9.1)
Unemployed
Japanese 13.0 (10.0–16.0) 11.0 (8.2–13.8) 14.8 (11.7–17.9) 15.6 (12.4–18.8)
Australian 4.4 (3.0–6.2) 4.6 (3.5–6.2) 5.4 (4.1–7.1) 5.5 (4.1–7.5)
Divorced/separated
Japanese 12.2 (9.3–15.1) 13.6 (10.6–16.6) 14.0 (10.9–17.1) 16.0 (12.8–19.2)
Australian 4.1 (3.0–5.7) 5.5 (4.2–7.3) 5.2 (3.9–6.9) 6.7 (5.1–8.7)
Single
Japanese 19.2 (15.7–22.7) 13.0 (10.0–16.0) 13.6 (10.6–16.6) 20.4 (16.9–23.9)
Australian 21.4 (18.8–24.4) 19.2 (16.6–22.2) 17.7 (15.2–20.4) 17.1 (14.9–19.6)
Discussion
The present findings from Japan and Australia support earlier work from several countries showing a predominant belief in social causes. These causes include day-to-day problems, death of someone close, traumatic events, and problems from childhood. The findings on risk factors are generally consistent with those on causes, with unemployment and divorce/separation widely seen to be risk factors in both countries. While a belief in social causes and risk factors was found in both countries, it was generally more common in Australia than in Japan. As far as depression is concerned, this belief is realistic because there is substantial evidence supporting social factors in the causation of depression [14]. However, for schizophrenia, social factors are of less importance, having an influence only on those who are genetically vulnerable [15].
The findings on poverty as a risk factor were an exception to the general trend of beliefs in social factors. While the Australians tended to see the poor as having higher risk, particularly for depression, for the Japanese the trend was in the opposite direction, with more people believing that the poor would have lower risk. The reason for this difference is unknown. It may reflect inaccurate beliefs in one country or else a true difference in risk factors between countries. In Australia, poverty is known to be associated with both depression and psychotic disorders [16,17], although there is debate about the causal pathways. However, in one Japanese study of depression in the workplace, poor economic status was not associated with increased risk [18]. Furthermore, a Japanese incidence study of schizophrenia found that features of residential areas that are associated with higher incidence in Western countries do not necessarily have the same association in Japan [19].
As well as a frequent public belief about social causes in both countries, there was also a common belief in personal vulnerability factors. However, the endorsement of personal vulnerability factors tended to take a different form in each country. The Japanese were more likely to believe in the role of the trait characteristics of nervous person and weakness of character, while Australians were more likely to endorse the role of genetics. Being a nervous person could be regarded as a lay description of the personality trait of neuroticism, which is a major risk factor for depression. There is also some evidence that neuroticism is a risk factor for schizophrenia [20]. However, the belief in weakness of character is of more concern, because this is a more stigmatizing explanation which could make people less likely to disclose that they are experiencing a mental disorder and to seek professional help. It has been noted that "Japanese patients are reluctant to openly discuss disturbances of mood, since these are considered to be indicative of personal weakness rather than treatable medical conditions" [21]. For Japanese people, the implication is that the disorder is the person's fault. In Australia, the belief in weakness of character as a cause has declined since the earlier survey in 1995, particularly for schizophrenia [22]. This change may have been affected by efforts to reduce the stigma of mental disorders.
The strong endorsement of genetics by Australians represents a major change from 8 years earlier, from around half the population in 1995 to around two-thirds in 2003–04 [22]. A possible reason for this change is the publicity surrounding the human genome project and the role of genes in health generally. Why this belief is weaker in Japan is unclear. However, in both countries genetics was seen to be more important for the chronic schizophrenia vignette than for the other vignettes, suggesting a greater genetic attribution for severe or chronic mental disorders.
Another difference between the two countries was that Australians were more likely to believe in the role of virus or infection and allergy. It is not clear why these beliefs are more prominent in Australia. However, such beliefs appear to be stable over time, because the percentages endorsing these causes are very similar to an Australian national survey carried out 8 years earlier [22]. These beliefs were most common for the vignette of depression without suicidal thoughts, which is the least severe of the cases presented. It may be that they reflect interpretations of the vignette as being a physical disorder or a secondary reaction to a physical disorder.
Taking all the findings together, there is some broad similarity between public and professional beliefs about causation, in that mental disorders are seen to be influenced by a combination of personal vulnerability and environmental triggers. The major difference would appear to be the greater use of morally judgmental attributions of personal vulnerability by the public compared to the more objective attributions of professionals. In this regard, the Australian public's views appear closer to those of professionals than do the Japanese public's.
Limitations
We have previously discussed some of the limitations of this work [13]. These relate to the methodology of the surveys, in particular the non-contact and refusal rate in the Australian survey and the lack of truly national coverage of the Japanese one. Furthermore, both surveys lack data on the characteristics of refusers. We also recognise the problems of making cross-national comparisons between two very different cultures. There will inevitably be subtleties of meaning and cultural factors operating within a structured household survey which could affect the results in unknown ways. Finally, the survey used closed rather than open-ended questions, which may have suggested responses that the participants would not have thought of spontaneously.
Conclusion
In both Japan and Australia, the public have a predominant belief in social causes and risk factors for mental disorders. However, there are also some major differences between the countries, with Australians having a stronger belief in infections, allergies and genetics, while Japanese have a stronger belief in being a nervous person and weakness of character. The latter belief is of particular interest because is may be associated with greater stigma and reduce the likelihood of seeking professional help and support from others. Reducing the belief in weakness of character as a cause would be a suitable target for mental health literacy campaigns. This is probably easier to achieve for depression, where there are both contemporary and historical figures who have suffered from depression, yet are perceived as being strong in character.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YN provided overall supervision of the research and provided comments on the manuscript.
AFJ co-designed the Australian survey, analyzed the Australian data, and co-wrote the manuscript.
YK provided specific guidance on the Japanese survey, including participation in survey interviewer training, and co-wrote the manuscript.
HC co-designed the Australian survey and provided comments on the manuscript.
HN provided specific guidance on the Japanese survey, including participation in survey interviewer training, and co-wrote the manuscript.
KMG co-designed the Australian survey and provided comments on the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study is part of the Australia-Japan Partnership, which is an agreement between the governments of the two countries for joint projects in areas of health. The Japanese research team wishes to thank the Ministry of Health, Labor, and Welfare for the Health and Labor Science Research Grants (Research on Psychiatric and Neurological Diseases and Mental Health) which allowed us to conduct our research. The Australian research team wishes to thank the Australian Department of Health and Aging, a National Health and Medical Research Council Program Grant, and "beyondblue: the national depression initiative" for support of the Australian survey and Kelly Blewitt for research assistance.
==== Refs
Jorm AF Korten AE Jacomb PA Christensen H Rodgers B Pollitt P Public beliefs about causes and risk factors for depression and schizophrenia Soc Psychiatry Psychiatr Epidemiol 1997 32 143 148 9130866
McKeon P Carrick S Public attitudes to depression: a national survey Ir J Psychol Med 1991 8 116 121
Matschinger H Angermeyer MC Lay beliefs about the causes of mental disorders: a new methodological approach Soc Psychiatry Psychiatr Epidemiol 1996 31 309 315 8952369 10.1007/BF00783418
Lauber C Falcato L Nordt C Rössler W Lay beliefs about causes of depression Acta Psychiatr Scand 2003 108 96 99 10.1034/j.1600-0447.108.s418.19.x
Priest RG Vize C Roberts A Roberts M Tylee A Lay people's attitudes to treatment of depression: results of opinion poll for Defeat Depression Campaign just before its launch BMJ 1996 313 858 859 8870574
Link BG Phelan JC Bresnahan M Stueve A Pescosolido BA Public conceptions of mental illness: labels, causes, dangerousness, and social distance Am J Public Health 1999 89 1328 1333 10474548
Magliano L Fiorillo A De Rosa C Malangone C Maj M Beliefs about schizophrenia in Italy: a comparative nationwide survey of the general public, mental health professionals, and patients' relatives Can J Psychiatry 2004 49 322 30 15198469
Taskin EO Sen FS Aydemir O Demet MM Ozmen E Icelli I Public attitudes to schizophrenia in rural Turkey Soc Psychiatry Psychiatr Epidemiol 2003 38 586 592 14564386 10.1007/s00127-003-0655-y
Kurumatani T Ukawa K Kawaguchi Y Miyata S Suzuki M Ide H Seki W Chikamori E Hwu HG Liao SC Edwards GD Shinfuku N Uemoto M Teachers' knowledge, beliefs and attitudes concerning schizophrenia: a cross-cultural approach in Japan and Taiwan Soc Psychiatry Psychiatr Epidemiol 2004 39 402 409 15133598 10.1007/s00127-004-0758-0
Furnham A Chan E Lay theories of schizophrenia: a cross-cultural comparison of British and Hong Kong Chinese attitudes, attributions and beliefs Soc Psychiatry Psychiatr Epidemiol 2004 39 543 552 15243692 10.1007/s00127-004-0787-8
Dietrich S Beck M Bujantugs B Kenzine D Matschinger H Angermeyer M The relationship between public causal beliefs and social distance toward mentally ill people Aust N Z J Psychiatry 2004 38 348 354 15144513 10.1111/j.1440-1614.2004.01363.x
Mandiberg JM The Japanese mental health system and law: social and structural impediments to reform Int J Law Psychiatry 1996 19 413 435 8968819 10.1016/S0160-2527(96)80010-0
Jorm AF Nakane Y Christensen H Yoshioka K Griffiths KM Wata Y Public beliefs about treatment and outcomes of mental disorders: a comparison of Australia and Japan BMC Medicine 2005 3 12 16004615 10.1186/1741-7015-3-12
Paykel ES Life events, social support and depression Acta Psychiatr Scand Suppl 1994 377 50 58 8053367
Tsuang M Schizophrenia: genes and environment Biol Psychiatry 2000 47 210 220 10682218 10.1016/S0006-3223(99)00289-9
Mackinnon A Jorm AF Hickie IB A national depression index for Australia Med J Aust 2004 181 S52 S56 15462643
Jablensky A McGrath J Herrman H Castle D Gureje O Evans M Carr V Morgan V Korten A Harvey C Psychotic disorders in urban areas: an overview of the Study on Low Prevalence Disorders Aust N Z J Psychiatry 2000 34 221 236 10789527 10.1046/j.1440-1614.2000.00728.x
Tokuyama M Nakao K Seto M Watanabe A Takeda M Predictors of first-onset major depressive episodes among white-collar workers Psychiatry Clin Neurosci 2003 57 523 531 12950708 10.1046/j.1440-1819.2003.01158.x
Ohta Y Nakane Y Nishihara J Takemoto T Ecological structure and incidence rates of schizophrenia in Nagasaki City Acta Psychiatr Scand 1992 86 113 120 1529733
Van Os J Jones PB Neuroticism as a risk factor for schizophrenia Psychol Med 2001 31 1129 1134 11513380 10.1017/S0033291701004044
Radford MH Transcultural issues in mood and anxiety disorders: a focus on Japan CNS Spectr 2004 6 13 15181380
Jorm AF Christensen H Griffiths KM Public beliefs about causes and risk factors for mental disorders: changes in Australia over 8 years Soc Psychiatry Psychiatr Epidemiol 2005 40 764 767 16133745 10.1007/s00127-005-0940-z
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Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-3-291616805810.1186/1476-7120-3-29Case ReportThe fusion band in V1: a simple ECG guide to optimal resynchronization? An echocardiographic case report Gianfranchi Lorella [email protected] Katia [email protected] Federico [email protected] Giorgio [email protected] Paolo [email protected] Division of Cardiology, Ospedale Civile, Cento Via Vicini 2 Cento (Fe) Italy2005 16 9 2005 3 29 29 31 8 2005 16 9 2005 Copyright © 2005 Gianfranchi et al; licensee BioMed Central Ltd.2005Gianfranchi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Patients with left bundle branch block have a preserved right bundle branch conduction and the efficacy of left ventricular pacing could be explained with the fusion between artificial pulse delivered in the left lateral wall and the spontaneous right ventricular activation. Moreover, the efficacy of left ventricular pacing could be enhanced with an optimal timing between the spontaneous right ventricular activation and the left ventricular pulse.
Case presentation
We evaluated a patient (male, 47 yrs) with surgically corrected mitral regurgitation, sinus rhythm and left bundle branch block, heart failure (NYHA class III) despite medical therapy and low ejection fraction (25%): he was implanted with a biventricular device.
We programmed ventricular pacing only through the left ventricular lead.
We defined what we called electrocardiographic "fusion band" as follow: programming OFF the stimulator, we recorded the native electrocardiogram and measured, through the device, the intrinsic atrioventricular interval. Then, atrioventricular interval was progressively shortened by steps of 20 ms down to 100 ms. Twelve leads electrocardiogram was recorded at each step. The fusion band is the range of AV intervals at which surface electrocardiogram (mainly in V1 lead) presents an intermediate morphology between the native left bundle branch block (upper limit of the band) and the fully paced right bundle branch block (lower limit).
The patient underwent echocardiographic examination at each atrioventricular interval chosen inside the fusion band. The following parameters were evaluated: ejection fraction, diastolic filling time, E wave deceleration time, aortic velocity time integral and myocardial performance index.
All the echocardiographic parameters showed an improvement inside the fusion band, with a "plateau" behaviour. As the fusion band in this patient ranged from an atrioventricular delay of 200 ms to an atrioventricular delay of 120 ms, we chose an intermediate atrioventricular delay of 160 ms, presuming that this might guarantee the persistence of fusion even during any possible physiological (autonomic, effort) atrioventricular conduction variation.
Conclusion
In this heart failure patient with left bundle branch block, tailoring of the atrioventricular interval resynchronized myocardial contraction with left ventricular pacing alone, utilizing a sensed right atrial activity and the surface electrocardiographic pattern.
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Background
Patients with heart failure (HF), left ventricular dysfunction and left bundle branch block (LBBB) can be treated with cardiac resynchronization therapy (CRT) [1], which can be obtained with both biventricular pacing or left ventricular (LV) pacing. Good results have been reported with the two types of pacing [2-12].
Patients with LBBB generally have a preserved right bundle branch conduction and the efficacy of LV pacing can be explained with a fusion between the artificial pacing pulse delivered in the left lateral wall and the spontaneous right ventricular (RV) activation. Moreover, the efficacy of LV pacing could be enhanced with an optimal timing between the spontaneous RV activation and the LV pulse [13-15].
Patients with sinus rhythm (SR), HF, left ventricular dysfunction and LBBB can provide a specific trigger for a good timing of ventricular stimulation, namely atrial activity, sensed or paced. In addition, as the atrioventricular (AV) interval can be programmed, the morphology of the QRS complex consequently varies, showing different degrees of fusion. In this context electrocardiogram (ECG) could be used as a marker reflecting the mechanical synchrony of the ventricles.
This case report suggests that a properly timed fusion between the spontaneous RV activation and LV pacing corresponds to a good hemodynamic response of the heart and, therefore, the optimal timing of LV pacing could be determined simply by programming the AV interval on the basis of surface ECG pattern.
Case presentation
We evaluated a patient (male, 47 yrs) with surgically corrected mitral regurgitation, HF (NYHA class III), SR, LBBB and left ventricular dysfunction (ejection fraction 25%) despite optimal medical therapy; he was implanted with a biventricular device with independent ventricular outputs (Contak Renewal ICD-Guidant).
We programmed ventricular pacing only through the LV lead. The device was tracked by the spontaneous atrial activity because of a preserved sinus node function.
We defined what we called electrocardiographic "fusion band" as follows: the stimulator was first programmed OFF to assess the basal ECG of the patient and the intrinsic AV interval, namely the interval between atrial and RV sensing measured through the implanted device, was measured as well. Then, AV interval was progressively shortened by steps of 20 ms down to the lower limit of 80 ms. Twelve leads ECG was recorded at each step. The fusion band is the range of AV intervals at which surface ECG (mainly V1 lead) shows an intermediate morphology between the native LBBB pattern (upper limit of the band) and the fully paced right bundle brunch block (RBBB) pattern (lower limit).
In order to investigate the "fusion band", we chose the following AV values: 200, 180, 160, 140, 120 ms (figure 1).
Figure 1 ECG morphology at different AV intervals. Different ECG morphology corresponding to each programmed AV interval, including the basal ECG of the patient without ventricular pacing. With the progressive shortening of the programmed AV interval, QRS morphology (mainly in V1 lead) changes from LBBB to a RBBB-like trace at the shortest AV interval, corresponding to a complete capture of both ventricles by LV pacing. Different degrees of ventricular fusion between RV spontaneous activation and LV pacing can be seen for AV intervals programmed in the range 200–120 ms.
The patient underwent echocardiographic examination at each AV interval. The following parameters were evaluated: ejection fraction (EF%), diastolic filling time (DFT), E wave deceleration time (DT), aortic velocity time integral (VTI) and myocardial performance index (MPI).
All the echocardiographic parameters showed an improvement at each programmed AV interval with a "plateau" behavior. Within an AV interval of 200 and 120 ms we choose to program the AV interval at an intermediate value of 160 ms (figure 2).
Figure 2 Echocardiographic parameters at each step of AV interval. Ejection fraction (EF%), diastolic filling time (DFT), E wave deceleration time (DT), Aortic Velocity Time Integral (Aortic VTI), Myocardial performance index (MPI).
Discussion
In this patient LV pacing seems to produce an improvement of the echocardiographic parameters at every AV interval inside the fusion band. It is not possible, however, to identify an AV value at which there is a full concordance of all the parameters. As the band of fusion ranges from an AV interval of 200 to 120 ms, we chose an intermediate AV value of 160 ms supposing that this might enable the persistence of fusion even during any possible physiological (autonomic, effort, etc) AV conduction variation. Data from literature support this hypothesis [9] since LV pacing alone seems effective in the mid-term in this patient population. This suggests that the acutely tailored AV interval is a valid option also during the spontaneous variation of the AV conduction. For this reason, LV pacing does not seem to be a good option in patients with AV block, due to the lack of spontaneous RV activation.
The improvement of every echocardiographic parameter inside the fusion band, as compared to the basal conditions, shows a plateau pattern. This has to be confirmed by a large-scale trial. We hypothesize that our method could be applied in the patients undergoing CRT, representing a simple and effective way to program the proper AV interval using only surface and intracardiac ECG analysis.
If further studies will confirm this hypothesis, the programming of the devices would be simplified and made faster without any loss in effectiveness. Moreover this method saves the energy of RV pacing.
The hypothesis of tailoring myocardial resynchronization with LV pacing alone from ECG criteria seems attractive and LV pacing could be proposed as a valid option in patients with sinus rhythm. This approach does not necessarily exclude the presence of RV lead as only patients with LBBB and stable sinus rhythm may do without RV pacing. In fact the possibility of new development of atrial fibrillation in HF patient must be considered: in this case biventricular pacing may be useful as during atrial fibrillation episodes fusion is not constantly present, due to the loss of the reference a trial trigger for LV pacing.
Moreover, patients with indication to implantable defibrillators need RV lead for the appropriate sensing and defibrillation of the arrhythmias.
Conclusion
Our HF patient with SR and LBBB showed an improvement of echocardiographic variables during the ECG "fusion band" obtained with LV pacing. This simple method, only based on ECG criteria, needs to be tested on a large group of patients.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All the authors contributed to the definition of the proposed method. The paper has been written by L.G. The draft has been discussed by all the authors and the final version approved.
Acknowledgements
Written consent was obtained from the patient for publication of the study.
==== Refs
Gregoratos G Abrams J Epstein AE Freedman RA Hayes DL Hlatky MA Kerber RE Naccarelli GV Schoenfeld MH Silka MJ Winters SL ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices – Summary Article: a Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (ACC/AHA/NASPE Committee to Update the 1998 Pacemaker Guidelines) J Am Coll Cardiol 2002 40 1703 1719 12427427 10.1016/S0735-1097(02)02528-7
Cazeau S Leclercq C Lavergne T Walker S Varma C Linde C Garrigue S Kappenberger L Haywood GA Santini M Bailleul C Daubert JC Effects of multisite biventricular pacing in patients with heart failure and intraventricular conduction delay N Engl J Med 2001 344 873 880 11259720 10.1056/NEJM200103223441202
Abraham WT Fisher WG Smith AL Delurgio BD Leon A Loh E Kocovic DZ Packer M Clavell AL Hayes DL Ellestad M Trupp RJ Underwood J Pickering F Truex C McAfee P Messenger J for the MIRACLE Study Group Multicenter InSync Randomized Clinical Evaluation. Cardiac resynchronization in chronic heart failure N Engl J Med 2002 346 1845 1853 12063368 10.1056/NEJMoa013168
Linde C Leclercq C Rex S Garrigue S Lavergne T Cazeau S McKenna W Fitzgerald M Deharo JC Alonso C Walker S Braunschweig F Bailleul C Daubert JC Long-Term Benefits of Biventricular Pacing in Congestive Heart Failure: Results From the Multisite STimulation In Cardiomyopathy (MUSTIC) Study J Am Coll Cardiol 2002 40 111 118 12103264 10.1016/S0735-1097(02)01932-0
Bristow MR Saxon LA Boehmer J Krueger S Kass DA De Marco T Carson P DiCarlo L DeMets D White BG DeVries DW Feldman AM for the Comparison of Medical Therapy Pacing and Defibrillation in Heart Failure (COMPANION) Investigators Cardiac-Resynchronization Therapy with or without an Implantable Defibrillator in Advanced Chronic Heart Failure N Engl J Med 2004 350 2140 2150 15152059 10.1056/NEJMoa032423
Cleland JGF Daubert JC Erdmann E Freemantle N Gras D Kappenberger L Tavazzi L for the Cardiac Resynchronization-Heart Failure (CARE-HF) Study Investigators The Effect of Cardiac Resynchronization on Morbidity and Mortality in Heart Failure N Engl J Med 2005 352 1539 1549 15753115 10.1056/NEJMoa050496
Nelson GS Curry CW Bradley TW Kramer A Declerck J Talbot M Douglas MR Berger RD McVeigh ER Kass DA Predictors of Systolic Augmentation From Left Ventricular Preexcitation in Patients With Dilated Cardiomyopathy and Intraventricular Conduction Delay Circulation 2000 101 2703 2709 10851207
Blanc JJ Etienne Y Gilard M Mansourati J Munier S Boschat J Benditt DG Lourie KG Evaluation of Different Ventricular Pacing Sites in Patients With Severe Heart Failure Results of an Acute Hemodynamic Study Circulation 1997 96 3273 3277 9396415
Blanc JJ Bertault-Valls V Fatemi M Gilard M Pennec PY Etienne Y Midterm Benefits of Left Univentricular Pacing in Patients With Congestive Heart Failure Circulation 2004 109 1741 1744 15023885 10.1161/01.CIR.0000124479.89015.64
Riedlbauchova L Fridl P Kautzner J Peichl P Performance of Left Ventricular Versus Biventricular Pacing in Chronic Heart Failure Assessed by Stress Echocardiography PACE 2004 27 626 631 15125719
Touiza A Etienne Y Gilard M Fatemi M Mansourati J Blanc JJ Long-Term Left Ventricular Pacing: Assessment and Comparison With Biventricular Pacing in Patients With Severe Congestive Heart Failure J Am Coll Cardiol 2001 38 1966 1970 11738301 10.1016/S0735-1097(01)01648-5
Auricchio A Stellbrink C Butter C Sack S Vogt J Misier AR Bocker D Block M Kirkels JH Kramer A Huvelle E Clinical Efficacy of Cardiac Resynchronization Therapy Using Left Ventricular Pacing in Heart Failure Patients Stratified by Severity of Ventricular Conduction Delay J Am Coll Cardiol 2003 42 2109 2116 14680736 10.1016/j.jacc.2003.04.003
Verbeek XA Vernooy K Peschar M Cornelussen RN Prinzen FW Intra-Ventricular Resynchronization for Optimal Left Ventricular Function During Pacing in Experimental Left Bundle Branch Block J Am Coll Cardiol 2003 42 558 567 12906989 10.1016/S0735-1097(03)00641-7
Vogt J Krahnefeld O Lamp B Hansky B Kirkels H Minami K Korfer R Horstkotte D Kloss M Auricchio A Electrocardiographic Remodeling in Patients Paced for Heart Failure Am J Cardiol 2000 86 152K 156K 10.1016/S0002-9149(00)01347-3
Auricchio A Pacing the Left Ventricle: Does Underlying Rhythm Matter? J Am Coll Cardiol 2004 43 239 240 14736443 10.1016/j.jacc.2003.10.028
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Cell ChromosomeCell & Chromosome1475-9268BioMed Central London 1475-9268-4-21616228310.1186/1475-9268-4-2ResearchNon-obstructive azoospermia and maturation arrest with complex translocation 46,XY t(9;13;14)(p22;q21.2;p13) is consistent with the Luciani-Guo hypothesis of latent aberrant autosomal regions and infertility Sills Eric Scott [email protected] Joseph Jinsuk [email protected] Michael A [email protected] Gianpiero D [email protected] Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Atlanta Medical Center, Atlanta USA2 San Francisco Xavier School of Medicine, Kralendijk, Netherlands Antilles3 Reproductive Biology Associates, Atlanta USA4 Cornell Institute for Reproductive Medicine, Weill Medical College of Cornell University, New York USA2005 14 9 2005 4 2 2 17 8 2005 14 9 2005 Copyright © 2005 Sills et al; licensee BioMed Central Ltd.2005Sills 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
To describe clinical and histological features observed in the setting of an unusual complex translocation involving three autosomes (9, 13, and 14) identified in an otherwise healthy male referred for infertility consultation.
Materials and methods
The patient was age 30 and no family history was available (adopted). Total azoospermia was confirmed on multiple semen analyses. Peripheral karyotype showed a 46,XY t(9;13;14)(p22:q21.2;p13) genotype; no Y-chromosome microdeletions were identified. Cystic fibrosis screening was negative. Bilateral testis biopsy revealed uniform maturation arrest and peritubular fibrosis.
Results
Formal genetic counseling was obtained and the extant literature reviewed with the couple. Given the low probability of obtaining sperm on testicular biopsy, as well as the high risk of any retrieved sperm having an unbalanced genetic rearrangement, the couple elected to proceed with fertility treatment using anonymous donor sperm for insemination.
Conclusion
Although genes mapped to the Y-chromosome have been established as critical to normal testicular development and spermatogenesis, certain autosomal genes are now also recognized as important in these processes. Here we present clinical evidence to support the Luciani-Guo hypothesis (first advanced in 1984 and refined in 2002), which predicts severe spermatogenic impairment with aberrations involving chromosomes 9, 13, and/or 14, independent of Y-chromosome status. Additional study including fluorescent in situ hybridization and molecular analysis of specific chromosomal regions is needed to characterize more fully the contribution(s) of these autosomes to male testicular development and spermatogenesis.
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Background
The higher observed frequency of chromosomal abnormality among infertile men compared to the general population provided early evidence that male fertility is influenced by genetic factors impacting gamete development [1]. Many of these genes have been localized to the Y-chromosome, including SRY, AZF, RBM, DAZ, USP9Y, TSPY, DFFRY, and others. A smaller number of genes important to spermatogenesis do not reside on the Y-chromosome [2,3], such as WT1 (chromosome 11), SOX9 (chromosome 17), and DAZLA (chromosome 3), although these autosomal genetic inducers/regulators of gamete development remain less understood. Both Luciani et al [4] and Guo et al [5] have suggested roles for several autosomes in spermatogenesis, although their hypothesis has proven difficult to test. In this report, we present an unusual complex translocation involving portions of chromosomes 9, 13, and 14 associated with azoospermia and discuss its relevance to the Luciani-Guo hypothesis.
Case report
A healthy Caucasian couple was referred for reproductive endocrinology consultation after attempting to achieve pregnancy for >1 yr. The female was age 33 and her evaluation was entirely normal. The husband's semen analysis showed total azoospermia and this was verified on subsequent studies. Cystic fibrosis testing showed no mutation and no Y-chromosome microdeletions were identified (20 cell analysis). Serum testosterone was 415 ng/dl; prolactin was also normal at 6.7 ng/ml. Serum LH and FSH were 2.4 and 2.6 mIU/ml, respectively. Peripheral blood karyotype of 20 cells revealed a non-mosaic 46,XY t(9;13;14)(p22;q21.2;p13) result [Fig. 1]. The patient was adopted and therefore family history was noninformative. Reproductive urology evaluation was sought and outpatient bilateral testis biopsy proceeded without complication. Testicular histology showed peritubular fibrosis and a uniform pattern of Leydig cell hyperplasia with spermatogenic maturation arrest [Fig. 2].
Figure 1 Peripheral karyotype of a male with azoospermia. Complex translocation of chromosomes 9, 13, and 14 was observed; Y-chromosome microdeletion analysis was normal.
Figure 2 Testicular histology observed in 46,XY t(9;13;14)(p22;q21.2;p13) patient. Seminiferous tubules containing only early germ cell forms were noted, but no fully developed sperm (maturation arrest). Peritubular fibrosis and scattered collections of Leydig cells were also present within the interstitium.
Genetic counseling was engaged and the couple was informed about the exceedingly rare nature of the husband's chromosomal aberration. Mathematical modeling to predict possible permutations for gamete recombination [6] confirmed that even if sperm were recovered via testicular biopsy for use with intracytoplasmic sperm injection, the risk for abnormal offspring was unacceptable. Based on these considerations, the couple elected to undergo ovulation induction and intrauterine insemination using anonymous donor sperm.
Discussion
Initial generalizations that male gamete organization and function were directed exclusively by genes on the Y-chromosome required revision when it was observed that certain autosomal anomalies caused gonadal failure, even when the Y-chromosome was intact. In 1984 Luciani et al described a male with non-obstructive azoospermia having a 13q;14q translocation in the absence of any Y-chromosome aberration. From this it was hypothesized that regions specifically involving chromosomes 13 and/or 14 might be required for normal testicular development [4]. A later investigation by Guo et al [5] studied >100 infertile Chinese males and extended the roster of candidate autosomes to include chromosomes 9 and 21. While our patient did not manifest an abnormality of chromosome 21, for many years absent spermatogenesis was considered typical for males with non-mosaic trisomy 21. Additional investigation demonstrated this was not always the case, however [7]. From this it may be concluded that some autosomes are more critical to gonadal structure and/or function than others.
Complex translocations involving more than two chromosomes are highly unusual events in humans [8]. In some carriers of chromosomal aberrations resulting in pachytene configurations with terminal asynaptic autosomal segments, there is a gradual association of asynaptic segments with the X-Y body [9]. Meiotic studies have suggested disordered spermatogenesis may result from such abnormal chromosome synapsis, as any condition interfering with X-Y bivalent formation or X-chromosome inactivation is critical in meiosis. Indeed, the asynapsed regions themselves may trigger developmental arrest of spermatocytes harboring meiotic error [1]. Since simple reciprocal translocations affecting just two chromosomes have deleterious effects on male fertility, it is not surprising that complex translocations involving three or more chromosomes would have drastic and severe consequences for gametogenesis. Even if fertility were maintained for such patients, the risk of genetic error in the offspring would be unacceptably high for most patients [10].
An ideal opportunity to test the Luciani-Guo hypothesis regarding chromosomes 9, 13, and 14 would present whenever these rearrangements occur, although until now there has been no description of rearrangements involving more than two of the implicated autosomes in the same individual. As a further complicating matter, tissue from gonadal biopsy is not always present to complete the evaluation.
This is the first published case of complex translocation involving chromosomes 9, 13, and 14. In association with this exceptional genetic rearrangement, our patient was azoospermic and testis biopsy showed maturation arrest. After a formal consultation with a geneticist, the clinical significance of this genotype was discussed and previous reports describing similar chromosomal abnormalities were reviewed. Based on these data the couple elected to pursue ovulation induction and intrauterine insemination with anonymous donor sperm. While relationships between specific chromosome aberrations and disease conditions should be confirmed by additional study, our data suggest that at present males with this particular complex translocation are poor candidates for surgically retrieved sperm for use with intracytoplasmic sperm injection, since the risk of obtaining genetically abnormal sperm from such individuals is high.
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Antonelli A Gandini L Petrinelli P Marcucci L Elli R Lombardo F Dondero F Lenzi A Chromosomal alterations and male infertility J Endocrinol Invest 2000 23 677 83 11097433
Okabe M Ikawa M Ashkenas J Male infertility and the genetics of spermatogenesis Am J Hum Genet 1998 62 1274 81 9644029 10.1086/301895
Kent-First M Muallem A Shultz J Pryor J Roberts K Nolten W Meisner L Chandley A Gouchy G Jorgensen L Havighurst T Grosch J Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection Mol Reprod Dev 1999 53 27 41 10230814 10.1002/(SICI)1098-2795(199905)53:1<27::AID-MRD4>3.0.CO;2-W
Luciani JM Guichaoua MR Mattei A Morazzani MR Pachytene analysis of a man with a 13q;14q translocation and infertility. Behavior of the trivalent and nonrandom association with the sex vesicle Cytogenet Cell Genet 1984 38 14 22 6200272
Guo J-H Zhu P-Y Huang Y-F Yu L Autosomal aberrations associated with testicular dysgenesis or spermatogenic arrest in Chinese patients Asian J Androl 2002 4 3 7 11907622
Benito C Gallego A A simple method for the estimation of recombination frequencies and genetic distances Cell Mol Biol Lett 2004 9 617 34 15647785
Sheridan R Llerena J JrMatkins S Debenham P Cawood A Bobrow M Fertility in a male with trisomy 21 J Med Genet 1989 26 294 8 2567354
Siffroi JP Benzacken B Straub B Le Bourhis C North MO Curotti G Bellec V Alvarez S Dadoune JP Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI Mol Hum Reprod 1997 3 847 51 9395262 10.1093/molehr/3.10.847
Solari AJ Synaptonemal complex analysis in human male infertility Eur J Histochem 1999 43 265 76 10682264
Grasshoff U Singer S Liehr T Starke H Fode B Schoning M Dufke A A complex chromosomal rearrangement with a translocation 4;10;14 in a fertile male carrier ascertainment through an offspring with partial trisomy 14q13→q24.1 and partial monosomy 4q27→q28 [corrected] Cytogenet Genome Res 2003 103 17 23 15004458 10.1159/000076282
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Cerebrospinal Fluid ResCerebrospinal Fluid Research1743-8454BioMed Central London 1743-8454-2-71617430010.1186/1743-8454-2-7ResearchCerebrospinal fluid may mediate CNS ischemic injury Wang Yanming F [email protected] Judith K [email protected] Guorong [email protected] Sulpicio G [email protected] Shunli [email protected] Yanguang [email protected] Neuroprotection Inc. 100 Cummings Center, Suite 439-C, Beverly, MA 01915 USA2 The Department of Anesthesia, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 USA3 Harvard Medical School and Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston MA 02115 USA and Gwathmey Inc. 763 Concord Avenue, Building E, Cambridge, MA 02138 USA2005 20 9 2005 2 7 7 29 3 2005 20 9 2005 Copyright © 2005 Wang et al; licensee BioMed Central Ltd.2005Wang 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 central nervous system (CNS) is extremely vulnerable to ischemic injury. The details underlying this susceptibility are not completely understood. Since the CNS is surrounded by cerebrospinal fluid (CSF) that contains a low concentration of plasma protein, we examined the effect of changing the CSF in the evolution of CNS injury during ischemic insult.
Methods
Lumbar spinal cord ischemia was induced in rabbits by cross-clamping the descending abdominal aorta for 1 h, 2 h or 3 h followed by 7 d of reperfusion. Prior to ischemia, rabbits were subjected to the following procedures; 1) CSF depletion, 2) CSF replenishment at 0 mmHg intracranial pressure (ICP), and 3) replacement of CSF with 8% albumin- or 1% gelatin-modified artificial CSF, respectively. Motor function of the hind limbs and histopathological changes of the spinal cord were scored. Post-ischemic microcirculation of the spinal cord was visualized by fluorescein isothiocyanate (FITC) albumin.
Results
The severity of histopathological damage paralleled the neurological deficit scores. Paraplegia and associated histopathological changes were accompanied by a clear post-ischemic deficit in blood perfusion.
Spinal cord ischemia for 1 h resulted in permanent paraplegia in the control group. Depletion of the CSF significantly prevented paraplegia. CSF replenishment with the ICP reduced to 0 mmHg, did not prevent paraplegia. Replacement of CSF with albumin- or gelatin-modified artificial CSF prevented paraplegia in rabbits even when the ICP was maintained at 10–15 mmHg.
Conclusion
We conclude that the presence of normal CSF may contribute to the vulnerability of the spinal cord to ischemic injury. Depletion of the CSF or replacement of the CSF with an albumin- or gelatin-modified artificial CSF can be neuroprotective.
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Background
The central nervous system (CNS) including brain and spinal cord is extremely susceptible to hypoxic-ischemic insults compared with peripheral organ systems such as the liver, kidney, lung, or intestines. The mechanism underlying this susceptibility is not completely understood. Many theories have been proposed and intensively investigated, including the involvement of oxygen free radicals, calcium overloading, excitatory amino acid release and nitric oxide [1-3]. These various mechanisms, however, have not been proven conclusively to mediate the vulnerability of the brain and spinal cord to ischemic injury.
In peripheral organ systems, capillaries are relatively permeable to proteins. The associated lymphatic system maintains lymph and interstitial fluid (ISF) protein concentration at about 2 g/dl, and ensures a negative interstitial pressure at about -3 mmHg [4]. In the CNS, the brain and spinal cord are bathed by the cerebrospinal fluid (CSF). Although the CSF system has similarities with the lymphatics, the CSF contains only about 25 mg/dl of protein [5]. In addition, intracranial pressure (ICP) averages about 10 mmHg, resulting in a positive interstitial fluid pressure [4], in contrast to the negative pressure in peripheral tissues. Hence, the CSF system may predispose the CNS to edema. Swelling of cerebral tissue can compress blood vessels inside the Virchow-Robin space leading to a persistent deficit in blood perfusion even after the restoration of blood perfusion, termed a 'no-reflow' or 'low reflow' phenomenon [6]. We therefore hypothesized that the presence of CSF and a positive ICP may explain why the CNS is more vulnerable to ischemia than peripheral organ systems.
The spinal cord has a linear contour, it is therefore possible to create a CSF free environment for the lumbar spinal cord by removing the CSF and adjusting body position. Using a rabbit model of spinal cord ischemia, this investigation distinguished the influence of the CSF from that of ICP, and demonstrated that the addition of albumin or gelatin to the CSF can prevent injury and may be neuroprotective.
Methods
Male New Zealand white rabbits, n = 77, weighing 2.5–3 kg were used (Harlan, Indiana). Animal studies were approved by the institutional animal care and use committee (permission number 2249) and complied with the 'Principles of Laboratory Animal Care' (Guide for the Care and Use of Laboratory Animals, National Institute of Health publication 86-23, 1985).
Pre-ischemia preparation
Each rabbit was anesthetized with isoflurane in 100% oxygen (5% for induction, 1% for maintenance) by facemask. Body temperature was maintained at 37 ± 1°C with a heating blanket. The right femoral artery was catheterized for monitoring mean arterial pressure (MAP) and for acquiring samples for blood gas analysis during surgical procedures. To remove the CSF and to monitor the ICP, a small silicon tube (OD = 0.025, ID = 0.012 inches, Braintree Scientific, MA) was placed in the cisterna magna. Briefly, an incision was made on the dorsal neck to expose the nuchal ridge and dorsal cervical musculature. The atlanto-occipital membrane was exposed by blunt dissection and pierced with a 30 gauge needle. The silicon tube was twisted gently through the hole on the membrane until CSF was noted to pulsate in the catheter. The tube was fixed in position and immobilized to the adjacent muscle by an instant adhesive cyanoacrylate gel (Plastics One, VA). To facilitate removal of the CSF and to administer albumin- and gelatin-modified artificial CSF another silicon tube was placed in the lumbar subarachnoid space. Briefly, a laminectomy was carried out at sacrococcygeal level to expose the lumbar subarachnoid space. The dura was then pierced with a 30 gauge needle. The tube was twisted gently into subarachnoid space through the dura and immobilized with the same adhesive to the adjacent muscle.
Experimental design
Rabbits were randomly divided into 7 groups.
Group 1. Control group (n = 12)
No treatment was given. The ICP was monitored through the small silicon tube in the cisterna magna and maintained at 10 – 15 mmHg by withdrawing or infusing CSF through the tube in lumbar subarachnoid space. Rabbits were subjected to 1 h spinal cord ischemia.
Group 2. CSF depleted group (n = 12)
The objective was to provide the ischemic spinal cord with a relatively CSF free environment. Prior to spinal cord ischemia, the CSF was removed as completely as possible; usually 0.8 – 1.2 ml CSF could be withdrawn. To assist in maintaining a CSF free environment below the lumbar spinal cord, two additional procedures were used. First, while the tube in the cisterna magna was withdrawing CSF, the tube in lumbar subarachnoid space was kept open. Second, after removing the CSF, rabbits were kept in a tilted position with the head down (10 degrees) to prevent any remaining CSF from flowing to the lumbar region during the period of ischemia. After CSF depletion, rabbits were subjected to 1 h spinal cord ischemia. The ICP was monitored and maintained at 0 mmHg during the period of ischemia.
Group 3. CSF replenished group (n = 12)
The objective was to provide the ischemic spinal cord with a normal CSF environment while maintaining ICP at 0 mmHg. Prior to spinal cord ischemia, the CSF was removed as completely as possible as described above. To ensure that CSF was present in the lumbar spinal cord ischemic region, two procedures were used. First, 0.1 ml CSF was immediately replenished through the tube in the lumbar subarachnoid space. This volume was previously determined by withdrawing CSF post mortem from a rabbit weighing 2.5 kg. Second, rabbits were kept in a tilted position with head up (10 degrees) during the period of ischemia to allow any remaining CSF inside cranium to flow into the ischemic region of the spinal cord. After CSF replenishment, the rabbits were subjected to 1 h spinal cord ischemia. The ICP was monitored and maintained at 0 mmHg.
Group 4. Albumin-modified artificial CSF group (n = 12)
The objective of this group was to replace the CSF with albumin-modified artificial CSF in the spinal subarachnoid space. Albumin-modified artificial CSF was made by dissolving 8% bovine albumin (A6003, Sigma-Aldrich) in artificial CSF (Na+ 150 mEq/L, K+ 3.0 mEq/L, Mg++ 0.9 mEq/L, Ca++ 1.4 mEq/L, P 1.0 mEq/L, Cl- 155 mEq/L, glucose 60 mg/dl). Prior to spinal cord ischemia, 2 ml of albumn-modified artificial CSF was quickly flushed in through the silicon tube in the lumbar subarachnoid space and allowed to flow out through the tube in the cisterna magna. Rabbits were then subjected to 1 h spinal cord ischemia, during which time albumin-modified artificial CSF was continuously infused at rate of 2 ml/h. The ICP was monitored and maintained at 10–15 mmHg during the infusion by withdrawing or infusing the albumin-modified artificial CSF from the lumbar subarachnoid space.
Group 5. Gelatin-modified artificial CSF treatment group (n = 12)
The objective of this group was to replace the CSF with gelatin-modified artificial CSF in the spinal subarachnoid space and to compare it with albumin-modified artificial CSF. Gelatin-modified artificial CSF was made by dissolving 1% porcine skin gelatin (G1890, molecular weight between 50,000 – 100,000, Sigma-Aldrich) in artificial CSF. The experiment was performed as described for group 4.
Group 6. (n = 10)
Treatment was the same as group 2, except that all rabbits were subjected to 2 h spinal cord ischemia and the CSF removal procedure was repeated every h.
Group 7. (n = 7)
Treatment was the same as group 2, except that all rabbits were subjected to 3 h spinal cord ischemia and the CSF removal procedure was repeated every h.
Induction of ischemia
After the treatment according experimental design, spinal cord ischemia was induced according to Zivin [7]. Under aseptic operative techniques, a midline abdominal laparotomy of 6–7 cm long was made and the intrarenal aorta was isolated. A Diethrich bulldog clamp (50 mm length, closing force 50 g) was used to cross-clamp the aorta just caudal to the left (lower) renal artery. In the rabbit, this corresponds to the second lumbar vertebrae (L 1–2) level. The aorta was cross-clamped for 1, 2 and 3 h according to the experimental design. Immediately after the removal of the clamp, the abdominal incision was closed in layers. After ischemia, all silicon tubes were disconnected, and the wounds were closed. Rabbits were returned to their cages, provided food and water and allowed to recover for seven days.
Neurological deficit evaluation
After ischemic injury, each rabbit was tested daily for seven days. Motor function of the hind limbs was scored from 0 – 5 according to modified Tarlov's score [8] by an investigator blinded to the experimental group. Score 0: complete recovery, able to hop normally (the hind limbs were able to leave the ground simultaneously when hopping). Score 1: able to hop but wobbly and might fall on their side occasionally. Score 2: able to stand, but unable to hop; Score 3: good movement of the hind limbs, but unable to stand; Score 4: spastic paraplegia with slight movement of the hind limbs; Score 5: spastic paraplegia with no movement to the hind limbs. An animal with a motor function (Tarlov score) ≥ 2 was considered paraplegic.
Histopathological evaluation
After seven days of neurological evaluation, rabbits were euthanized with an overdose of pentobarbital sodium (200 mg/kg) injection via the heart. The spinal cord was perfused with 10% buffered formalin. The spinal cords from lower thoracic level to the lower lumbar level were harvested by multiple laminectomies and fixed in a 10% buffered formalin solution for one week. Spinal cord segments at the lumbar enlargement were embedded in paraffin. Transverse sections were cut at 6 μm and stained with hematoxylin-eosin. The spinal cord lesions at L5 level were scored under the microscope at a magnification of 40× and 100× by an investigator blinded to experimental procedures according to the following criteria [9-11]:
Score 1: Number of vacuolations < 5 in grey matter of the hemi-section.
Score 2: Number of vacuolations ≥ 5 in grey matter of the hemi-section.
Score 3: The architecture of grey matter was necrotic or completely destroyed or replaced by scar tissue. No neurons could be seen.
Visualization of the spinal cord microcirculation
At 23 h after spinal cord ischemia, lumbar spinal cord microcirculation was studied in two rabbits from groups 1–5. Each rabbit received 0.3 g/kg 15% fluorescein isothiocyante (FITC) albumin (Sigma-Aldrich) by i.v. bolus injection [12]. Rabbits were euthanized 2 min after FITC injection by the method described above. Spinal cords were immediately removed and fixed in 10% formalin overnight and transferred to 25% sucrose for 24 h. The tissues were frozen with dry ice. Transverse sections, 100 μm thick, were cut with a microtome at the level of L5, transferred to glass slides and investigated immediately by fluorescence microscopy at 40× magnification.
Statistical analysis
The physiological parameters were analyzed with one-way repeated measures ANOVA (analysis of variance) followed by Tukey test. The neurological deficit scores and the histopathological scores were analyzed with Kruskal-Wallis ANOVA on Ranks followed by the Tukey test. A p value < 0.05 was considered statistically significant.
Results
Physiological parameters at baseline and reperfusion were similar for all experimental groups and there were no significant differences between groups. In addition, there were no significant differences between baseline and reperfusion values for MAP, PO2, PCO2, pH and glucose in any group (Table 1).
Table 1 Physiologic parameters during rabbit spinal cord ischemia. Values are mean ± SD. Baseline physiological values were determined 5 minutes before cross-clamping the aorta. Reperfusion physiological values were determined 5 minutes after releasing the clamp around abdominal aorta. None of the physiological parameters at baseline and reperfusion differed significantly between the various experimental groups. There were no significant differences in MBP, PO2, PCO2 and glucose between baseline and reperfusion in any group. The pH value was slightly decreased after ischemia in each group, however, there were no significant differences either.
Group
1 2 3 4 5 6 7
Variable n = 10 n = 10 n = 10 n = 10 n = 10 n = 10 n = 7
MAP (mmHg)
Baseline 82 ± 12 83 ± 11 86 ± 12 76 ± 7 84 ± 14 79 ± 11 81 ± 15
Reperfusion 80 ± 8 81 ± 11 81 ± 10 76 ± 11 78 ± 14 74 ± 11 73 ± 9
pH
Baseline 7.40 ± 0.03 7.41 ± 0.03 7.40 ± 0.03 7.39 ± 0.02 7.39 ± 0.02 7.40 ± 0.03 7.40 ± 0.03
Reperfusion 7.35 ± 0.02 7.34 ± 0.03 7.35 ± 0.05 7.34 ± 0.03 7.35 ± 0.04 7.33 ± 0.02 7.33 ± 0.03
PaO2 (mmHg)
Baseline 212 ± 19 221 ± 18 225 ± 24 208 ± 22 213 ± 20 226 ± 18 223 ± 24
Reperfusion 225 ± 26 209 ± 20 223 ± 20 212 ± 20 225 ± 21 230 ± 14 234 ± 31
PaCO2 (mmHg)
Baseline 38 ± 3 37 ± 4 38 ± 2 37 ± 3 39 ± 2 37 ± 2 37 ± 3
Reperfusion 35 ± 7 35 ± 3 36 ± 5 35 ± 5 36 ± 3 35 ± 4 34 ± 5
Glucose (mg/dl)
Baseline 131 ± 22 133 ± 27 123 ± 26 128 ± 29 141 ± 25 121 ± 30 126 ± 25
Reperfusion 134 ± 22 143 ± 25 117 ± 24 138 ± 30 144 ± 25 128 ± 26 135 ± 30
The neurological deficit score, taken 24 h after ischemia did not differ from the score obtained seven days after ischemia. Control animals subjected to 1 h of spinal cord ischemia demonstrated permanent paraplegia (100% paraplegic rate, Fig. 1A, group 1-closed circles). The CSF depletion with the ICP maintained at 0 mmHg prevented the development of paraplegia (0% paraplegic rate, Fig. 1A, group 2-open circles). However, replenishment of CSF around ischemic spinal cord (returning a small amount of CSF while maintaining a tilted, head-up position) did not prevent paraplegia, even when the ICP was controlled at 0 mmHg (100% paraplegic rate, Fig. 1A, group 3-closed triangles). Replacing the CSF with albumin- or gelatin-modified artificial CSF under physiological ICP conditions (10–15 mmHg) significantly prevented the development of paraplegia (~20% paraplegic rate, Fig. 1A, groups 4-open triangles, and 5-closed squares).
Figure 1 Effect of CSF removal and replacement of CSF with albumin- and gelatine-modified artificial CSF on rabbit spinal cord ischemia. Group 1 (closed circles). 1 h ischemia; prior treatment: none; ICP: 10–15 mmHg. Group 2 (open circles). 1 h ischemia; prior treatment: CSF depleted; ICP: 0 mmHg. Group 3 (closed triangles). 1 h ischemia; prior treatment: CSF replenished; ICP: 0 mmHg. Group 4 (open triangles). 1 h ischemia; prior treatment: 8% albumin in artificial CSF; ICP: 10–15 mmHg. Group 5 (closed squares). 1 h ischemia:; prior treatment: 1% gelatin in artificial CSF; ICP: 10–15 mmHg. Group 6 (open squares). 2 h ischemia; prior treatment: CSF depleted; ICP: 0 mmHg. Group 7 (closed diamonds). 3 h ischemia; prior treatment: CSF depleted; ICP: 0 mmHg. A. Neurological deficit (Tarlov score) determined at 7 d after spinal cord ischemia. Group 1 vs. groups 2, 4, 5 and 6; Group 3 vs group 2, 4 and 5; Group 7 vs. group 2 and 5 are significantly different when analyzed by Kruskal Wallis ANOVA followed by Dunn's test (p < 0.05). B. Histopathological score determined at 7 d after spinal cord ischemia by H&E staining. Groups 1, 3 and 7 vs. Groups 2, 4, 5 and 6 were significantly different analyzed by Kruskal Wallis ANOVA followed by Dunn's test (p < 0.05).
Similarly, the CSF depletion significantly prevented paraplegia in rabbits subjected to 2 h of spinal cord ischemia (~30% paraplegic rate, Fig. 1A, group 6-open squares), but did not significantly prevent paraplegia after 3 h of ischemia (100% paraplegic rate, Fig. 1A, group 7-closed diamonds).
In all groups, the scores for the severity of the histopathological damage paralleled that for the neurological deficits (Fig 1B). Typical histopathological findings following spinal cord ischemia included necrosis and vacuolation of the neuropil in the grey matter. Without CSF removal, the severe necrosis often destroyed normal architecture of the grey matter resulting in liquefaction (Fig 2A). The depletion of CSF, replacement of CSF with albumin- or gelatin-modified artificial CSF prevented signs of histopathological damage (Fig 2B&C). In contrast, the replenishment of CSF around ischemic spinal cord resulted in necrosis or severe vacuolation of grey matter (Fig 2D&E).
Figure 2 Transverse sections of rabbit lumbar spinal cord after 1 h ischemia (H&E stain). A. Histopathological score 3, from a rabbit of Group 1. Severe necrosis destroys the entire structure of grey matter indiscriminately. B. Histopathological score 1, from a rabbit of Group 2. No apparent spinal cord damage, the grey matter is well preserved. C. Higher magnification of B showing normal morphology of grey matter and neurons. D. Histopathological score 2, from a rabbit of Group 3. The grey matter is lightly stained with many vacuolations of the neuropil. E. Higher magnification of D. Marked vacuolations of the neuropil in the grey matter, and some neurons are triangular with darkly stained shrunken nuclei (arrow).
Normal microcirculation of the spinal cord when visualized by FITC-albumin demonstrated good capillary filling with clear contrasting of fluorescein signals between grey and white matter (stronger in grey matter than in white matter). After ischemia the 'no-reflow' phenomenon was characterized by the absence of capillary filling in both the grey matter and white matter. A 'no-reflow' phenomenon of lumbar spinal cord was demonstrated in control rabbits (Fig 3A). In contrast, normal microcirculation of lumbar spinal cord was observed after ischemia in rabbits with the depletion of CSF, or replacement of CSF by albumin- or gelatin-modified artificial CSF (Fig 3B,D&E). The 'low-reflow' phenomenon was also noticed in rabbits with the replenishment of CSF (Fig 3C).
Figure 3 Transverse sections of rabbit lumbar spinal cord to show the microcirculation after 1 h of ischemia and 23 h reperfusion, revealed by FITC-albumin. A. No CSF removed (Group 1) – the spinal cord demonstrates extremely faint fluorescein signal with absence of capillary filling in both grey matter and white matter, indicating a 'no-reflow' phenomenon. B. Depletion of the CSF (Group 2) – spinal cord section demonstrates intensive fluorescein signals, which are much stronger in grey matter than in white matter, indicating good blood perfusion. C. Replenishment of the CSF (Group 3) – grey matter demonstrates faint fluorescein signal similar to white matter, indicating a marked blood perfusion deficit, i.e. 'low-reflow' phenomenon. D. Albumin-modified artificial CSF replacement (Group 4), and E. Gelatin-modified artificial CSF replacement (Group 5) – capillary filling in both grey matter and white matter are clearly demonstrated by good fluorescein signal, albeit slightly less than that of Group 2, indicating some preservation of blood perfusion.
Discussion
The CNS lacks a lymphatic system; instead it is surrounded by the CSF. The CSF is very different from the lymph in peripheral tissues in at least two aspects: protein concentration and the resultant interstitial fluid pressure.
In peripheral tissues, capillaries are relatively permeable and as a result the ISF contains about 2 g/dl of plasma proteins. It is believed that interstitial proteins and hyaluronic acids form a dense network of proteoglycan filaments that impede fluid flow through the interstitium. Normally the amount of free-flowing fluid, present in the interstitium is small. A low interstitial protein concentration results in an increased amount of free ISF. An elevated concentration of interstitial protein may reduce the free ISF, but it also attracts more fluid, resulting in increased volume. The lymphatic system is the scavenging pathway for interstitial proteins. By regulating the removal of excess protein, the lymphatic system keeps the interstitial protein concentration around 2 g/dl. This ensures limited free fluid and also regulates the ISF volume. Lymph flow reduces ISF volume resulting in negative interstitial pressure. Therefore, the movement of proteins from plasma to ISF and finally to lymph is important for maintaining extracellular homeostasis.
In the CNS, the CSF is secreted by the choroid plexuses that line the cerebral ventricles. Tight junctions linking the adjacent choroidal epithelial cells form the blood-CSF barrier and prevent most large molecules from passing into the CSF from the blood. Therefore the CSF contains an extremely low protein concentration. The choroid plexuses may not be the only sites for CSF production. Milhorat reported that in monkeys with choroid plexuses removed, up to 60% of the CSF is produced from ISF flow out of the brain [13]. The ISF has a bulk flow rate of 0.1–0.3 μl/min/g in rat brain along preferential pathways especially the Virchow-Robin spaces and axon tracts [14]. Formation of ISF is thought to occur by active transport processes at the cerebral capillary [15]. The blood-brain barrier (BBB) prevents proteins from entering the interstitium. Therefore, it is speculated that the ISF in brain, just like the CSF, has a low protein concentration. It is estimated that intracellular protein concentration averages about 16 g/dl in mammalian cells [4]. Therefore water and Na+ in the ISF have the potential to move easily into cells. More importantly, the CSF is contiguous with the ISF, with the Virchow-Robin spaces, serving as a conduit. To make matters worse, the ICP averages about 10 mmHg leading to a positive interstitial fluid pressure. Taken together, these factors make the CNS prone to edema formation. As a result cells in the CNS constantly consume energy to remove excess intracellular fluid in physiological condition. When cell energy is compromised, such as in ischemia, or when the cell membrane is damaged by direct trauma, cells rapidly become swollen, i.e. cytotoxic edema (Fig 4).
Figure 4 Overview of mechanisms underlying the CNS vulnerability to ischemia. Blood brain barrier and Blood-CSF barrier determine the low protein concentration in the cerebrospinal fluid and the interstitial fluid. A large amount of free fluid causes the CNS cells to consume more energy to maintain normal intracellular environment. The positive intracranial pressure facilitates the edema formation. Edema results in 'no-reflow' phenomenon.
By taking advantage of the linear contour of the spinal cord, we were able to verify the role of CSF independently of the influence of ICP. All rabbits in the control group developed permanent paraplegia, necrosis of grey matter and associated 'no-reflow' phenomenon. Depletion of the CSF around the ischemic spinal cord significantly prevented paraplegia and associated pathological changes (group 2). Depletion of the CSF creates a partially CSF free environment in the ischemic spinal cord region. Although depletion of the CSF does not eliminate ISF, it limits the extent to which the ISF can be replenished from the CSF and limits occurrence of edema. It is estimated that the total volume of CSF is about 2.3 ml or 0.67 ml/kg for an adult rabbit, and the CSF production rate is reportedly to be between 7.8–8.8 μl/minute [16-18]. We were only able to remove 0.8–1.2 ml CSF. We believe that the remainder of the CSF may reside inside the cranium as we took several steps to approximate the CSF free environment in the ischemic spinal cord region. The depletion of CSF also reduced the ICP to 0 mmHg. Therefore, in group three, we tested the presence of the CSF around the ischemic spinal cord while maintaining a 0 mmHg ICP. This allowed us to examine the role of CSF without the influence of ICP. The data demonstrated that the CSF itself is detrimental to ischemic spinal cord.
Using albumin-modified artificial CSF to replace the CSF significantly prevented paraplegia even when the ICP was maintained within the physiological range. These data indicate that detrimental effect of the CSF may be largely ascribed to its low protein concentration. A high concentration of protein present in artificial CSF not only provides limited free fluid to ISF, but may also increase the protein concentration in ISF resulting in reduced amount of the free fluid in interstitium. The increase of protein concentration in ISF was less likely to result in volume expansion in the interstitium because of the protein concentration gradient between the CSF and the ISF; any ISF volume increase would be transferred to the protein-modified artificial CSF. We believe that the ultimate reason why protein is important for the extracellular environment may be due chiefly to its water and ion binding capacity. The water binding capacity for albumin is so large that it is estimated that one gram of albumin can bind 18 ml of water [19,20]. A number of physiological functions have been reported for albumin, such as an anti-inflammatory effect [21,22]. However, these alternative functions are less likely to be the cause of the beneficial effect because gelatin, which is mainly comprised of denatured proteins, was equally neuroprotective. Although the albumin and gelatin are both colloid osmotic agents, their efficacies in preventing the spinal cord damage were not likely to be due to their molecular weight, as we found that other colloid osmotic agents, such as 10% Dextran (molecular weight 40, 000–70,000 daltons) and 6% Hetastarch (molecular weight 400,000–550,000 daltons) which were known to create similar colloidal osmotic pressure were ineffective (Y. Wang, unpublished observations). In addition to low protein concentration, many nutrients are also lower in the CSF. For examples, the CSF contains about two third of plasma glucose concentration (CSF: 61 mg/dl; plasma: 92 mg/dl), and it contains about one fifteenth of plasma insulin concentration (CSF: 4 μU/ml; plasma: 20–30 μU/ml) [23-25]. Harkness and co-workers showed that the adenosine 5'-triphosphate (ATP) concentration was about 1 to 20 μmol/l in plasma, however, it was not measurable in the CSF [26]. Muñoz and co-workers reported that the ATP concentration in CSF is only about 16 nM/l [27]. Therefore, additional studies are needed to examine if the deficiency of these nutrients in normal CSF or the presence of any other microconstituents in CSF, may also contribute to vulnerability of the CNS.
Rabbits with albumin- or gelatin-modified artificial CSF replacement maintained with a physiological ICP still showed ~20% paraplegic rate, while depletion of the CSF showed 0% paraplegic rate, although the difference was not statistically significant by Fisher exact test. The effect of depleting the CSF may be partially due to ICP reduction, and the 20% paraplegic rate may be explained by the presence of physiological ICP. Elevated ICP has been known to be clinically detrimental. For example, CSF drainage has been used for almost 50 years to prevent paraplegia during aortic surgery when the aorta needs to be cross-clamped [28,29]. While this approach has been reported to be effective in many studies [30-38], in some examples positive outcomes were not achieved [39-41]. Crawford et al performed a randomized clinical study using CSF drainage in 98 patients that underwent cross-clamping of the aorta during surgery [39]. They controlled the ICP at 10–15 mmHg while draining the CSF, and did not find this approach effective in preventing paraplegia. We believe that this negative outcome might be attributed to the normal level of ICP and a large amount of CSF remaining in the ischemic areas of the spinal cord. These clinical outcomes in conjunction with our studies indicate that both the presence of CSF and physiological ICP are detrimental to ischemic spinal cord. The presence of CSF may be a fundamental issue.
Our experiments demonstrated a clear 'no-reflow' and 'low-reflow' phenomenon, which was accompanied by paraplegia and histopathological damage in the spinal cord. Depletion of the CSF or replacement with albumin- or gelatin-modified artificial CSF prevented the 'no-reflow' phenomenon, associated motor function deficit and histopathological spinal cord damage. Although many investigators reported blood perfusion deficits following brain and spinal cord injuries [42-44], the mechanism was not clear. Our findings suggest that this post-ischemia deficit in blood perfusion may be linked, at least in part, to the CSF associated edema and collapsing of blood vessels in the Vichow-Robin space. This blood perfusion deficit likely blocks collateral circulation and induces a feedback loop contributing irreversible cell death and tissue necrosis (Fig 4).
It has been shown that just 25 minutes of spinal cord ischemia in this rabbit model is enough to cause permanent paraplegia [10,15]. In our experiments, depletion of the CSF prevented spinal cord damage after 1 h of ischemia. With the extension of the ischemic period to 2 h, there was still significant protection of the spinal cord (group 6). Permanent damage of the spinal cord was only seen after 3 h of ischemia (group 7). These results indicate that a greater level of resistance to ischemic injury can be conferred to the spinal cord in a transiently CSF-free environment.
Conclusion
In summary, our findings suggest that the extracellular environment, consisting of a low protein concentration in the CSF and the ISF and a positive interstitial pressure, may determine the vulnerability of CNS to ischemia. This study has determined that depletion of the CSF or replacing the CSF with albumin-, or gelatin-modified artificial CSF in combination with a lowered ICP, provides protection in the setting of ischemic injury to the CNS.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YFW: conceived of the study, designed and coordinated all experiments, participated in spinal cord ischemia experiment in rabbits and drafted the manuscript.
JKG: helped in the preparation of the manuscript and data analysis.
GZ: participated in spinal cord ischemia experiment in rabbits.
SGS: participated in data analysis and helped in the revision of the manuscript.
SH: participated in spinal cord ischemia experiment in rabbits.
YW: participated in spinal cord ischemia experiment in rabbits.
All authors have read and approved the final manuscript.
Acknowledgements
This work was supported in part by NIH grant R43 NS046858 to Yanming F. Wang.
==== Refs
Dirnagl U Iadecola C Moskowiz MA Pathobiology of ischaemic stroke: an integrated view Trends Neurosci 1999 22 391 397 10441299 10.1016/S0166-2236(99)01401-0
Fisher M Schaebitz W An overview of acute stroke therapy. Past, present, and future Arch Intern Med 2000 160 3196 3206 11088079 10.1001/archinte.160.21.3196
De Keyser J Sulter G Luiten PG Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing? Trends Neurosci 1999 22 535 540 10542428 10.1016/S0166-2236(99)01463-0
Guyton AC Hall JE Textbook of Medical Physiology 1998 Philadelphia: WB Saunders Company
Brown PD Davies SL Speake T Millar ID Molecular mechanisms of cerebrospinal fluid production Neuroscience 2004 129 957 970 15561411 10.1016/j.neuroscience.2004.07.003
Ames A Wright RL Kowada M Thurston JM Majno G Cerebral ischemia: II. The no-reflow phenomenon Am J Pathol 1968 52 437 453 5635861
Zivin JA DeGirolami U Spinal cord infarction: a highly reproducible stroke model Stroke 1980 11 200 202 7368250
Tarlov IM Acute spinal cord compression paralysis J Neurosurg 1972 36 10 20 5007267
Yamashita A Matsumoto M Matsumoto S Itoh M Kawai K Sakabe T A comparison of the neurotoxic effects on the spinal cord of tetracaine, lidocaine, bupivacaine, and ropivacaine administered intrathecally in rabbits Anesth Analg 2003 97 512 9 12873946 10.1213/01.ANE.0000068885.78816.5B
Jacobs TP Shohami E Baze W Burgard E Gunderson C Hallenbeck JM Feuerstein G Deteriorating stroke model: histopathology, edema, and eicosanoid changes following spinal cord ischemia in rabbits Stroke 1987 18 741 50 3603601
Weir CJ Zivin JA Lyden PD Inter-relationships between spinal cord blood flow, neuronal death and neurological function in rabbit spinal cord ischemia Brain Research 2002 946 43 51 12133593 10.1016/S0006-8993(02)02822-6
Bottiger BW Krumnikl JJ Gass P Schmitz B Motsch J Martin E The cerebral "no-reflow" phenomenon after cardiac arrest in rats. Influence of low-flow reperfusion Resuscitation 1997 34 79 87 9051828 10.1016/S0300-9572(96)01029-5
Milhorat TH Hammock MK Fenstermacher JD Levin VA Cerebrospinal fluid production by the choroids plexus and brain Science 1971 173 330 332 4997797
Abbott NJ Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology Neurochemistry international 2004 45 545 552 15186921 10.1016/j.neuint.2003.11.006
Rapoport SI Blood-brain barrier in physiology and medicine 1976 New York: Raven Press
Thomas SA Davson H Segal MB Quantification of efflux into the blood and brain of intraventricularly perfused [3H] thymidine in the anaesthetized. Rabbit Exp Physiol 1997 82 139 148 9023512
Pollay M Davson H The passage of certain substances out of the cerebrospinal fluid Brain 1963 86 137 150 13972012
Welch K Secretion of cerebrospinal fluid by choroids plexus of the rabbit Am J Physiol 1963 205 617 624 14065919
Lewis RT Albumin: role and discriminative use in surgery Can J Surg 1980 23 322 328 6998548
Granger DN Gabel JC Drke RE Taylor AE Physiologic basis for the clinical use of albumin solutions Surg Gynecol Obstet 1978 146 97 104 337546
Lang JD JrFigueroa M Chumley P Aslan M Hurt J Tarpey MM Alvarez B Radi R Freeman BA Albumin and hydroxyethyl starch modulate oxidative inflammatory injury to vascular endothelium Anesthesiology 2004 100 51 8 14695724 10.1097/00000542-200401000-00012
Hammel HT Evolving ideas about osmosis and capillary fluid exchange FASEB J 1999 13 213 231 9973310
Ratzmann KP Hampel R Glucose and insulin concentration patterns in cerebrospinal fluid following intravenous glucose injection in humans Endokrinologie 1980 76 185 188 7004864
Ono T Steffens AB Sasaki K Influence of peripheral and intracerebroventricular glucose and insulin infusions on peripheral and cerebrospinal fluid glucose and insulin levels Physiology & Behavior 1983 30 301 306 6342012 10.1016/0031-9384(83)90023-9
Gorden P Roth J Circulating insulins Arch Intern Med 1969 123 237 247 5766249 10.1001/archinte.123.3.237
Harkness RA Coade SB Webster DB ATP, ADP and AMP in plasma from peripheral venous blood Clinica Chimica Acta 1984 143 91 98 10.1016/0009-8981(84)90216-X
Muñoz DJB Thorne PR Housley GD Billett TE Adenosine 5'-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea Hearing Research 1995 90 119 125 8974988 10.1016/0378-5955(95)00153-5
Miyamoto K Ueno A Wada T Kimoto S A new and simple method of preventing spinal cord damage following temporary occlusion of the thoracic aorta by draining the cerebrospinal fluid J cardiovasc surg 1960 16 188 197 13771492
Blaisdell FW Cooley DA The mechanism of paraplegia after temporary thoracic aortic occlusion and its relationship to spinal fluid pressure Surgery 1962 51 351 355 13869747
Juvonen T Biancari F Rimpilainen J Satta J Rainio P Kiviluoma K Strategies for spinal cord protection during descending thoracic and thoracoabdominal aortic surgery: Up-to-date experimental and clinical results – a review Scand Cardiovasc J 2002 36 136 160 12079635
McCullough JL Hollier LH Nugent M Paraplegia after thoracic aortic occlusion: influence of cerebrospinal fluid drainage. Experimental and early clinical results J Vasc Surg 1988 7 153 160 3336121 10.1067/mva.1988.avs0070153
Azizzadeh A Huynh TT Miller CC 3rdSafi HJ Reversal of twice-delayed neurologic deficits with cerebrospinal fluid drainage after thoracoabdominal aneurysm repair: a case report and plea for a national database collection J Vasc Surg 2000 31 592 598 10709075 10.1016/S0741-5214(00)90323-9
Hill AB Kalman PG Johnston KW Vosu HA Reversal of delayed-onset paraplegia after thoracic aortic surgery with cerebrospinal fluid drainage J Vasc Surg 1994 20 315 317 8040958
Widmann MD DeLucia A Sharp J Richenbacher WE Reversal of renal failure and paraplegia after thoracoabdominal aneurysm repair Ann Thorac Surg 1998 65 1153 1155 9564954 10.1016/S0003-4975(98)00040-X
Heller LB Chaney MA Paraplegia immediately following removal of a cerebrospinal fluid drainage catheter in a patient after thoracoabdominal aortic aneurysm surgery Anesthesiology 2001 95 1285 1287 11685001 10.1097/00000542-200111000-00036
Ortiz-Gomez JR Gonzalez-Solis FJ Fernandez-Alonso L Bilbao JI Reversal of acute paraplegia with cerebrospinal fluid drainage after endovascular thoracic aortic aneurysm repair Anesthesiology 2001 95 1288 1289 11685002 10.1097/00000542-200111000-00037
Tsusaki B Grigore A Cooley DA Collard CD Reversal of delayed paraplegia with cerebrospinal fluid drainage after thoracoabdominal aneurysm repair. [Letter] Anesthesia & Analgesia 2002 94 1674 12032060 10.1097/00000539-200206000-00073
Bower TC Murray MJ Gloviczki P Yaksh TL Hollier LH Pairolero PC Effects of thoracic aortic occlusion and cerebrospinal fluid drainage on regional spinal cord blood flow in dogs: correlation with neurologic outcome J Vasc Surg 1989 9 135 144 2911133 10.1067/mva.1989.vs0090135
Crawford ES Svensson LG Hess KR Shenaq SS Coselli JS A prospective randomized study of cerebrospinal fluid drainage to prevent paraplegia after high-risk surgery on the thoracoabdominal aorta J Vasc Surg 1991 13 36 46 1987395 10.1067/mva.1991.25385
Wadouh F Lindemann EM Arndt CF Hetzer R Borst HG The arteria radicularis magna anterior as a decisive factor influencing spinal cord damage during aortic occlusion J Thorac Cardiovasc Surg 1984 88 1 10 6738091
Svensson LG Von Ritter CM Groeneveld HT Richards ES Hunter SJ Robinson MF Hinder RA Cross-clamping of the thoracic aorta. Influence of aortic shunts, laminectomy, papaverine, calcium channel blocker, allopurinol, and superoxide dismutase on spinal cord blood flow and paraplegia in baboons Ann Surg 1986 204 38 47 3729582
Fischer M Hossmann KA No-reflow after cardiac arrest Intensive Care Med 1995 21 132 141 7775694 10.1007/BF01726536
Follis F Miller K Seremin OU Pett S Kessler R Temes T Wernly JA Experimental delayed postischemic spinal cord hypoperfusion after aortic cross-clamping Can J Neurol Sci 1995 22 202 207 8529172
Yamada T Morimoto T Nakase H Hirabayashi H Hiramatsu KI Sakaki T Spinal cord blood flow and pathophysiological changes after transient spinal cord ischema in cats Neurosurgery 1998 42 626 634 9526997 10.1097/00006123-199803000-00033
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Chiropr OsteopatChiropractic & Osteopathy1746-1340BioMed Central London 1746-1340-13-201616805310.1186/1746-1340-13-20Case ReportA multi-modal treatment approach for the shoulder: A 4 patient case series Pribicevic Mario [email protected] Henry [email protected] Macquarie Injury Management Group Department of Health and Chiropractic Macquarie University, 2109, Sydney Australia2 Macquarie Injury Management Group Department of Health and Chiropractic Macquarie University, 2109, Sydney Australia2005 16 9 2005 13 20 20 6 9 2005 16 9 2005 Copyright © 2005 Pribicevic and Pollard; licensee BioMed Central Ltd.2005Pribicevic and Pollard; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
This paper describes the clinical management of four cases of shoulder impingement syndrome using a conservative multimodal treatment approach.
Clinical Features
Four patients presented to a chiropractic clinic with chronic shoulder pain, tenderness in the shoulder region and a limited range of motion with pain and catching. After physical and orthopaedic examination a clinical diagnosis of shoulder impingement syndrome was reached. The four patients were admitted to a multi-modal treatment protocol including soft tissue therapy (ischaemic pressure and cross-friction massage), 7 minutes of phonophoresis (driving of medication into tissue with ultrasound) with 1% cortisone cream, diversified spinal and peripheral joint manipulation and rotator cuff and shoulder girdle muscle exercises. The outcome measures for the study were subjective/objective visual analogue pain scales (VAS), range of motion (goniometer) and return to normal daily, work and sporting activities. All four subjects at the end of the treatment protocol were symptom free with all outcome measures being normal. At 1 month follow up all patients continued to be symptom free with full range of motion and complete return to normal daily activities.
Conclusion
This case series demonstrates the potential benefit of a multimodal chiropractic protocol in resolving symptoms associated with a suspected clinical diagnosis of shoulder impingement syndrome.
ShoulderImpingement SyndromeMulti-modal TreatmentChiropractic
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Background
Practitioners of manual therapy commonly encounter patients presenting with shoulder pain and symptoms associated with rotator cuff pathology. Shoulder pain is the most common extraspinal complaint encountered in primary care clinics, and in clinical frequency is exceeded only by low back and neck pain [1]. Many shoulder conditions are associated with dysfunction of the rotator cuff [2-4].
Rotator cuff disorders represent a complex clinical entity requiring a thorough understanding and knowledge of shoulder anatomy, biomechanics and the functional relationship of the shoulder to nearby spinal structures including the cervical and thoracic spines.
Rotator cuff disorders commonly occur secondary to repetitive overuse (occupational or overhead throwing sports), which contributes to micro traumatic changes within rotator cuff tissue [5]. In addition, a single macro traumatic episode (fall on outstretched hand) can cause injury to rotator cuff tissue [5]. The normal aging process will also negatively influence the rotator cuff mechanism [2].
The most common source of shoulder pain originates from the rotator cuff tendons, with the most prevalent clinical diagnosis being impingement syndrome of the supraspinatus tendon [2-4,6].
Before discussing our case series it is important to review some important elements of taking a history and performing a shoulder physical examination. Certain clinical features may alert the practitioner to potentially serious causes (red flags) of shoulder pain, which constitute possible contra-indication to manual therapy [7,8] (Table 1). Other (yellow flag) features of the clinical history may affect the outcome of manual therapy and therefore recovery [7,8] (Table 2). A differential diagnosis list for shoulder pain [9] is seen in Table 3.
Table 1 Alerting features of a possible serious condition (red flag), which may present with shoulder pain [7,8].
POSSIBLE SERIOUS CAUSES OF SHOULDER PAIN (RED FLAGS)
Signs of infection (fever) Violent trauma
History of drug abuse Swelling
Weight loss Pain at rest
Age over 50 Night sweats
History of previous malignancy History of fall
Constant, non mechanical pain No precipitating event (for onset)
Palpable deformities of bone/tissue HIV
Widespread neurological symptoms/signs
Table 2 Possible features that may affect manual therapy outcome and ultimate patient recovery for patients presenting with shoulder pain (yellow flags) [7,8].
YELLOW FLAGS
Previous history of shoulder pain
Personal problems (alcohol, financial, marital)
Compensable injury
Unrealistic expectation of therapy
Long term absence from sport work
Belief that shoulder pain is dangerous
Dissatisfaction
Table 3 Describes the differential diagnosis for shoulder pain [9].
Referred pain from musculoskeletal sources Cervical facet joints
Thoracic facet joints
Myofascial pain syndromes
Referred pain from visceral sources Lungs
Gallbladder
Heart
Diaphragm
Neuropathies Brachial plexus neuropathies
Peripheral neuropathies
Radicular pain Cervical nerve root compression
Table 4[9] shows sources of shoulder pain mostly derived from local structures within the shoulder, whether due to trauma, overuse, arthritides or disease.
Table 4 Describes the sources of shoulder pain derived from local structures [9].
Trauma Fracture
Dislocation
Tendon rupture
Overuse Inflammation (tendinitis, bursitis)
Capsular sprains
Arthritides Osteoarthritis
Rheumatoid variants
Other Infection
Neoplasm
This paper will discuss a common cause of shoulder pain and its largely unreported multi-modal conservative management in a chiropractic setting. This management will include pertinent aspects of the patient history, physical examination, differential diagnosis for shoulder pain as well as its management in 4 cases.
Case Presentations
Four presentations
A case of shoulder pain in a fit 42-year-old Caucasian male is presented. The pain was located diffusely in the postero-lateral aspect of the right shoulder and started gradually 4–6 weeks prior to presentation. No causative event was reported, although workplace activities required the patient to repetitively lift files above the shoulder level onto a shelf. Of note was the mention of a particularly busy period (increased intensity and duration) at work prior to the onset of pain.
The patient described the pain as being of a constant nagging and aching sensation with an intensity of 3/10 on the visual analogue scale (VAS). He also reported an intermittent sharp and catching sensation in the same location on shoulder abduction, with an intensity of 6/10 (VAS scale). No referred pain, or other neurological symptoms were reported, although he did report subjective weakness of the shoulder during elevation above shoulder level and inability to use the right arm comfortably.
Holding his arm on top of the steering wheel aggravated the pain when driving, as did sleeping on his right side, and also combing his hair. He described that heat packs provided short-term relief of pain. The patient reported no prior shoulder problems, no use of medication, and his medical, family and social history were otherwise unremarkable.
Physical examination of the right arm produced pain and restriction of movement at 50 degrees of right external rotation in the neutral position, with restriction and pain at 90 degrees of abduction. Both movements were guarded. An impingement sign was present, as confirmed by a positive Hawkins test. Hawkins test involves positioning the arm at 90 degrees of flexion with subsequent internal rotation. In addition Neers impingement test gave slight discomfort. Neer's impingement test is performed with the patient sitting as the practitioner stands behind the patient with one hand supporting the scapula to prevent scapula rotation and the other hand holding the forearm. The shoulder is brought into maximum flexion with a small degree of internal rotation. The test is considered positive if there is pain in the last 10–15 degrees of flexion. Pain is produced because the greater tuberosity is compressed against the anterior acromion or coracoacromial ligament, hence this test may aggravate an inflamed bursa (subacromial), the supraspinatus tendon or the anterior structures of the coracoacromial arch [10].
Muscle testing revealed slight weakness of the right infraspinatus muscle (Grade lV of V) and also right latissimus dorsi. Other routine shoulder tests revealed no abnormal findings (including instability testing, glenoid labrum testing, lateral slide test and muscle tests).
On palpation muscle spasm was noted in the right infraspinatus muscle and to a lesser extent the right rhomboid, supraspinatus and upper trapezius when compared to the other side. Significant focal tenderness was palpated over the rotator cuff insertion on the greater tuberosity of the humerus. Specific joint motion palpation revealed likely lateral flexion restriction of the right C5/6 lower cervical facet joint and left T2/3 thoracic facet joint with immobility of the right acromio-clavicular joint in an inferior direction.
The patient presented with X-rays, revealing no abnormalities.
A likely working diagnosis of a Primary Grade 2 Posterolateral Rotator Cuff Impingement (Neer classification-Table 5[11]) was determined.
Table 5 Neer classification of impingement [11].
STAGE l Involving oedema and haemorrhage
STAGE ll Involving fibrosis and tendonitis
STAGE lll Involving degeneration (bone spurs) and tendon rupture
A second patient presenting was a slightly overweight 32 years old caucasian female with right-sided shoulder pain located superior, and in the postero-lateral aspect of the shoulder. The pain started 2 weeks prior to presentation after practising certain manual therapy manoeuvres of the lumbar spine at university. The patient was practising lumbar spine and sacro-iliac pisiform contact posterior-anterior manipulation. During this the shoulder is placed repetitively in a combined position of adduction, flexion and internal rotation. The patient described the pain as being a sharp, shooting sensation, intermittent, dependent on motion, with an intensity of 7/10 (VAS scale).
A diffuse aching sensation was also reported in the right upper deltoid region (so-called "military badge"). The pain was aggravated by elevation of the arm and sleeping on the right side. Relief was obtained by applying ice and taking anti-inflammatory/analgesic medication (Ibuprofen). The patient reported no prior shoulder problems, no general use of medication; her medical, family and social history were otherwise unremarkable.
Physical examination of the right shoulder revealed slight postero-lateral pain in the shoulder on external rotation and abduction. External rotation was restricted at 60 degrees and abduction at 90 degrees. Impingement was elicited with the Hawkins test and with the Neer's test. Other routine shoulder tests revealed no abnormal findings.
On palpation muscle spasm was notionally present in the right rhomboid major, upper trapezius, supraspinatus and particularly the infraspinatus. Trigger points were noted in the infraspinatus muscle with reproduction of the upper arm pain upon specific pressure. Motion palpation revealed likely right acromio-clavicular and sternoclavicular joint fixation, left T3/4 and right C5/6 lateral flexion restriction. The patient presented with plain film radiographs, which revealed no abnormality.
A likely working diagnosis of Grade 2 Primary Impingement of the rotator cuff (Neer classification-Table 5[11]) was determined. The working diagnosis also included the presence of an active infraspinatus myofascial pain syndrome.
The third patient was a slightly apprehensive 29-year-old Caucasian male with right-sided diffuse anterior and superior shoulder pain. The pain started gradually over an 8–10 week period, with the intensity being most prevalent during the 2 weeks prior to presentation. The patient was employed as a factory worker; a job that required combined repetitive shoulder movements and periods of administrative keyboard work.
The pain was described as a constant, deep, dull and nagging ache with an intensity of 5/10 (VAS scale). No neurological symptoms were reported, there were no dermatomal/sclerotomal pain referral patterns, although a slight diffuse aching sensation was mentioned in the right elbow and more prominently right "military badge" area. Together with the shoulder pain the patient reported a less intense (4/10) dull sensation specifically at the base of the cervical spine on the right and a vague headache like sensation at the base of the skull.
The right shoulder felt subjectively weaker with inability to lift the arm above shoulder level without pain. The pain was aggravated by specific arm postures and lying on the right side. There was no pertinent medical/family/social history.
Examination revealed a painful arc with onset of pain at 70 degrees abduction, external rotation being restricted at 70 degrees with a catching sensation at the end of motion. Reproduction of the pain was elicited with a Hawkins test and on supraspinatus muscle testing ("Empty can" test) revealing a grade 4 weakness and pain. Other routine shoulder tests revealed no abnormal findings.
Right cervical rotation restriction (65 degrees) was noted on the right, with a right Kemps joint stress test (combined right cervical rotation, lateral flexion and extension) reproducing the low cervical pain but no shoulder pain.
Palpation revealed muscle tenderness in the right supraspinatus, upper trapezius, levator scapulae and infraspinatus muscle groups. A trigger point was palpated in the infraspinatus muscle, which upon applying pressure reproduced the right upper arm diffuse ache. Palpating the rotator cuff insertion on the humerus and coracoacromial ligament caused significant tenderness. Motion palpation revealed likely joint restriction at the right C5/6 cervical facet joint, T2/3 and acromio-clavicular joint. Of interest was the postural presentation of a "rounded shoulder" and increased thoracic kyphosis.
A likely primary working diagnosis of a Grade ll Primary Rotator Cuff Impingement (Neer classification-Table 5[11]) with Supraspinatus tendonosis was determined, with secondary involvement of the cervical and thoracic spines.
The fourth patient presenting was a 40-year-old Caucasian female. She presented with right-sided anterior shoulder pain, which was nagging, aching and accompanied by a catching sensation on specific movements. The aching pain was constant with an intensity of 6.5/10 (VAS scale), while the catching pain was slightly more intense at 8/10. No neurological sensations were reported. The patient reported a diffuse aching pain in the posterior aspect of the shoulder over the scapula. Nothing relieved the pain, while arm elevation, driving, prolonged sitting behind the computer and poor posture made the pain worse.
The pain started 4 days prior to presentation after spending most of the weekend cleaning the walls at home with a sponge prior to painting. The patient had not had this pain before although due to her work (accountant) she often complains of posterior shoulder tension. The patient had been treated previously for an unrelated complaint (right sided sacroiliac area pain). The medical, family and social histories were unremarkable.
The physical examination revealed restriction in external rotation (60 degrees), and abduction with pain/catching at 90 degrees. Internal rotation was also tight and sore especially with the Hawkins test. The impingement sign was present with reproduction of the anterior pain with a Hawkins and Neers test.
Scapula dysfunction was also noted with a positive right-sided lateral glide test. It should be noted that no major difference was seen with the lateral glide test on the previous 3 patients.
Of importance was the postural presentation of anteriorly rotated shoulders, increased thoracic kyphosis and forward head carriage. A scoliotic curve was also noted with an apex convex to the right in the mid thoracic region. Palpation revealed muscle spasm in the right posterior shoulder girdle muscles with increased muscular tension and sensitivity to palpation in the right supraspinatus and infraspinatus compared to the left. Infraspinatus palpation revealed local muscle spasm with a reproduction of the posterior ache on specific pressure. Increased tenderness was noted whilst palpating the coracoacromial ligament and supraspinatus insertion on the humerus. Specific joint motion palpation revealed likely restriction in the right C5/6 joint, T3/4 and acromioclavicular joint.
A likely working diagnosis of a Grade ll, Primary Shoulder Rotator Cuff Impingement (Neer classification- refer to Table 5[11]) was determined. Of note was the secondary contribution of the scapula to this process. The working diagnosis also included the presence an active infraspinatus myofascial pain syndrome.
The interventions
The 4 patients were admitted to a multimodal treatment protocol, which included the following interventions: soft tissue therapy, ultrasound phonophoresis, manipulation and exercise.
All of the patients received soft tissue therapy that involved the application of ischaemic pressure to the supraspinatus and infraspinatus muscles, as well as the rhomboids, upper trapezius and levator scapulae. The application involved palpating the muscle bellies and applying a sustained pressure into areas of muscle spasm until a release of the barrier of resistance was felt. Release meaning the relaxation of the point of muscle spasm with a decrease in the sensitivity and muscle tone after re-palpating the area. The pressure was applied repetitively, using a myofascial T-bar (a plastic, T shaped hand held tool with a rubber tip attached to the end in contact with the skin). Care was taken not to cause increased discomfort to the patient (to the level of pain tolerance).
Longitudinal and transverse friction massage was applied to the posterior tenomuscular junction of the infraspinatus muscle, the coracoacromial ligament (postero-inferior aspect) and the insertion of the supraspinatus on the greater tuberosity of the humerus. The friction massage application was achieved by palpating the capsular or tendinous adhesions and frictioning over its surface with the practitioner's index finger. This was maintained until friction anaesthesia was achieved and the patient could not feel any discomfort. A new point was then chosen and the process repeated. Once again care was taken to not cause excessive discomfort to the patient. At the end of the treatment sessions ice application was advised at a frequency of three applications of 15 minutes with two 20-minute breaks.
Ultrasound phonophoresis was applied to the areas that previously underwent friction massage with a topical corticosteroid [1% sigmacort]. Ultrasound was applied with a continuous wave form for 7 minutes at a setting of 2.2 W/cm2 to the rotator cuff insertion on the anterior-inferior aspect of the humerus and posterior inferior aspect of the acromioclavicular joint.
Peripheral thrust manual manipulation was applied to the glenohumeral joints in external rotation (progressive) and inferiorly to the acromioclavicular joint and anterior to posterior to the sternoclavicular joint in all of the patients where a likely motion restriction was detected.
Mechanically assisted manipulations were also used with the Activator 2 apparatus in humeral external rotation or inferior through the AC joint. This particular technique was chosen for one of the female patients (fourth patient as an alternative) who expressed concern with peripheral manual manipulation after the first treatment session as an alternative technique.
Diversified spinal manipulations were used to manipulate the thoracic and cervical spines at the level of T3/4 and C5/6. All patients were given a basic exercise program with initial emphasis on isometric strengthening of the supraspinatus and infraspinatus muscles. This was implemented once a reduction in pain and improved range of motion was noted at a frequency of 4 sets of 10 repetitions, 2–3 times per day. Theraband (extendable elastic) exercises were also implemented at the same frequency after the initial isometric strengthening period. This also included shoulder shrugs, wall push-ups and scapula retraction exercises.
Patient 1 was treated for a total of 5 visits, patient 2 was treated 4 times, patient 3 was treated 5 times, and patient 4 was treated 4 times.
At the end of the last treatment session (5 and 4 treatments respectively) a repeat physical examination revealed a full and painless range of motion with no subjective symptoms, and negative orthopaedic testing (Hawkin's and Neer's). Patient 1 was seen 4 weeks later for a new and unrelated complaint, who after questioning reported no shoulder complaints (pain). Full range of motion was maintained. Patient 2 was contacted via the phone and upon questioning also reported no subjective pain and full return to normal activities at 1 month post treatment. Patient 3 was followed at 4 and 8 weeks after the last treatment revealing no subjective and objective symptoms. Patient 4 was seen at 4 and 8 weeks with no symptoms of impingement reported and no objective findings.
Discussion and Conclusions
Rotator cuff impingement and or tendonosis is a common disorder encountered in a primary health care setting [12-15]. Perhaps, less in chiropractic practises as opposed to medical and physiotherapy. To date, there are no data investigating the prevalence of shoulder pain in the chiropractic setting. This may be due to the lack of general public awareness about the scope and capabilities of chiropractors to be involved in management of non-spinal disorders or simply the public making another choice. This condition presents a challenge to the chiropractor due to its prevalence, and its possible close interrelationship with the spine.
A major reason for documenting this treatment protocol is to encourage the development of future clinical guidelines for chiropractors and to encourage the expansion of their treatment range to include peripheral disorders.
Another goal of this report is to highlight that multimodal management is often required to address the painful shoulder and not to determine or show which treatment approach or particular therapy was more effective. The four patients in this paper were managed with a treatment protocol that included a number of therapies. The literature [16-22] suggests that the multimodal approach is an appropriate method for the successful conservative management of shoulder problems.
The cervical and thoracic spines should be reviewed as a possible factor associated with rotator-cuff dysfunction. As an example consider the slumping posture in a competitive swimmer. Others and we hypothesise that the rounded shoulders and increased thoracic kyphosis places increased demands on the rotator cuff and contributes to the impingement process [23]. A possible mechanism for this hypothesis is as follows: the posture may alter the mechanical function (orientation) of the scapula and humerus, leading to muscular imbalances, abnormal movement patterns during glenohumeral elevation with associated weakness of the posterior cuff muscles. Therefore this may lead to a loss of force couple at the glenohumeral joint with resultant repetitive humeral head impingement [23-25].
The outcome measures for the study included improvement of pain, return to pre-treatment activities, and restoration of full active and passive movements. The outcome measures were mainly subjective in nature and dependent on the response of the patients and the practitioner's skill in conducting the orthopaedic reassessment, therefore allowing an element of examination bias. This particular shortcoming may be improved by using more sensitive scoring systems that can be accurately reproduced by different observers such as the subjective shoulder rating system [26], UCLA scoring system [27], or the highly sensitive Constant/Murley functional assessment of the shoulder [28].
Although frequently advocated for outcomes based assessment, goniometric measurement for the shoulder remains questionable. Williams et al [29] studied 22 observers who used three different types of goniometers to assess the range of abduction and visual estimation. The results demonstrated visual estimation to be the most reliable method. Moderate inter-observer reliability was also demonstrated in a study by Bostrom et al [30] where range of motion was measured using a goniometer.
This report presents an approach that combines aspects of traditional forms of chiropractic, physiotherapy and medicine in the conservative management of certain shoulder pain.
The individual therapies that were used in this multimodal treatment protocol have been shown to be useful in the management of shoulder pain both singularly and in combination [18,19,31-36].
Of the electro-modalities the apparatus used was ultrasound. Some authors routinely advocate the usage of ultrasound in conjunction with other modalities and report positive outcomes [3,16,35]. The physiologic benefits of ultrasound have been attributed to its thermal actions; these involve an increase in peripheral blood flow, increased tissue metabolism and greater tissue extensibility [37].
The use of ultrasound for shoulder pain and its effect on soft tissue structures of the shoulder has been studied extensively in the literature. A recent study by Nykanen [36] investigating pulsed ultrasound treatment of the painful shoulder in a randomised, double blind and placebo controlled study, showed no differences in outcome between the treatment and placebo groups at the end of the trial period. However, when the ultrasound was used to complement treatment the patients reported a significant subjective improvement in symptoms. A further study by Downing [35], and Perron et al [38], also showed no apparent benefit from ultrasound therapy. None of these studies demonstrated statistically significant results supporting ultrasound therapy. A recent review of the literature conducted by Van der Windt [39] also concluded that there is little evidence that ultrasound therapy is effective for soft tissue disorders of the shoulder. By contrast to the above studies the subjects in this paper were treated with a 3MHz setting plus phonophoresis that may have influenced the outcome measures. Nevertheless the efficacy and effectiveness of ultrasound for shoulder pain remains in doubt.
In this study the subjects were also treated with an ultrasound technique known as phonophoresis. Phonophoresis involves the movement of a medication through intact skin into the underlying soft tissue, by ultrasonic pertubation [37]. By using ultrasound a topical corticosteroid cream can be successfully delivered across the skin with a view to reducing the inflammation and pain associated with the more superficial soft tissue injuries and disorders [40]. Davick [40] showed in his study corticosteroid medication penetration through to the epidermal layer of skin, and further into the stratum corneum. The medication used to treat the subjects was a topical corticosteroid – Sigmacort 1%. This approach combined with the therapeutic effects of ultrasound appeared subjectively to have a beneficial effect as a treatment adjunct.
There is some evidence reporting the positive effects of phonophoresis. Griffin et al [41] conducted a double blind study comparing the effects of phonophoresis and ultrasound in 102 patients with various shoulder complaints. Of the subjects receiving phonophoresis 68% showed significant improvement in range of motion and pain as opposed to 28% in the ultrasound group.
In 1999 one paper by chiropractors investigated the benefits of phonophoresis. Gimblet et al [16], reported treating two subjects with calcific tendonitis by using soft tissue therapy, phonophoresis and manipulation. Both subjects at the end of the treatment protocol experienced complete resolution of symptoms.
Transverse friction massage has been advocated by a number of authors in the management of shoulder disorders [19,34]. Hammer describes friction massage as a technique where an involved muscle, tendon or ligament is massaged by applying pressure with a reinforced finger [19,34]. The transverse motion across the involved tissue and the resultant hyperaemia are said to be the chief healing factors of friction massage [19,34]. The transverse action is said to prevent the formation of scar tissue while longitudinal friction effects the transportation of blood and lymph [19].
The traumatic hyperaemia is postulated to release histamine and bradykinins resulting in vasodilation and reduction of oedema [34]. Friction massage is said to stimulate the proliferation of fibroblasts and collagen fibre realignment with cross linkages [39].
It is reported that up to two weeks are required for mature cross-links to form [24]. In the acute stage a light friction is suggested while in the chronic condition, a stronger pressure may be required [34]. Hammer [19] also describes the successful management of a chronic bursitis by the use of soft tissue friction massage.
The management of the subjects in this paper also included orthopaedic, motion assessment and treatment of spinal structures including the cervical and thoracic spines. Diversified spinal adjustments were directed at the identified hypo mobile motion segments of the cervical and thoracic spines. This included assessment and adjustment of the glenohumeral joint in restricted planes of motion.
It is postulated that abnormal thoracic and cervical spine postural alignment (with any associated spinal joint fixation) may alter the resting position of the scapula contributing to problems of the rotator cuff musculature [23]. In our cases changes in the lateral spinal curves were particularly noted in the third and fourth patients [23].
Abnormal spinal curves can result from chronic poor posture which may result in shoulder girdle muscle imbalance, altered muscle length tension relationships, joint incongruity, ligamentous laxity, changes in arthrokinematics and gross shoulder motion [23].
As noted by many clinicians a commonly related postural condition is that associated with anterior head carriage associated with rounded shoulders [19,23]. This type of postural deviation often causes a compensatory extension at the atlanto-occipital articulation, reversal or flattening of the cervical lordosis, thoracic kyphosis, protraction of the scapulae with the inferior angle of the scapula moving medially whilst the glenoid fossa moves anterior and inferior, and finally internal rotation of the humerus.
As a result, muscle imbalances of the shoulder girdle may occur. These potentially include parascapular muscle weakness, winging of the scapula, altered scapula position, and scapula dysrhythmia [10,23]. Also, weakness of the posterior rotator cuff muscles may influence the force couple mechanism at the glenohumeral joint causing a resultant upward shear of the humeral head during elevation of the arm.
During shoulder elevation the dominant force vector is provided by the deltoid muscle and in a superior direction. Under normal circumstances the cuff muscles will counter this superior shear in the opposite direction, creating a stabilizing and compressive action of the humeral head with respect to the glenoid during elevation. A diagrammatic representation of the gleno-humeral force couple [42] is seen in Figure 1. With cuff weakness (even slight) the force couple may be altered enabling an abnormal upward displacement of the humeral head and the impingement of the subacromial structures and the humeral head against the under surface of the acromion [10,23].
Figure 1 The glenohumeral force couple. The resultant force (action) of the rotator cuff muscles results in compression and inferior glide of the humeral head during elevation. (RA = resultant action, Deltoid, SS = Supraspinatus, SSc = Subscapularis, IS = Infraspinatus and TM = Teres Minor) [42].
Repetition of this process may cause irritation of pain producing structures creating shoulder pain syndromes. In order to address the abnormal force couple and its potentially causative mechanism, specific exercises were introduced to help restore strength and muscular functioning of the glenohumeral joint and scapula articulations. (That is, once motion was normalised).
It is acknowledged that a significant weakness in this case series is the lack of imaging using diagnostic ultrasound to confirm the diagnosis of impingement or indeed some other cause for the pain. We encourage a further study of the treatment protocol described above. This study should be a randomised controlled trial and include diagnostic ultrasound confirmed impingement.
Successful management of rotator cuff impingement and related shoulder pain syndromes should include the consideration of potential sources of shoulder pain. Also the function of the implicated structures in global shoulder function should be reviewed. This should include the associated structures of the scapulohumeral, scapulothoracic articulations, the cervical and the thoracic spine.
This paper highlights a successful outcome for 4 subjects with clinically diagnosed shoulder impingement syndrome after receiving a multimodal treatment approach in a chiropractic setting.
Authors' contributions
MP provided treatment to the subjects, participated in the design and helped draft the manuscript.
HP conceived of the study, participated in its design and helped to draft and edit the manuscript. All authors read and approved the manuscript.
Acknowledgements
No source of funding was used in the preparation of this manuscript. The authors have no conflict of interest that is directly relevant to the content of this manuscript.
==== Refs
Calliet R Shoulder Pain 1991 3 FA Davis Company-Philadelphia 1 50
Morison DS Greenbaum BS Einhorn A Shoulder impingement Orthop Clin Nth Am 2000 31 285 293 10.1016/S0030-5898(05)70148-6
Almekinders LC Impingement syndrome Clin Sports Med 2001 20 491 504 11494837 10.1016/S0278-5919(05)70265-9
Michener LA McClure PW Kardune AR Anatomical and biomechanical mechanisms of subacromial impingement syndrome Clin Biomech 2003 18 369 379 10.1016/S0268-0033(03)00047-0
Iannoti JP Evaluation of the painful shoulder J Hand Therapy 1994 7 77 83
Dalton S Clinical examination of the shoulder Baillieres Clinical Rheumatology 1989 3 453 474
Brox J Shoulder pain: Best Practise and Research Clinical Rheumatology 2003 17 33 56 12659820
Australian Cochrane Collaboration Acute Shoulder Pain Evidence Based Management of Acute Musculoskeletal Pain 2003 Australasian Acute Pain Guidelines Group. Australian Academic Press; Brisbane 119 155
Souza TA Shoulder girdle complaints Differential Diagnosis for the Chiropractor 1997 Gaithersberg. Aspen 141 172
Hammer WI The Shoulder Functional soft tissue examination and treatment by manual methods 1999 Gaithersberg. Aspen 35 135
Neer CS Impingement lesions Clin Orthop 1983 173 70 77 6825348
Gupta R Leggin BG Ianotti JP Results of surgical repair of full thickness tears of the rotator cuff Orthop Clin North Am 1997 28 241 248 9113719 10.1016/S0030-5898(05)70283-2
Van der Windt Koes BW de jong BA Bouter LM Shoulder disorders in general practise: incidence, patient characteristics, and management Ann Rheum Dis 1995 54 959 964 8546527
Almekinders LC Impingement syndrome Clin Sports Med 2001 20 491 504 11494837 10.1016/S0278-5919(05)70265-9
Michener LA McClure PW Kardune AR Anatomical and biomechanical mechanisms of subacromial impingement syndrome Clin Biomech 2003 18 369 379 10.1016/S0268-0033(03)00047-0
Gimblet PA Saville J Ebrall P A conservative management protocol for calcific tendinitis of the shoulder J Manipulative Physiol Ther 1999 22 622 627 10626706
Pink MM Tibone JE The painful shoulder in the swimming athlete Orthop Clin North Am 2000 31 247 261 10736394 10.1016/S0030-5898(05)70145-0
Shrode LW Treating shoulder impingement using the supraspinatus synchronization exercise J Manipulative Physiol Ther 1994 17 43 53 8138733
Hammer WI The use of transverse friction massage in the management of chronic bursitis of the hip or shoulder J Manipulative Physiol Ther 1993 16 107 111 8445352
Bang MD Deyle GD Comparison of supervised exercise with and without manual physical therapy for patients with shoulder impingement syndrome J Orthop Sports Phys Ther 2000 30 126 137 10721508
Conroy DE Hayes KW The effect of joint mobilization as a component of comprehensive treatment for primary shoulder impingement syndrome J Orthop Sports Phys Ther 1998 28 3 14 9653685
Leahy PM Altered biomechanics of the shoulder and the subscapularis Chiropractic Sports Med 1991 5 62 66
Greenfield B Catlin P Coats P Green E McDonald J North C Posture in patients with shoulder overuse injuries and healthy individuals J Orthop Sports Phys Ther 1995 21 287 295 7787853
Culham EC Peat M Functional anatomy of the shoulder complex J Orthop Sports Phys Ther 1993 18 342 350 8348135
Kelly BT Kadrmas WR Speer KP The manual muscle examination for rotator cuff strength: an electromyographic investigation Am J Sports Med 1996 24 581 588 8883676
Kohn D Geyer M The subjective shoulder rating system Arch Orthop Trauma Surg 1997 116 324 328 9266033
Soldatis JJ Moseley JB Etminan M Shoulder symptoms in healthy athletes: A comparison of outcome scoring systems J Shoulder Elbow Surg 1997 6 265 271 9219131 10.1016/S1058-2746(97)90015-X
Constant CR Murley AHG A clinical method of functional assessment of the shoulder Clin Orthop Rel Res 1987 214 160 164
Williams JG Callaghan M Comparison of visual estimation and goniometry in determination of a shoulder joint angle Physiotherapy 1990 76 655 657
Bostrom Charms-Ringdahl K Nordemar R Clinical reliability of shoulder function assessment in patients with rheumatoid arthritis Scand J Rheumatol 1991 20 36 48 2011714
Roodman WU Etiologies of shoulder impingement syndrome in competitive swimmers Chiropractic Sports Med 1989 3 27 31
Frieman BG Albert TJ Fenlin JM Rotator cuff disease: a review of diagnosis pathophysiology, and current trends in treatment Arch Phys Med Rehabil 1994 75 604 609 8185458
Morrison DS Frogameni AD Woodworth P Non-operative treatment of Subacromial Impingement Syndrome J Bone Jt Surg 1997 79 732 737
Hammer WI Friction massage; from Functional soft tissue examination and treatment by manual methods 1999 Gaithersberg: Aspen 463 478
Downing DS Weinstein A Ultrasound therapy of subacromial bursitis Phys Ther 1986 66 194 199 3511478
Nykanen M Pulsed ultrasound treatment of the painful shoulder a randomised, double-blind, placebo controlled study Scan J Rehab Med 1995 27 105 108
Mantone JK Burkhead WZ Noonan J Jr Non-operative treatment of rotator cuff tears Orthop Clin North Am 2000 31 295 311 10736398 10.1016/S0030-5898(05)70149-8
Perron M Malouin F Acetic acid iontophoresis and ultrasound for the treatment of calcifying tendinitis of the shoulder: a randomised controlled trial Arch Phys Med Rehabil 1997 78 379 384 9111457 10.1016/S0003-9993(97)90229-X
Van der Windt D Van der Heijden G Van der Berg S Gerben ter R de Winter AF Bouter LM Ultrasound therapy for musculoskeletal disorders: a systemic review Pain 1999 81 257 271 10431713 10.1016/S0304-3959(99)00016-0
Davick JP Martin RK Albright JP Distribution and deposition of tritated cortisol using phonophoresis Phys Ther 1988 68 1672 1675 3186792
Griffin JE Echternach JL Price RE Touchstone JC Patients treated with ultrasonic driven hydrocortisone and with ultrasound alone Phys Ther 1967 47 594 601 4859775
Donatelli RA Impingement syndrome and impingement related instability Physical therapy of the shoulder 1997 3 Churchill Livingstone 229 256
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Clin Mol AllergyClinical and molecular allergy : CMA1476-7961BioMed Central London 1476-7961-3-131615330510.1186/1476-7961-3-13ResearchSequence homology: A poor predictive value for profilins cross-reactivity Sankian Mojtaba [email protected] Abdolreza [email protected] Nazanin [email protected] Mahmoud [email protected] Immunobiochemistry Lab, Immunology Research Center, Bu-Ali Research Institute, Mashhad, Iran2 Molecular biology Lab, Immunology Research Center, Bu-Ali Research Institute, Mashhad, Iran2005 10 9 2005 3 13 13 28 6 2005 10 9 2005 Copyright © 2005 Sankian et al; licensee BioMed Central Ltd.2005Sankian 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
Profilins are highly cross-reactive allergens which bind IgE antibodies of almost 20% of plant-allergic patients. This study is aimed at investigating cross-reactivity of melon profilin with other plant profilins and the role of the linear and conformational epitopes in human IgE cross-reactivity.
Methods
Seventeen patients with melon allergy were selected based on clinical history and a positive skin prick test to melon extract. Melon profilin has been cloned and expressed in E. coli. The IgE binding and cross-reactivity of the recombinant profilin were measured by ELISA and inhibition ELISA. The amino acid sequence of melon profilin was compared with other profilin sequences. A combination of chemical cleavage and immunoblotting techniques were used to define the role of conformational and linear epitopes in IgE binding. Comparative modeling was used to construct three-dimensional models of profilins and to assess theoretical impact of amino acid differences on conformational structure.
Results
Profilin was identified as a major IgE-binding component of melon. Alignment of amino acid sequences of melon profilin with other profilins showed the most identity with watermelon profilin. This melon profilin showed substantial cross-reactivity with the tomato, peach, grape and Cynodon dactylon (Bermuda grass) pollen profilins. Cantaloupe, watermelon, banana and Poa pratensis (Kentucky blue grass) displayed no notable inhibition. Our experiments also indicated human IgE only react with complete melon profilin. Immunoblotting analysis with rabbit polyclonal antibody shows the reaction of the antibody to the fragmented and complete melon profilin. Although, the well-known linear epitope of profilins were identical in melon and watermelon, comparison of three-dimensional models of watermelon and melon profilins indicated amino acid differences influence the electric potential and accessibility of the solvent-accessible surface of profilins that may markedly affect conformational epitopes.
Conclusion
Human IgE reactivity to melon profilin strongly depends on the highly conserved conformational structure, rather than a high degree of amino acid sequence identity or even linear epitopes identity.
food allergymelonprofilincross-reactivityepitope
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Introduction
Profilins are well-known ubiquitous cytoskeleton proteins which are thought to be a link between the microfilament system and signal transduction pathways [1]. Profilin was first recognized as an allergen in birch pollen, called Bet v 2 [2]. Currently, plant profilins have been shown to be highly cross-reactive allergens that bind IgE antibodies of patients with food and tree pollen allergy [3]. Furthermore, profilins were recognized as causing allergic reaction to pear, peach, apple, melon, tomato, celery, pumpkin seeds, and peanut [4]. Several studies addressing the cross reactivity of IgE antibodies to conservative plant allergens have shown that profilins account for some of the fruit-fruit [5], fruit-plant pollen [6], and latex-food syndromes [7]. For example, it seems profilins are involved in the celery-mugwort-spice syndrome and cross-reactivity between ragweed pollen and cucurbitaceous family [8,9].
Recently, cDNA coding for a number of profilins were characterized and expressed as recombinant allergens. The tertiary structures of some of these profilins have been determined by x-ray crystallography [10]. These data provide new perspectives to the molecular basis of the cross-reactive epitopes of the profilins. Previously, we have identified, cloned and expressed melon (Cucumis melo) major allergen, and this allergen was introduced to the International Union of Immunological Society (IUIS) allergen nomenclature subcommittee as Cuc m 2. We observed melon-related fruits such as watermelon, cucumber and cantaloupe and found little clinical cross-reactivity with melon [11]. The aim of this study was to investigate cross-reactivity of rCuc m2 with other plant profilins and the role of the linear and conformational epitopes in these IgE cross-reactivities.
Materials and methods
Patient's sera
Individuals (n = 24) who complained of clinical symptoms after ingestion of melon were recruited at the Department of Immunology and Allergy of Ghaem Hospital Mashhad, Iran. Seventeen out of 24 patients (10 women, 7 men, mean age 34 years) were included. Diagnosis was established from clinical history and skin prick tests. The skin prick test (SPT) was performed according to the guideline of the subcommittee on skin tests of the European Academy of Allergology and Clinical Immunology [12]. The sera were collected from all of the subjects, which had a clinical history of allergic reaction to melon. A control group (n = 15) with no history of allergic disease and negative skin prick tests to melon was also selected.
Allergenic extracts
After washing the fruits, the seeds were removed and the inner part of pulp isolated. Homogenized in a blender and extracted in phosphate-buffer (1:10 w/v) 100 mM (pH 8.2) containing 1% (w/v) polyvinyl pyrrolidone, 10 mM ethelene diaminetetraceticacid (EDTA), and 10 mM diethyldithiocarbamate (DIECA). The slurry was centrifuged (15000 g) for 30 min at 4°C and fractionated in the range of 30% to 60% saturation of (NH4)2SO4 to enrich melon profilin. The pellet was dissolved and extensively dialyzed against phosphate-buffer 100 mM pH 7.4 (4°C, 72 h) and freeze-dried. Some of the lyophilized samples were reconstituted in distilled water (1/10 w/v) and glycerinated for skin testing. Cynodon dactylon (Bermuda grass) and Poa pratensis (Kentucky blue grass) pollens (Sigma) were extracted as described previously [13]. Allergen extract of kiwi and banana were prepared as described previously by Moller et al [14]. Presence of profilin in all of the extraction was proven by immunoblot analysis using peroxidase conjugated rabbit polyclonal antibody against saffron pollen profilin (kindly provide by F. Shirazi, Bu-Ali Research Institute, Mashhad, Iran) (data not shown).
Cloning, expression and purification of rCuc m 2
Total RNA was extracted from 1 g of fine powder from melon pulp grounded under liquid nitrogen by means of the Concert™ plant RNA purification kit (Invitrogen). First-strand cDNA was synthesized from 2 μg total RNA using a first-strand cDNA synthesis Kit (Fermentas) with a Oligo (dT)18 as primer. The Cuc m 2 coding region was amplified with Pfu DNA polymerase (Fermentas), using two specific primers. According to the sequence of Cuc m 2 (GenBank accession number: AY271295), the 5' primer (5'-TCACATATGTCGTGGCAAGTTTACGTCG-3') mimics the first six codons and introduces an NdeI restriction site (underlined). The 3' primer (5'-AAGCTCGAGGCCCTGATCAATAAGATAATC-3') mimics the last seven codons, excluding the stop translation codon, and introduces an Xho I restriction site (underlined). After PCR amplification, the 400-bp product was ligated into pET21b+ (Novagen). The fidelity of the cloned product was verified by sequencing. The resulting pET21b+/Cuc m 2 construct was transformed into BL21 (DE3) strain of Escherichia coli. Expression and purification of rCuc m 2 were carried out as described previously [15]. Purified rCuc m 2 was then subjected to reducing SDS-PAGE, and eletroblotted on PVDF membrane.
rCuc m 2-specific IgE an inhibition ELISA
The wells of the ELISA microplate (Nalgen Nunc International) were coated with 100 μl of recombinant melon profilin (rCuc m 2) at a concentration of 50 ng/well in coating buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6) at 4°C for 16 h. After blocking with 150 μl of 2% BSA in PBS at 37°C for 30 min., the plates were incubated with 100 μl of patients sera for three hours at RT followed by incubating with a goat biotinylated anti-human IgE (KirKeggard & Perry laboratories) diluted 1/1000 in PBS containing 1% BSA for 2 hours. The wells were then incubated for 1 h with strepavidin horseradish peroxidase-labeled (Sigma) diluted 1/1000 in PBS containing 1% BSA. Each incubation step was followed by 5 washes with PBS-T (PBS containing 0.05% Tween 20). Enzyme reaction was performed using tetramethyl benzidine (TMB)/H2O2 as the substrate. The reaction was stopped by 3 M HCl after 30 minutes at RT in darkness and the absorbance was read at 450 nm. Results were expressed as optical density (OD) units. Based on the mean value of 15 normal sera (<0.3 OD unites), OD value of greater than 0.6 were considered positive.
In order to assess relatedness of rCuc m 2 to profilins from other fruit and pollen, ELISA inhibition was carried out as follows: 100 μl of a pooled serum comprising five sera from subjects showing IgE antibodies to rCuc m 2 preincubated with 100 μl of different concentrations of extracts of Cynodon dactylon (Bermuda grass) and poa pratensis (Kentucky blue grass) pollen, melon, watermelon, banana, peach, cantaloupe, tomato and grape, rCuc m 2 and BSA for 2 hours at room temperature. This solution was then added to a flat-bottomed microtiter plate that had been coated with rCuc m 2 (50 ng/well). The ELISA procedure thereafter was the same as described for measurement of melon allergen-specific IgE.
SDS-PAGE and immunoblotting analysis
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of melon extract and rCuc m 2 was performed according to laemmli [16] using a separation gel of 15% acrylamide under reducing conditions. Separated protein bands were electro-transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore Corp., Bedford, MA, U.S.A.), essentially by the method of Towbin et al [17].
Immunodetection was carried out on PVDF after treatment with methanol for 15 sec and blocking with Superblock at 4°C for 16 h. Membranes were probed with individual sera from melon-allergic patients (diluted 1/5 in PBS containing 1:10 v/v blocking buffer) or with sera from non-allergic subjects for 4 h (overnight for IgE immunoblot of total extract) at room temperature. Stripes are then washed 4 times for 5 min with 0.05% Tween-20 in PBS and incubated for 2 h with a rabbit anti-human IgE polyclonal antibody conjugated with peroxidase (DAKO) diluted 1/2000 in PBS containing blocking buffer (1:10 v/v). After washing, the peroxidase reaction was developed with Super Signal West Pico Chemiluminescent substrate (Pierce) for 5 min, and IgE-binding proteins were detected by ECL-hyperfilm (Amersham Pharmacia Biotech) after exposure for 1 min.
Fragmentation of rCuc m 2 and immunoblotting analysis
To investigate the role of the linear and conformational epitopes in the IgE binding to the rCuc m 2, a combination of chemical cleavage and immunoblotting techniques was used. Amino acid sequence analysis of Cuc m 2 revealed an Asp-Pro site at the position of 57–58 that makes it susceptible to cleavage by pH 2.5. Partial acid hydrolysis was carried out according to the protocol described by Inglis [18]. Briefly, 50 μl of rCuc m 2 (1 mg/ml) was added to 150 μl formic acid and incubated for 24–48 h at 37°C and room temperature. The resulting fragments were separated by tricine-SDS-polyacrylamide gel and visualized by silver staining [19]. Immunodetection of separated protein bands was carried as described above. This immunoblotting analysis was performed with a pooled serum from five melon allergic patients (No: 2, 6, 8, 13 and 14) that showed IgE immunoblot reactivity with 14.5 kDa component of melon extract and r Cuc m 2.
Alternatively, the membrane was blocked with 5% skim milk and incubated with a peroxidase conjugated rabbit polyclonal antibody against saffron pollen profilin at a 1:1000 dilution in PBS containing 2.5% skim milk. The peroxidase reaction was developed as described above.
Structure prediction and modelling
The deduced amino acids sequence of Cuc m 2 was subjected to a BLAST similarity search. A mulitple alignment of the homologous allergens sequences was performed by BioEdit and modified manually when necessary [20]. The percentage identities were determined by comparison of the amino acid sequences after multiple sequence alignment (Fig. 3).
Solvent accessibility and charge distribution of an antigen surface may play prominent roles in immunoreactivity of a epitope. Therefore, in order to display the theoretical effect of amino acid differences between rCuc m 2 and other profilins on the solvent accessibility and charge distribution of the rCuc m 2 surface area, comparative models of tomato, watermelon and melon profilins were generated using the Internet server Swiss Model . The profilin models were built using the X-ray structure of 1g5uB as template. This protein has, respectively, 77.9%, 82.4% and 74.8% sequence identity with melon, tomato and watermelon profilin. The program ZMM was then used with the above constraints to minimize the conformational energy of the proteins [21]. The ZMM uses the Amber all-atom force field [22]. The AMBER force field with a cut-off distance of 8 Å has been used to minimize conformational energy in the space of generalized coordinates including torsion and bond angles. Low-energy conformations were searched by the Monte Carlo minimization method [23]. Monte Carlo trajectories were terminated when 500 sequential energy minimizations did not improve the lowest-energy conformation. Calculations and analysis of low-energy conformers were performed using the ZMM molecular modeling package. The essential accuracy and correctness of the models were evaluated using PROCHECK and WHAT-IF program from online Biotech Validation Suite . All molecular models were viewed and examined for accessible and electrostatic energy of the protein surface using the Swiss Pdb Viewer program. We have ignored solvating effects and used Coulomb law for the calculations of the electrostatic energy.
Results
The seventeen patients suffering from melon allergy were included in our study. Case histories in respect to melon allergy are summarized in Table 1. Oral allergy syndrom and rhinoconjunctivitis were the most prominent manifestations on ingesting melon (94 and 58%, respectively). Sera from 11 of 17 (64%) patients showed increased IgE reactivity to rCuc m 2. Therefore, the melon profilin, rCuc m 2, was identified as a major allergen. Melon allergic individuals also showed clinical features of allergic reaction to fruit from various botanical families such as grape (58%) and tomato (35%).
Table 1 Clinical data, rCuc m 2-specific IgE levels and SPT responses of the selected patients with allergy to melon
Patient No. Age (years) Sex Symptoms* Allergy to other fruits rCuc m 2 Specific IgE (ODξ) SPT with melon extract (mm)
1 29 F R Grape, Kiwi 0.68 5
2# 52 M RC, OAS, D, U, G Grape 0.97 12
3 24 F RC, OAS Tomato 0.72 5
4 30 M RC, E, SI, OAS, U Grape 0.84 8
5 31 M OAS, C Kiwi,Tomato 0.63 5
6# 28 M RC, OAS, SI Tomato, grape, peach, zucchini, cantaloupe, 1.2 8
7 44 M RC, OAS, SI, G Cantaloupe, Kiwi 0.65 5
8# 43 F RC, OAS, SI, C Walnut, Spice 1.02 8
9 30 F R, OAS Grape <0.3 4
10 24 F RC, OAS, SI, C Grape, Tomato, zucchini, Cantaloupe 1.32 4
11 46 M R, OAS, C, D ND <0.3 5
12 39 F RC, OAS, U, SI Grape, Tomato <0.3 3
13# 28 F RC, OAS, SI, C, E Tomato 0.83 10
14# 27 F R, OAS Fig, grape, zucchini 0.94 5
15 45 F OAS Zucchini, watermelon <0.3 4
16 21 M R, OAS Zucchini, grape, watermelon <0.3 8
17 39 F RC, OAS, U, SI, D Grape, garlic <0.3 10
* C, cough; D, dyspnea; E, eczema; R, rhinitis; RC, rhinoconjunctivitis; G, gastrointestinal symptoms; SI, Skin itching; U, urticaria; OAS, Oral allergy syndrome (OAS; defined as the onset of immediate oral itching with or without angioedema of the lips and oral mucosa); ND, not determined. #; Patients' sera were selected for inhibition assays. ξ; OD, Optical density.
Sera from patients no; 2–4, 6, 8,13 and 14 that indicated highest level of specific IgE against rCuc m 2 in ELISA were selected for melon extract immunoblotting. Patients' sera no. 2, 6, 8, 13 and 14 (Table 1) reacted only with the 14.5 kDa component of melon extract (Data not shown). To prepare the inhibition assay pool of sera, reactivity of all of these sera with melon profilin was confirmed by a positive IgE immunoblot reactivity to rCuc m 2. Inhibitions of IgE binding to rCuc m 2 by other plant profilins are represented in Fig. 1. All of the melon, Bermuda grass pollen, peach, tomato and grape extracts revealed significant inhibition of IgE binding to rCuc m 2, and cantaloupe extract showed less significant inhibition. In contrast, watermelon, banana and poa pratensis indicated no notable inhibition.
Figure 1 Inhibition of the binding of IgE antibodies in sensitized serum to immobilized rCuc m 2. Inhibition was assayed by a competitive ELISA method. The pooled sera (1: 5 dilution) was preincubated for 1 h with an equal volume of various concentrations from each extraction solution which was made in PBS before adding to the plate coated with rCuc m 2 (50 ng/well). Sample concentrations are expressed as those in preincubation mixture. Inhibition with BSA was used as negative control (not shown).
The best result for acid hydrolysis was achieved by 24 hours incubation at 37°C. The fragments of acid hydrolyzed rCuc m 2 were resolved into two distinct bands (10 and 6 kDa). In addition, a 14.5 kDa protein band appeared as a complete rCuc m 2 molecule that was not affected by acid hydrolysis (Fig. 2, at the left). The hydrolyzed rCuc m 2 was assessed with the rabbit polyclonal antibody against saffron pollen profilin and a pooled serum of five melon-sensitive individuals in immunoblotting analysis. Immunoblotting analysis with rabbit polyclonal antibody shows the reaction of the antibody to the 14.5, 9, and 6 kDa protein bands (Fig. 2, on the right). In contrast, IgE-blotting displayed only a major IgE-binding band at approximately 14.5 kDa to which pooled serum reacted (Fig. 2, in the middle).
Figure 2 SDS-PAGE and immunoblot analysis of acid hydrolyzed rCuc m2. silver stained-SDS gel electrophoresis of rCuc m2 after incubation with 75% formic acid for 24 h and 48 h at room temperature (F24) and 37°C (F*24 and F*48). "A" and "B" arrow indicate protein band with molecular mass of approximately 6 and 9 kDa. Figure in the middle shows IgE-immunoblotting of two concentration of F*48 using a pooled serum of melon profilin-sensitized individuals (in the middle) and figure on the right display immunoblotting of the same sample with rabbit polyclonal anti-saffron antibody.
Figure 3 Comparison of Cuc m 2 with different plant profilins, including watermelon (Citrullus lanatus), tomato (Lycopersicon esculentum), Bermuda grass (Cynodon dactylon), banana (Musa acuminate), peach (Prunus persica) and latex (Hevea brasiliensi). Amino acid sequence identity of Cuc m 2 with other members of profilin family are indicated at the end of each amino acid sequence. Areas covering experimentally determined sequential IgE-reactive epitopes are underlined.
The deduced protein sequence of Cuc m 2 was subjected to a BLAST similarity search that showed the highest degrees of identity with profilins from the following sources: Citrullus lanatus (Watermelon), Ricinus communis (Castor bean), Phaseolus vulgaris (Green bean), Hevea brasiliensis (Latex), Lycopersicon esculentum (Tomato), Capsicum annuum (Pepper), Prunus persica (Peach), Cynodon dactylon (Bermuda grass), respectively. Figure 3 shows an alignment of the Cuc m 2 amino acid sequence with profilins of other plants.
Three-dimensional structure of the tomato, watermelon and melon profilins are shown in figure 4 and 5. The models were evaluated in terms of stereochemical and geometric parameters such as bond lengths, bond angles, torsion angles, G-factor and packing environment, and they were found to satisfy all stereochemical and geometric criteria. No residue was located in the disallowed regions of the Ramachandran map. After energy minimization of the models, the overall conformational energy of comparative models of tomato, watermelon and melon profilins are -765, -792 and -709 kcal/mol, respectively. Main-chain Cα atoms of 1g5uB, melon, watermelon and tomato profilin superimpose with an RMS deviation of 0.80, 0.77 and 0.82 Å, respectively. Superimposing of the 3-dimensional models of the melon, watermelon, tomato and latex profilin (1g5uB) showed nearly the same tertiary structure. Alignment of the three-dimensional model of watermelon and melon indicated most of the alignment diversity located on the accessible area of watermelon profilin (Fig. 4). In addition to accessible area of molecule surface, amino acid differences among profilins influence – the electric potential of the solvent-accessible surface of profilins (Fig. 5).
Figure 4 Three-dimensional view of melon profilin model. (A) Amino acid differences with watermelon profilin indicated in red on the ribbon diagram of Cuc m 2 model, H2 shows second α-helix. (B) Most of these amino acid differences located at the solvent accessible area of the Cuc m 2 surface and displayed in light blue color.
Figure 5 (A) Superimposing of tomato (red ribbon), watermelon (green ribbon) and melon (yellow ribbon) profilin models. Electrostatic potentials at the surface of melon (B) watermelon (C) and tomato (D) profilin models. Blue represents positive potentials and red represents negative potentials. Orientations of B, C and D models are the same as in part A.
Discussion
Allergen immunotherapy and diagnosis rely on the use of high quality natural allergenic products. However, apart from improved standardization and quality control, there have been few significant innovations in allergen immunotherapy in recent years. In the last decade, There has been remarkable progress in the molecular biology of allergens and more than a hundred food allergens have been cloned and expressed in the prokaryotic, yeast and eukaryotic expression systems. More over, classification of allergens in to the groups based on similarity provides an optimistic prospective to diagnosis and treatment with a small panel of cross-reactive allergens which reflect a high number of allergens. The cross-reactivity of allergens has to be well characterized to define panels of cross-reactive allergens, the pattern of clinical sensitivities and the probability of novel foods being allergen [24,25].
Several studies have focused on establishing the actual patterns of allergen cross-reactivity. In some cases high sequence homology is related to pan-allergenicity as in lipid transfer proteins [26], while in other cases high homology does not result in cross reactivity as in birch and carrot cyclophilins [27]. In this study, we aimed to define cross-reactivity rules in profilins. The result of a sequence homology search reveals high similarity among profilins. Despite high sequence similarity among profilins, our study indicated that high homology between two profilins does not necessarily results in their cross reactivity. Alignment of amino acid sequences of Cuc m 2 and watermelon showed up to 89 percent identity (Fig 3). However, there were only two patients with history of allergy to watermelon in 17 allergic individuals to melon (Table 1). This lack of clinical cross-reactivity between melon and watermelon was confirmed by inhibition experiments (Fig. 1). Although the profilin sequence of cantaloupe, the other fruit belonging to the same family as melon is not available, it seems that only a little cross reactivity can be found between these two according to our results (Fig. 1, Table 1). Interestingly, extracts of peach, tomato, grape and Cynodon dactylon inhibit IgE binding to Cuc m 2 nearly the same as melon extract. The rCuc m 2 showed lower identity with profilins of these plants than with the watermelon profilin. According to continuous epitope mapping and structural analysis of birch and sunflower pollen profilins, the amino acid composition of each B cell epitope was located at the 1–7, 39–46, 98–107 and 105–114 positions [28-30]. Comparison of melon and watermelon profilin amino acid sequences revealed no significant differences at the continuous epitope sites (Fig. 3). Therefore, it would be advisable to assess if conformational epitopes are involved in IgE binding to rCuc m 2. In order to define the role of the continuous and discontinuous epitopes in IgE binding to melon profilin, we used a combination of chemical cleavage and immunoblotting techniques. Cleavage of melon profilin into two fragments destroyed human IgE binding of both fragments and only whole Cuc m 2 showed IgE-binding activity. In contrast, rabbit polyclonal anti-Cuc m 2 showed similar binding activity to Cuc m 2 fragments (Fig. 2). It seems rabbit polyclonal antibody and human IgE recognize distinct epitopes on the profilin molecules. These experiments confirmed findings that indicated sunflower pollens and melon profilins lost their reactivity with the pooled sera of patients with melon allergy after treatment with pepsin [31]. The study of Rihs et al. also demonstrated that only the full-length soybean profilin was able to bind with IgE antibodies and any of the three overlapping recombinant fragments of soybean profilin comprising amino acid residues 1–65, 38–88, and 50–131 did not show significant binding reactivity [32].
We used 3D structural modelling to construct models of profilin allergens and explain these results. Most of amino acid differences between watermelon and melon profilins were located at the accessible site of α-Helix (especially H2) and β-turns (Fig. 4). It seems that these residues dramatically alter solvent accessibility (Fig. 4) and the electric potential of the protein surface area (Fig. 5). Both could result in changes in IgE-binding capacity of conformational epitopes, despite similar folding patterns of the plant profilins. This is mainly due to this fact that protein folding is liberal with respect to amino acid substitutions for many positions in the sequence. Such substitutions may markedly affect the protein outer surface or directly involve contact residues important for the antigen-antibody interaction, thus reducing or abolishing antibody reactivity [33]. Fortunately, these alterations will not always influence IgE binding activity of an epitope. Nuclear magnetic resonance studies indicate that only a small number of residues within an epitope are functionally important for antibody binding [34]. It could be the reason for melon profilin cross-reactivity with tomato, peach, Cynodon dactylon and grape profilins. This evidence led to the suggestion that a shared topology and conformational epitope is the presumed basis for extensive IgE cross-reactivity between Cuc m 2 and other plant profilins. On the other hand, earlier studies on Cuc m 2 oligomerization showed multimer forms of Cuc m 2 had more IgE activity than monomer Cuc m 2 (data not published). If we assume that polymerization patterns of profilins are similar to human profilin [35], most of the reported sequential epitopes will be located at the inaccessible site of multimeric profilins.
In conclusion, The presence of IgE cross-reactivity among profilins strongly depends on the highly conserved conformational structure, rather than the percentage of amino acid sequence identity. Clarifying conformational and sequential epitopes of profilin may open up novel ways to improve our knowledge about cross-reactivity among profilins. It would be useful to define cross-reactive clusters of profilin and other allergen molecule families in order to reach a diagnosis and treatment strategy based on a small set of cross-reactive allergens.
Acknowledgements
The authors thank Anna Pomes for her valuable comments and the research administration of Mashhad University of Medical Sciences for its support.
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Machesky LM Pollard TD Profilin as a potential mediator of membrane-cytoskeleton communication Trends Cell Biol 1993 3 381 385 14731655 10.1016/0962-8924(93)90087-H
Valenta R Duchêne M Pettenburger K Sillaber C Valent P Bettelheim P Breitenbach M Rumpold H Kraft D Scheiner O Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitized individuals Science 1991 253 557 560 1857985
Wensing M Akkerdaas JH van Leeuwen WA Stapel SO Bruijnzeel-Koomen CA Aalberse RC IgE to Bet v 1 and profilin: crossreactivity patterns and clinical relevance J Allergy Clin Immunol 2002 110 435 42 12209091 10.1067/mai.2002.126380
Breiteneder H Ebner C Atopic allergens of plant foods Curr Opin Allergy Clin Immunol 2001 1 261 7 11964699 10.1097/01.all.0000011024.76416.01
Scheurer S Wangorch A Nerkamp J Skov PS Ballmer-Weber B Wütrich B Cross-reactivity within the profilin panallergen family investigated by comparison of recombinant profilins from pear (Pyr c 4), cherry (Pru ar 4) and celery (Api g 4) with birch pollen profilin Bet v 2 J Chromatogr B 2001 756 315 25
Van Ree R Voitenko V van Leeuwen Aalberse RC Profilin is a cross-reactive allergen in pollen and vegetable foods Int Arch Allergy Immunol 1992 98 97 104 1643445
Vallier P Ballard S Harf R Valenta R Deviller P Identification of profilin as an IgE-binding component in latex from Hevea brasiliensis: clinical implications Clin Exp Allergy 1995 25 332 9 7600379
Ebner C Jensen-Jarolim E Leitner A Breitender H Characterization of allergens in plant-derived spices: Apiaceae spices, pepper (Piperaceae), and paprika (bell peppers, Solanaceae) Allergy 1998 53 52 4 9825999
Garcia Ortiz JC Cosmes Martin P Lopez Asunolo A Melon sensitivity shares allergens with Plantago and grass pollens Allergy 1995 50 269 73 7545882
Thorn KS Christensen HE Shigeta R Huddler D Shalaby L Lindberg U Chua NH Schutt CE The crystal structure of a major allergen from plants Structure 1997 5 19 32 9016723 10.1016/S0969-2126(97)00163-9
Sankian M Varasteh AR Esmail N Moghadam M Pishnamaz R Mahmoudi M Melon allergy and allergenic cross reactivity of melon with other allergens Iranian J Basic Med Sci 2004 4 330 323
Dreborg S Skin tests used in type I allergy testing Allergy 1984 39 596 601 6528954
Calabozo B Barber D Polo F Purification and characterization of the main allergen of Plantago lanceolata pollen, Pla l Clin Exp Allergy 2001 31 322 330 11251634 10.1046/j.1365-2222.2001.00985.x
Moller M Kayma M Vieluf D Steinhart H Determination and characterization of cross-reacting allergens in latex, avocado, banana, and kiwi fruit Allergy 1998 53 289 296 9542609
Wallner M Gruber P Radauer C Maderegger B Susani M Hoffmann-Sommergruber K Ferreira F Lab scale and medium scale production of recombinant allergens in Escherichia coli Methods 2004 32 219 226 14962755 10.1016/j.ymeth.2003.08.004
Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970 277 680 685 5432063 10.1038/227680a0
Towbin H Staehlin I Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc Natl Acad Sci USA 1979 776 4350 4 388439
Inglis AS Cleavage at aspartic acid Meth Enzymol 1983 91 324 332 6304451
Schagger H von Jagow G Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 1987 166 368 379 2449095 10.1016/0003-2697(87)90587-2
Hall TA BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucl Acids Symp Ser 1999 41 95 98
Zhorov BS Vector method for calculating derivatives of energy of atom-atom interactions of complex molecules according to generalized coordinates J Struct Chem 1981 22 4 8 10.1007/BF00745970
Weiner SJ Kollman PA Case DA Singh UC Chio C Alagona G Profeta S Weiner PK A new force field for molecular mechanical simulation of nucleic acids and proteins J Am Chem Soc 1984 106 765 784 10.1021/ja00315a051
Li Z Scheraga HA Monte Carlo-minimization approach to the multiple-minima problem in protein folding Proc Natl Acad Sci USA 1987 84 6611 6615 3477791
Chapman MD Smith AM Vailes LD Arruda LK Dhanaraj V Pomés A Recombinant allergens for diagnosis and therapy of allergic disease J Allergy Clin Immunol 2000 106 409 418 10984358 10.1067/mai.2000.109832
Valenta R Vrtala S Laffer S Spitzauer S Kraft D Recombinant allergens Allergy 1998 53 552 561 9689336
Asero R Mistrello G Roncarolo D de Vries SC Gautier MF Ciurana CL Lipid transfer protein: a panallergen in plant-derived foods that is highly resistant to pepsin digestion Int Arch Allergy Immunol 2001 124 67 69 11306929 10.1159/000053671
Fujita C Moriyama T Ogawa T Identifcation of cyclophilin as an IgE binding protein from carrots Int Arch Allergy Immunol 2001 125 44 50 11385287 10.1159/000053795
Fedorov AA Ball T Mahoney NM Valenta R Almo SC The molecular basis for allergen cross-reactivity: crystal structure and IgE-epitope mapping of birch pollen profilin Structure 1997 5 33 4 9016715 10.1016/S0969-2126(97)00164-0
Wiedemann P Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody J Biol Chem 1996 271 29915 29921 8939935 10.1074/jbc.271.47.29915
Asturias JA Gomez-Bayon N Arilla MC Sanchez-Pulido L Valencia A Martinez A Molecular and structural analysis of the panallergen profilin B cell epitopes defined by monoclonal antibodies Int Immunol 2002 14 993 1001 12202397 10.1093/intimm/dxf070
Rodriguez-Perez R Crespo JF Rodriguez J Salcedo G Profilin is a relevant melon allergen susceptible to pepsin digestion in patients with oral allergy syndrome J Allergy Clin Immunol 2003 111 634 9 12642849 10.1067/mai.2003.74
Rihs HP Chen Z Rueff F Petersen A Rozynek P Heimann H Baur X IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes J Allergy Clin Immunol 1999 104 1293 301 10589015
Crameri R Correlating IgE reactivity with three-dimensional structure Biochem J 2003 376 e1 e2 14602045 10.1042/BJ20031363
Mueller GA Smith AM Chapman MD Rule GS Benjamin DC Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2 J Biol Chem 2001 276 9359 9365 11134039 10.1074/jbc.M010812200
Nodelman MI Bowman GD Lindberg U Schutt CE X-ray Structure determination of Human Profilin II: A Comparative Structural Analysis of Human Profilins J Mol Biol 1999 294 1271 1285 10600384 10.1006/jmbi.1999.3318
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Clin Pract Epidemiol Ment HealthClinical Practice and Epidemiology in Mental Health : CP & EMH1745-0179BioMed Central 1745-0179-1-161618535510.1186/1745-0179-1-16ResearchAn epidemiological survey of psychiatric disorders in Iran Mohammadi Mohammad-Reza [email protected] Haratoon [email protected] Ahmad Ali [email protected] Hossein [email protected] Hamid Reza [email protected] Hamid Reza [email protected] Seyed Abbas Bagheri [email protected] Mehdi [email protected] Javad [email protected] Homayoon [email protected] Emran Mohammad [email protected] Bita [email protected] Hamid [email protected] Mohammad [email protected] Ahmad [email protected] Department of Psychiatry, Psychiatry and Psychology Research Center, Tehran University of Medical Sciences, Roozbeh Hospital, South Kargar AV., 13185/1741, Tehran, Iran2 Director of National Research Center for Medical Sciences of Iran, No. 26, 1st Al., Kooh-e-Noor St., Motahhari Ave., Tehran, Iran3 Department of Biostatistics and Epidemiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran4 Department of Psychology, Shahid Beheshti University, Tehran, Iran5 Department of Mental Health, Ministry of Health and Medical Education, Tehran, Iran6 Department of statistics and Computer, Social Welfare and Rehabilitation University, Tehran, Iran7 Iranian Center for Addiction, Tehran University of Medical Sciences, Tehran, Iran8 Department of Community Medicine, Medical School, Ahvaz University of Medical Sciences, Ahvaz, Iran9 Department of Psychiatry, Hafez Hospital, Shiraz University of Medical Sciences, Shiraz, Iran2005 26 9 2005 1 16 16 18 6 2005 26 9 2005 Copyright ©2005 Mohammadi et al; licensee BioMed Central Ltd.2005Mohammadi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The nation-wide epidemiological survey of psychiatric disorders in term of lifetime prevalence is not adequately known in Iran. The prevalence of lifetime psychiatric disorders was estimated among the population of aged 18 and over on gender, age group, educational level, occupational status, marital status, and residential area.
Methods
The subjects were 25,180 individuals selected through a clustered random sampling method. The psychiatric disorders were diagnosed on the bases of Diagnostic and Statistical Manual of Mental Disorders-IV criteria. It is the first study in which the structured psychiatric interview administered to a representative sample of the Iranian population age 18 and over by the 250 trained clinical psychologist interviewers. The data was entered through EPI-Info software twice in an attempt to prevent any errors and SPSS-11 statistical software was also used for analyses. The odds ratios and their confidence intervals estimated by using logistic regression.
Results and Discussion
The prevalence of psychiatric disorders was 10.81%. It was more common among females than males (14.34% vs. 7.34%, P < 0.001). The prevalence of anxiety and mood disorders were 8.35% and 4.29% respectively. The prevalence of psychotic disorders was 0.89%; neuro-cognitive disorders, 2.78% and dissociative disorders, 0.77%. Among mood disorders, major depressive disorder (2.98%) and among anxiety disorders, phobic disorder (2.05%) had the higher prevalence. The prevalence of psychiatric disorders among divorced and separated 22.31%; residents of urban areas 11.77%; illiterates 13.80%; householders 15.48%; unemployed 12.33% that were more than other groups.
Conclusion
The mental health pattern in Iran is similar to the western countries, but it seems that the prevalence of psychiatric disorders in Iran may be lower than these countries.
It is estimated that at least about 7 millions of Iranian population suffer from one or more of the psychiatric disorders. It shows the importance of the role of the psychiatric disorders in providing preventive and management programs in Iran.
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Background
In order to make policies and strategies for control and prevention of psychiatric disorders, their prevalence in each specific community should be determined [1]. Population-based studies of psychiatric morbidity in Iran are rare. Many studies have been conducted in primary health care settings, or on specific populations and disorders [2], but these studies did not allow estimation of population prevalence.
In Iran, the previous epidemiologic studies on psychiatric disorders using Diagnostic and Statistical Manual of Mental Disorders forth version (DSM-IV) criteria have not carried out nationwide. The studies conducted in limited populations and in a few numbers of the cities that reported the prevalence of the disorders vary from 11.9% to 23.8% [3].
The only study that has been conducted is the study of the health status in Iran in 1999 [4]. 35,014 individuals at 15 years of age and over were assessed by General Health Questionnaire (GHQ-28). It showed that 21% of the subjects suffered from psychiatric symptoms. Four groups of psychiatric symptoms including depression, anxiety, somatization and social functions were studied. 879 subjects from Tehran were interviewed by psychiatric registrars based on DSM-IV criteria. The prevalence of psychiatric disorders, epilepsy, and mental retardation was estimated 21.5%. Predicted estimations of prior studies in other countries, disregard to their diversity of goals and purposes, reported up to 46% of general population [5]. These variations may be due to diversity of screening methods, diagnostic tools, sampling methods and cultural differences between populations sampled.
By estimating the nationwide prevalence of different types of psychiatric disorders, it will be useful for planning of preventive programs by health, treatment and educational authorities. This study was conducted under the title of "The national plan for epidemiologic study of psychiatric disorders in Iran."
Current study was approved by the national council for scientific research and deputy for research of Ministry of Health and was conducted by National Research Center for Medical Sciences of Iran.
The aim of the study was to estimate the lifetime prevalence rate of mental disorders and its distribution by gender, age, educational level, occupational status, marital status, and residential area in the population aged 18 years and over living in Iran.
Methods
The lifetime prevalence of psychiatric disorders was assessed according to DSM-IV [6] by means of the Schedule for Affective Disorders and Schizophrenia (SADS) [7,8]. It is the first study ever in which a full, structured psychiatric interview has been administered to a representative sample of the Iran population aged 18 and over by 250 trained clinical psychologists. Lifetime prevalence is the proportion of the sample who reported having experienced a given disorder at some time in their lives.
Sample
Overall, 25,180 subjects of Iranian residents, aged 18 years and over from urban and rural areas of Iran, were selected by clustered random sampling method. The country's population according to statistics provided by the health system was 63,042,188 in 1999, of which 64.5% lived in urban areas and 35.5% in rural areas. From 12,398,235 households of the country, 7,795 households were chosen. It was 1559 clusters that each one included 5 households. The choice of cluster size was based on the daily performance capacity of the data collecting group. Out of 1,559 clusters, 977 were from urban and 582 were from rural areas. The sampling framework was based on the household lists available from the department of health in the provinces. The response rate was 90%.
Diagnostic Assessment and Instrument
The screening and diagnosis of disorders could be made based on the findings of the schedule (SADS) in one stage. The SADS questions were translated to Farsi, and then converted into English by two translators. After confirming of translation, the questionnaire was ready for the result of the test performance. To validate of the content of the questions, the test questionnaire was studied by a number of psychiatric authorities. It was applied after eliminating the defects, adjusting the main form, conducting the constructing validity, and predictive validity tests on 200 existing patients of Roozbeh hospital in Tehran, who already had a psychiatric diagnosis. On the co-rated interviews, there was a strong agreement between interviewers for diagnosis of the disorders. For test- retest interviewers, the Cohen Kappa coefficient was 0.87. The Cohen Kappa coefficients for anxiety disorders, mood disorders, and psychotic disorders were 0.79, 0.88, and 0.91 respectively. The Cohen Kappa coefficient was about 0.45 [8], in cases of substance use disorder and personality disorder. More materials about reliability, validity and specificity are in the previous articles (2, 7, 8 and 11).
Training the use of the instrument and procedure of collecting data
The subjects were interviewed face-to-face at home by expert clinical psychologists. The interviewers were employed by the Prevention Deputy of Welfare Organization and the medical Universities in each of the Provinces. Our clinical psychologist used SADS in their interviews. In addition, they attended a three day training and role playing workshop in Tehran in order to develop proper interviewing and decision-making skills. Every clinical psychologist had to interview at least five clients and deal with the complications and questions presented in the workshop. Data gathering was precisely supervised by either a psychiatrist or psychologist and one representative of prevention deputy of each province as the supervisor of the project. The data was entered through EPI-Info software twice in an attempt to prevent any errors. The SPSS for Windows (version 11.0) used for analyzing the data. The odds ratios and their confidence interval estimated by using logistic regression.
Results
Overall, 10.81% of subjects suffered from at least one psychiatric disorder. Major depressive disorder, phobic disorder, obsessive-compulsive disorder and epilepsy were the top four common psychiatric disorders in Iran.
The prevalence for women was about two time that of men (14.34% versus 7.34, P < 0.001). Data regarding prevalence of the psychiatric disorders in terms of age group, gender, marital status, educational level, occupation, and residential area are presented in table 1.
Table 1 Prevalence (%) distribution of psychiatric disorders of the overall population by the socio-demographic factors (N = 25180)
Demographic variables Disorders
Sample(n) Cases(n) (%) 95% CI P. value
Sex Men 12660 929 7.34 6.9–7.8 <0.001
Women 12520 1807 14.34 13.8–15.0
Age Groups 18–25 7730 701 9.07 8.4–9.7 <0.001
26–40 8196 904 11.03 10.4–11.7
41–55 5604 707 12.62 11.7–13.5
56–65 1932 196 10.14 8.8–11.5
>65 1659 206 12.42 10.8–14.0
Marital Status Single 7334 641 8.74 8.1–9.4 <0.001
Married 16982 1925 11.34 10.9–11.8
Divorced or separated 121 27 22.31 14.9–29.7
Dead spouse 640 129 20.16 17.0–23.3
Residential area Rural 9466 886 9.36 8.8–9.9 <0.001
Urban 15714 1850 11.77 11.3–12.3
Education High Education 2513 203 8.08 7.1–9.2 <0.001
Diploma 4774 447 9.36 8.5–10.2
High or Guidance School 4972 459 9.23 8.4–10.0
Elementary 6715 784 11.68 10.9–12.4
Illiterate 5839 806 13.80 12.9–14.7
Occupation Worker 2263 172 7.60 6.5–8.8 <0.001
Officers 2730 191 7.00 6.1–8.0
Student 1713 148 8.64 7.4–10.1
Private sector 5245 319 6.08 5.4–6.7
Retired 710 79 11.13 8.8–13.4
Housewife 9592 1485 15.48 14.8–16.2
Unemployed 1947 240 12.33 10.9–13.8
Others 909 99 10.89 8.9–12.9
Total 25180 2736 10.81 10.5–11.3
The prevalence of psychiatric disorders in the age group 18–25 was less than the other groups. Married respondents reported higher rates of lifetime psychiatric disorder than singles. Divorced, separated, and widowed respondent had higher rates of lifetime psychiatric disorder. Additionally, the rate was higher in urban areas and in the illiterate subjects (P < 0.001). In the category of occupation, housewives had the highest rate of the disorders (P < 0.001) (Table 1).
1- The prevalence of psychiatric disorders in females was more than in males except for mental retardation.
2- Married subjects had a higher prevalence rate of psychiatric disorders when compared with singles, except mental retardation.
3- The prevalence of psychiatric disorders showed a different pattern among the occupations of the subjects.
4- Educational background had no significant in prevalence of psychiatric disorder except for mood disorders, epilepsy and mental retardation.
5- The prevalence of all types of psychiatric disorders except epilepsy was higher in urban areas when compared to rural areas.
The logistic regression analysis showed on Table 2.
Table 2 Demographic correlation of lifetime psychiatric disorders of Iranian adults (N = 25180)
Variable Mood Disorders Anxiety Disorders Psychotic Disorders Epilepsy Mental Retardation Dementia
OR 95% CI OR 95% CI OR 95% CI OR 95% CI OR 95% CI OR 95% CI
Sex
Male 0.60 0.47–0.77 0.41 0.33–0.51 0.59 0.46–0.76 1.19 0.83–1.71 3.17 1.2–7.79 1.18 0.61–2.28
Female 1 1 1 1 1 1
Age
18 – 25 0.99 0.74–1.30 1.19 0.94–1.50 0.99 0.74–1.35 1.52 1.01–2.29 3.18 1.29–7.79 0.19 0.09–0.43
26 – 40 1.35 1.07–1.69 1.27 1.02–1.58 1.37 1.07–1.74 1.19 0.84–1.68 6.61 2.89–15.13 0.24 0.14–0.43
41 – 55 1.56 1.24–1.96 1.50 1.20 – 1.87 1.57 1.24–1.98 1.27 0.92–1.77 2.10 0.83–5.31 0.29 0.17–0.49
56+ 1 1 1 1 1 1
Marital Status
Single 0.57 0.06–5.17 0.73 0.63–0.84 0.99 0.69–0.44 0.90 0.73–0.12 6.57 4.23–10.20 0.57 0.35–0.91
Widow/Widower/Separated 0.01 0.00–3.71 1.45 1.08–1.95 1.89 0.91–3.90 1.50 0.96–2.35 3.31 1.16–9.50 6.97 4.50–0.79
Married 1 1 1 1 1 1
Occupation
Employed 0.60 0.45–0.82 0.86 0.64–1.15 0.46 0.25–0.83 0.55 0.40–0.76 0.24 0.15–0.40 0.41 0.23–0.73
Student 0.70 0.46–1.07 0.95 0.66–1.36 0.86 0.39–1.87 0.64 0.39–1.08 0.05 0.01–0.40 0.53 0.17–1.70
Retired 0.99 0.61–1.60 1.40 0.87–2.25 0.69 0.22–2.11 0.42 0.20–0.90 0.00 0.00–18.34 0.72 0.33–1.57
Housewife 0.98 0.71–1.35 1.23 0.93–1.64 1.03 0.58–1.80 0.77 0.56–1.05 0.17 0.10–0.30 0.64 0.38–1.05
Unemployed 1 1 1 1 1 1
Education
University 0.59 0.45–0.76 0.88 0.65–1.18 0.88 0.65–1.20 0.50 0.33–0.78 0.02 0.01–.06 0.42 0.17–1.01
Primary and High School 0.80 0.92–0.70 1.105 0.93–1.31 0.98 0.83–1.17 0.70 0.57–0.87 0.03 0.02–0.05 0.49 0.31–0.78
Uneducated 1 1 1 1 1 1
Residential Area
Urban 1.26 1.10–1.45 1.48 1.29–1.70 1.43 1.00–2.06 0.99 0.81–1.21 2.05 1.34–3.16 1.71 1.16–2.52
Rural 1 1 1 1 1 1
The odds ratios were based on logistic regression analysis.
The Prevalence rate of different types of psychiatric disorders by sex is presented in Table 3.
Table 3 The Prevalence of different types of psychiatric disorders by sex (N = 25180)
Type of psychiatric disorders Males (n = 12660) Females (n = 12520) Total (n = 25180)
Number (%) Number (%) Number (%)
Mood psychiatric disorders
Major depressive disorder 201 1.59 549 4.38 750 2.98
Minor Depressive disorder 28 0.22 54 0.43 82 0.33
Bipolar mood disorder 23 0.18 15 0.12 38 0.96
Mood disorder with psychotic feature 3 0.02 2 0.02 5 0.02
Total 255 2.01 620 4.95 875 4.29
Psychotic disorders
Schizophrenia 23 0.18 39 0.31 62 0.25
Schizoaffective 9 0.07 16 0.13 25 0.10
Short term psychotic disorder 6 0.05 5 0.04 11 0.04
Schizopherniform 4 0.03 2 0.02 6 0.02
Other psychotic disorders 43 0.34 78 0.62 121 0.48
Total 85 0.67 140 1.12 225 0.89
Anxiety disorders
Panic disorder 100 0.79 274 2.19 374 1.49
Generalized anxiety disorder 93 0.73 243 1.94 336 1.33
Obsessive compulsive disorder 91 0.7 353 2.8 444 1.8
Agoraphobia 34 0.27 141 1.13 175 0.70
Phobic disorders 113 0.89 403 3.22 516 2.05
Post traumatic stress disorder 98 0.77 150 1.20 248 0.98
Total 529 4.15 1564 12.48 2093 8.35
The neuro-cognitive disorders
Epilepsy 199 1.57 255 2.04 454 1.80
Mental retardation (Severe) 56 0.44 51 0.41 107 0.42
Dementia 65 0.51 75 0.60 140 0.56
Total 320 2.52 381 3.05 701 2.78
Dissociative and Somatization disorders
Somatization Disorder 16 0.13 30 0.24 46 0.18
Dissociative Fugue 3 0.02 5 0.04 8 0.03
Dissociative Amnesia 65 0.51 61 0.49 126 0.50
Depersonalization Disorder 7 0.06 7 0.06 14 0.06
Total 91 0.72 103 0.83 194 0.77
Total 1280 10.07 2808 22.43 4088 17.08
Each subjects may suffered from more than one of the above psychiatric disorders.
Discussion
Most of the subjects (89.2%) lack any psychiatric disorder. The prevalence of subjects with no lifetime DSM-III-R diagnosis in a study in Oslo was 47.7% [9]. Also, the rate in the National Co-morbidity Study in the USA was 52% [10]. Although the two study samples were taken from the general population, they used the Composite International Diagnostic Interview [11] and DSM-III-R criteria. This difference may be based on the instrument and method used in their studies. For example, the Norway study focused on the population of Oslo, a large city with different social problems when compared to small cities or villages. This generalization cannot be applied to the whole country. It is possible that some depressive and anxiety disorders are more common in larger cities with a higher rate of disorder. The National Co-morbidity Survey in the United States [9] was a large cross-sectional population study with a design that was not comparable to our study. Also, the US study was limited to respondents aged 15–54. Plus, they used a paper and pencil version. These differences in the psychiatric interview may be responsible for the differences in the findings.
A cross national study showed that one third of the subjects experienced at least one disorder at some time in their life in Brazil (36.3%), Canada (37.5%), Germany (38.4%), Netherlands (40.9%), and the USA (48.6%). Lifetime prevalence estimates were considerably lower in Mexico (20.2%) and Turkey (12.2%) [12]. The current study shows that psychiatric disorders are not infrequent in Iran. As many as 10.81% of the subjects reported having experienced one or more psychiatric disorder at some time in their lives. However, it is lower than the rate reported in the study that used GHQ-28 [4]. The difference may be due to the method and tool used for screening and diagnosis of the disorders. The SADS included 16 groups of psychiatric disorders whereas the GHQ-28 questionnaire only studies the symptoms of anxiety, depression, somatisization and social dysfunctions.
The present study shows that the rate of psychiatric disorders in women is higher than men (14.34% versus 7.34%) which are consistent to the results of some studies conducted in Iran and other countries [4,9,13,14]. However, a study in Netherlands, reported that more than four out of ten respondents (41.2%) reported a lifetime prevalence of at least one DSM-III-R disorder. The most common psychiatric disorders were major depression, alcohol abuse and phobias with no significant difference between men and women (42.5% versus 39.9%). More than 15% of the respondents had major depression in their history. Though they did not find gender differences in the overall prevalence of mental disorders, differences did emerge when the disorders were examined separately. Obsessive-compulsive disorder was the only exception among the anxiety disorders [15] that was more prevalent among men. In the current study, all types of the psychiatric disorders were more common in females than males except BMD, acute brief psychotic disorder, somatoform disorder, and amnestic disorder.
In a study in Brazil based on ICD-10, it showed that nearly 46% of the sample had at least one lifetime mental disorder, i.e., almost one in two respondents reported a given disorder at some time in their lives [5]. They reported that women were more likely than men to have mood disorders (with the exception of bipolar disorder, for which there were no gender differences), and anxiety disorders (except for obsessive-compulsive disorder, social phobia, and generalized anxiety disorder). There were no gender differences in the rates of somatoform disorders.
The results of the some previous studies showed a different pattern, with the highest prevalence typically occurring among the youngest age groups [[12,16], and [17]]. The current study shows that the prevalence of psychiatric disorders in the ages of 41 and over is more than the age group 18–40. A study in Norway showed that the most common age group was 30–39 [9]. The rate in the USA was more in the age group 25–34 [10].
The highest estimated prevalence was found among respondents at the lowest level of educational attainment. This is in accordance with results of six of the seven surveys of the cross national study (Canada, USA, Brazil, Mexico, Germany, Netherlands, and Turkey). Germany was the exception (with an insignificant relationship) [12]. Also, it was consistence with the study in Norway [9].
Some studies have found an occupational gradient in the prevalence of common psychiatric disorders [18]; others have failed to find such association [19]. Our result is similar to the later study. A study in Brazil using ICD-I0 classification showed that except for anxiety disorders, unemployed respondents were more likely to have any lifetime disorder. Students, homemakers, and retired persons were all less likely to have any psychiatric morbidity [5] when compared with the employed.
The rates of most types of psychiatric disorders in urban areas are higher, when compared to rural areas. These findings are reinforced by the previous study [3]. Recently, a study showed that the difference was not statistically meaningful [20]. It is possible that other factors that are related to location of residence are more important, such as: poverty, unemployment, lower socioeconomic status, and sex.
The differences in the methods of selecting the samples, operational definition of variables, data gathering methods, and tools are considered as important factors in inconsistencies with the results. In particular, the validity and reliability of the tool should be considered. The validity and reliability of SADS was well reflected in the previous studies [21,22]. However, the validity and reliability of western societies does not confirm its validity in countries with deep cultural difference. The effects of cultural factors on estimating the prevalence of psychiatric disorders through diagnostic interviews has been shown in previous studies [23]. The present study shows that the prevalence rate of bipolar mood disorder (BMD) was 0.96%, the results of the other studies showed a different pattern. Findings reported by the recent multi-center European study prevalence rate of BMD reveal even lower frequencies under 1%. Data from surveys of large samples showed the lifetime prevalence rates of BMD around 1.5%. A main question is whether the low prevalence rates of BMD are not an artifact of the over diagnosis of depression [24].
Limitations
The community epidemiological surveys of psychiatric disorders have been carried out in different parts of the world in the absence of a common format for diagnostic interviews. Therefore, cross-national syntheses or comparisons of the results of these surveys could not be made.
Substance use is one of the major concerns of mental health in Iran, and was not addressed in the current study. In cases of substance use disorder, the Cohen Kappa coefficient was low. It may be due to the expensive penalty for substance use in Iran e.g. loss of job. Another limitation includes difficulty in recalling the past, which may cause under reporting. The study relied only on self-reporting to make diagnosis. As well, it is possible that some respondents in community surveys did not disclose information about psychiatric disorders to interviewers. Also, we know that a substantial portion of people with relatively uncommon disorders like schizophrenia are already long-term residents of mental hospitals, and thus may have not been included in the study. Unfortunately, there has not been any survey of people with schizophrenia in Iranian hospitals. This unwillingness of disclosure may not take a form of high rates of refusal to participate in epidemiological surveys, but rather of high rates of agreement accompanied by intentionally low reported rates of disorders. A noticeable population of Iranian people has immigrated to the other countries during last 3 decades and we don't know how this could be influencing the results. Prevalence rate may be affected by recall bias for episodes of nervous problems years before. Less severe episodes of symptoms earlier in life may be forgotten. Forgetting episodes may not be just a matter of forgetting, but also a result of processing the significance and meaning of symptoms with time. It is questionable whether symptoms that today are considered medical would now be understood and reported as nervous symptoms. These types of biases could be effective in interpretation of the results. It is possible that the diagnostic assessment and the instrument was not the most accurate method for this study. This instrument (SADS) was chosen over others (SCAN, DISC, CIDI, SCID and etc.) because of its wide use of it in Iran (for example; Mohammadi et al. [2,8]). The experts are more familiar with SADS which is used by our clinical psychologist in their daily clinical practices while K-SADS is used in Iran for children and adolescents. Without any bias the lifetime prevalence should increase clearly by increasing age. In contrary, the prevalence seems to be lowest in those over 56 years of age. Finally, this article does not address the one year or six month prevalence of the disorders that might have been more useful for making policies and strategies for the prevention of the psychiatric disorders.
Conclusion
The pattern of prevalence of mental health in Iran is similar to the western countries, but it seems that the prevalence of psychiatric disorders in Iran may be lower than these countries.
Women along with retired and unemployed men had more types of psychiatric disorders, mandating a necessity to plan programs in prevention and treatment of these disorders.
On the bases of the results of the current study, it is estimated that at least about 7 millions of Iranian population suffer from one or more of the psychiatric disorders and they need mental health services. It shows the importance of incorporating psychiatric disorder in planning preventive and management programs in Iran.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Mohammad-Reza Mohammadi (principal investigator, conception, designer, country coordinator and final approval, psychiatrist)
Haratoun Davidian (designer, psychiatrist)
Ahmad Ali Noorbala (conception and design, psychiatrist)
Hossein Malek Afzali (statistical consultant, biostatistician)
Hamid Reza Naghavi (designer, province coordinator and investigator, psychiatrist)
Javad Alaghebandrad (designer, coordinator and investigator, psychiatrist)
Seyed abbas Bagheri Yazdi (interpretation and revising, clinical psychologist)
Mehdi Rahgozar (statistical analysis, programmer, interpretation and revising, biostatistician)
Hamid Reza Pouretemad (province coordinator and investigator, clinical Psychologist)
Homayoon Amini (province coordinator and investigator, psychiatrist)
Emran Mohammad Razzaghi (investigator, psychiatrist)
Bita Mesgarpour (coordinating and revising, pharmacist)
Hamid Soori (statistical consultant and revising, epidemiologist)
Ahmad Ghanizadeh (interpretation and revising, psychiatrist)
All authors read and approved the final manuscript.
Acknowledgements
First of all we appreciate the participation of the individuals and families in the study.
We also appreciate the contributions of Dr. Kazem Mohammad the project consultant, Mohammad Reza Rostami, Saeed Madani, Dr. Tavakoli, Dr. Mohammad-Taghi Joghatai, all the headquarters, the medical commission of national scientific research council, National Research Center for Medical Sciences of Iran, Deputy for Research and Technology, Deputy of Health of Iran, Medical Commission of National Research Council, The Welfare and Rehabilitation Sciences University, deputy for Prevention of Welfare Organization, psychiatric department of Roozbeh hospital, and all 250 clinical psychologists and experts who assisted us in conducting this national study.
==== Refs
Kebede D Alem A Rashid E The prevalence and socio-demographic correlates of mental distress in Addis Ababa, Ethiopia Acta Psychiatr Scand Suppl 1999 397 5 10 10470348
Mohammadi MR Rahgozar M Bagheri Yazdi SA Naghavi HR Pouretemad HR Amini H Rostami MR Khalajabadi Farahani F Mesgarpour B An epidemiological study of psychiatric disorders in Tehran Province J Andisheh Va Raftar 2003 2 4 13
Noorbala AA Mohammad K Bagheri Yazdi SA The epidemiological study of psychiatric disorders in Tehran J Hakim 1998 4 212 23
Noorbala AA Bagheri Yazdi SA Yasamy MT Mohammad K Mental health survey of the adult population in Iran Br J Psychiatry 2004 184 70 3 10.1192/bjp.184.1.70 14702230
Andrade L Walters EE Gentil V Laurenti R Prevalence of ICD-10 mental disorders in a catchment area in the city of Sao Paulo, Brazil Soc Psychiatry Psychiatr Epidemiol 2002 37 7 316 25 10.1007/s00127-002-0551-x 12111023
American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) 1994 4 Washington DC
Endicott J Spitzer RL A diagnostic interview: the schedule for affective disorders and schizophrenia Arch Gen Psychiatry 1978 35 7 837 44 678037
Mohammadi MR Ghanizadeh A Rahgozar M Noorbala AA Davidian H Malekafzali H Naghavi HR Baghery Yazi SA Saberi SM Mesgarpour B Akhondzadeh S Alaghebandrad J Tehranidoost M Prevalence of obsessive-compulsive disorder in Iran BMC Psychiatry 2004 4 2 15018627 10.1186/1471-244X-4-2
Kringlen E Torgersen S Cramer V A Norwegian psychiatric epidemiological study Am J Psychiatry 2001 158 7 1091 8 10.1176/appi.ajp.158.7.1091 11431231
Kessler RC McGonagle KA Zhao S Nelson CB Hughes M Eshleman S Wittchen HU Kendler KS Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey Arch Gen Psychiatry 1994 51 1 8 19 8279933
Robins LN Wing J Wittchen HU Helzer JE Babor TF Burke J Farmer A Jablensky A Pickens R Regier DA Sartorius N Towle LH The Composite International Diagnostic Interview: An epidemiologic instrument suitable for use in conjunction with different diagnostic systems and in different cultures Arch Gen Psychiatry 1989 45 12 1069 77
WHO International Consortium in Psychiatric Epidemiology Cross-national Comparisons of the Prevalences and Correlates of Mental Disorders Bull World Health Organ 2000 78 4 413 26 10885160
Harding TW de Arango MV Baltazar J Climent CE Ibrahim HH Ladrido-Ignacio L Murthy RS Wig NN Mental disorders in primary health care: a study of their frequency and diagnosis in four developing countries Psychol Med 1980 10 231 41 7384326
Barrett JE Barrett JA Oxman TE Gerber PO The prevalence of psychiatric disorders in a primary care practice Arch Gen Psychiatry 1988 45 1100 6 3264145
Bijl RV van Zessen G Ravelli A de Rijk C Langendoen Y The Netherlands Mental Health Survey and Incidence Study (NEMESIS): objectives and design Soc Psychiatry Psychiatr Epidemiol 1998 33 581 6 10.1007/s001270050097 9857790
Robins LN Regier DA Psychiatric disorders in America: the Epidemiologic Catchment Area Study 1991 New York: The Free Press
Lin TY A study of incidence of mental disorders in Chinese and other cultures Psychiatry 1953 16 315 335
Stansfeld SA Marmot MG Social class and minor psychiatric disorder in British Civil Servants: a validated screening survey using the General Health Questionnaire Psychol Med 1992 22 3 739 49 1410098
Lewis G Bebbington P Brugha T Farrell M Gill B Jenkins R Meltzer H Socioeconomic status, standard of living, and neurotic disorder Lancet 1998 352 9128 605 9 10.1016/S0140-6736(98)04494-8 9746021
Judd FK Jackson HJ Komiti A Murray G Hodgins G Fraser C High prevalence disorders in urban and rural communities Aust N Z J Psychiatry 2002 36 1 104 13 10.1046/j.1440-1614.2002.00986.x 11929446
Simpson SG McMahon FJ McInnis MG MacKinnon DF Edwin D Folstein SE DePaulo JR Diagnostic reliability of bipolar II disorder Arch Gen Psychiatry 2002 59 8 736 40 10.1001/archpsyc.59.8.736 12150650
Green RL Price TR Procedural validity of an abbreviated version of the SADS/RDC diagnostic process Psychiatry Res 1986 18 4 379 91 10.1016/0165-1781(86)90022-3 3749394
Compton WM 3rdHelzer JE Hwu HG Yeh EK McEvoy L Tipp JE Spitznagel EL New methods in cross-cultural psychiatry: psychiatric illness in Taiwan and the United States Am J Psychiatry 1991 148 12 1697 704 1957932
Carta MG Angst J Epidemiological and clinical aspects of bipolar disorders: controversies or a common need to redefine the aims and methodological aspects of surveys Clin Pract Epidemiol Ment Health 2005 1 4 10.1186/1745-0179-1-4
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Clin Pract Epidemiol Ment HealthClinical Practice and Epidemiology in Mental Health : CP & EMH1745-0179BioMed Central 1745-0179-1-181618536510.1186/1745-0179-1-18ReviewAsthma and depression: a pragmatic review of the literature and recommendations for future research Opolski Melissa [email protected] Ian [email protected] Department of General Practice, University of Adelaide SA 5005, Australia2005 27 9 2005 1 18 18 17 6 2005 27 9 2005 Copyright ©2005 Opolski and Wilson; licensee BioMed Central Ltd.2005Opolski and Wilson; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Although the association between asthma and psychosocial factors has long been recognised, it is only in the last decade that the impact of coexisting asthma and depression has become the focus of considerable research interest. However, the findings so far have been confusing and often contradictory. This paper sets out a methodical review and appraisal of the literature to date, including suggestions for future research.
Method
PubMed and PsycINFO databases were used to search for English-language articles relating to asthma and depression research. The resulting articles were then reviewed and summarised, creating a report that was used to develop research recommendations.
Results
The main findings from this review included: (a) results are mixed as to whether persons with asthma are more likely to be depressed than those without asthma; (b) asthma and depression may have an 'additive' adverse effect on the normal asthma-related quality of life reductions; (c) subjective measures of asthma severity may be more strongly related to depression than objective measures; (d) specific asthma symptoms appear to be linked to depression; (e) sadness and depression can produce respiratory effects consistent with asthma exacerbations; (f) depression appears to be negatively related to asthma treatment compliance; (g) corticosteroid use in asthma treatment has been associated with depression, though it is unclear how common this problem is in real life; (h) interventions that address the physical, psychological, and social consequences of asthma are likely to lead to the most successful treatment outcomes; (i) treating the depression of individuals with asthma is likely to minimise the negative effects of the coexistence; and (j) a number of common methodological problems were observed in the literature.
Recommendations
There is a large amount of research yet to be undertaken to clarify issues around asthma and depression, with the overdue next step being to design integrated treatment approaches, and carry out large-scale prospective studies to determine the impact of using such approaches to treat individuals with depression and asthma. Such studies will be the only way in which some fundamental questions about the development and coexistence of these two conditions will be answered.
asthmadepressiondepressive disorderresearch
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Although the pathophysiology of asthma is clearly defined, the psychological influences are currently less well understood [1]. Depression is one of the most prevalent psychological problems in allergic patients [2], though it is often regarded as a somewhat 'natural reaction' to the diagnosis of a significant illness such as asthma [3-5]. However, while sadness and mild depression may be considered a fairly normal response to a diagnosis of chronic illness, more severe, chronic depression can lead to serious consequences for persons with asthma [4].
Research history
The association between asthma and psychological factors has been recognised for centuries [6]. Asthma has long been considered a psychosomatic disease, and during the 1930s–50s, was even known as one of the 'holy seven' psychosomatic illnesses [7]. At that time, psychoanalytic theories described the aetiology of asthma as psychological [8], with treatment often primarily involving psychoanalysis and other 'talking cures' [9]. As the asthmatic wheeze was interpreted as the child's suppressed cry for his or her mother, psychoanalysts viewed the treatment of depression as especially important for individuals with asthma [10].
During the early 1970s, as the understanding of the aetiology and pathophysiology of asthma improved [11,12], the emphasis on psychological issues decreased. There were, however, increases in asthma-related morbidity and mortality in many first-world countries [13], which were eventually attributed to the inappropriate use of β-agonists without corticosteroids. This issue led to a resurrection of psychosocial research relating to non-medical factors in asthma, involving variables such as poor family functioning, poor doctor-patient relationships, and social difficulties [11]. In the 1980s–1990s the growing interest in psychosocial variables also led to a number of studies being conducted into the links between asthma and anxiety and variables such as 'negative affect' or 'psychological distress', and in the late 1990s the connections between asthma and depression came to the research forefront, as the potential importance of this relationship came to be recognised. Today, as a result of years of biopsychosocial research, asthma is considered a disease of the pulmonary system with genetic and allergic origins that is significantly affected by psychosocial factors.
Asthma, depression, and quality of life
It is well documented that individuals with asthma tend to experience reduced health-related quality of life (HRQOL) [14], and although HRQOL tends to be lower for individuals with severe asthma, the effect on those with more mild asthma can also be considerable [15,16]. Asthma of any severity may lead to reductions in each of the physical, psychological, and social domains of HRQOL [17], with most people with asthma reporting some restriction on their life and having poorer health status than individuals without asthma [17,18]. A large population-based study by Ford et al [14] demonstrated that individuals with asthma had significantly lower HRQOL than those who had never had asthma, and also experienced an average of 10 days per month of impaired physical or mental health, almost double that of those who had never had asthma.
The Medical Outcomes Study demonstrated that depression and chronic disease often have 'additive' adverse effects on wellbeing [19,20], and there is evidence to support this finding for asthma and depression. People with coexisting asthma and depression have significantly lower overall HRQOL, and also experience lower physical and mental health functioning than those with similar asthma activity but fewer depressive symptoms [21-23]. These findings of reduced asthma-related quality of life are similar to those found in studies of other chronic medical conditions, where a history of depression has also been associated with poorer functional health status [19,20,24,25].
Are people with asthma more likely to be depressed?
Investigations of the prevalence of depression in asthma have reported findings ranging from 1% to 45% of individuals with asthma also suffering from depression or depressive symptoms [22,23,26-31]. Unfortunately, this wide variation in the reported rates makes it difficult to draw conclusions, but most authors suggest an increased prevalence of depression in patients with asthma.
Various studies have investigated whether individuals with asthma are more likely to be depressed than those without asthma, and while a number of studies have supported this hypothesis [5,32-36], other investigators [1,27] have not found such evidence. In response to the differing findings in regards to this issue, Adams [37] theorised that differing sampling techniques may have been related to the discrepancies between findings.
Asthma severity and depression
While it seems logical that having more severe asthma would be associated with an increased risk of depression, studies have reported somewhat mixed results. In investigations utilising objective measures of asthma severity (i.e. clinician diagnoses, airway reactivity testing), Mrazek [34] found that more severe asthma was associated with increased depressive symptoms; however, studies by Afari et al [21] and Janson et al [27] did not find this association. Thus, the evidence so far does not strongly support a direct relationship between objective asthma severity and depression. However, two studies that used patients' subjective ratings of their own asthma severity found significant relationships between perceived asthma severity and depressive symptoms [5,38].
Taken as a whole, these investigations seem to suggest that subjective measures of asthma severity may be more strongly related to depression than objective severity measures. One potential explanation for this possibility is that the individual's perception of their own asthma severity may be more important than the 'real' severity of the individual's asthma in determining whether or not asthma leads to the person becoming depressed.
Specific asthma symptoms and depression
A small number of studies have investigated the links between specific asthma symptoms and depression. One of these, a population study by Goldney et al [39], examined the relationship between depression (as measured by the Prime-MD) and a number of symptoms known to be related to asthma severity, and found that dyspnoea, wakening at night, and morning symptoms were particularly strongly associated with depression. Another study by Janson et al [27] found significant positive correlations between depression (as determined by the Hospital Anxiety and Depression Scale (HADS)) and various, but not all, symptoms of asthma.
However, while specific symptoms of asthma may be linked to depression, it is also possible that the assessment of depression in the afore-mentioned studies could have been inaccurate due to the misinterpretation of asthma symptoms as symptoms of depression. Alternatively, it is possible that the findings and their interpretation are accurate, and certain symptoms of asthma may be related to depression due to their effect on the individual's quality of life [39]. The finding of Goldney et al [39] relating asthma symptoms to significant decrement in the quality of life also leads to the question of whether experiencing certain specific asthma symptoms might lead to person with asthma becoming depressed, rather than depression resulting from simply 'having' asthma.
Can depression have a direct effect on pulmonary functioning?
The influence of emotional states on pulmonary function in asthma has been studied extensively [40]. Recent findings indicate that airways are reactive to psychological states, with these reactions causing changes consistent with greater airway instability and asthma exacerbations [41]. Consistent with this finding, personal retrospective accounts of asthma exacerbations have also suggested that changes in emotional states often result in asthma exacerbations [42].
In the laboratory, Ritz et al [43] found increased total respiratory resistance in subjects with asthma (but not those without asthma) following exposure to depressing stimuli. Krommydas et al [44] reported that individuals with asthma and symptoms of depression (measured by the Personal Disturbance Scale (DSSI/sAD)) had significantly lower FEV1% than individuals with asthma who showed no symptoms of depression. It is not clear from this study, however, whether the results are due to the depression or the depression is due to the reduced lung function.
Further, a study of overall physical vulnerability to asthma by Miller and Wood [45] suggested that depressive emotions in children (evoked whilst viewing selected scenes from the film 'ET: The Extra Terrestrial') may be associated with greater cholinergic influence and instability of oxygen saturation, consistent with poorer airway function in asthma. In contrast, happiness appeared to be related to effects that were more likely to relieve airway constriction.
Ritz and Steptoe [40] conducted a study consisting of both laboratory and field measurements, and demonstrated a consistency between both situations. From the field measurements, episodes of strong positive and negative mood were compared with records of relatively neutral emotional state, with the results showing that for persons with asthma (but not those in the non-asthmatic control group), negative affect, and to a lesser degree, positive affect, were associated with a reduction in FEV1%. These findings of reduced FEV1% during negative mood in the 'real life' component of the study were also consistent with and significantly related to increases in respiratory resistance during the viewing of a depressing film in the study's laboratory component.
Overall, the research on affect and pulmonary function indicates that depressed or sad mood, even when only short-lived and mild, can produce respiratory effects that are consistent with heightened airway vulnerability or asthma exacerbations. Further investigations are needed to establish whether this effect commonly occurs in the everyday lives of persons with asthma.
Depression, asthma self-management, and treatment compliance
Self-management of asthma can be challenging, due to the often complicated and onerous nature of treatment [41,46]. The individual with asthma may need to avoid allergens; take preventive medication regularly; and decide when reliever medications are required and whether further medical assistance should be sought [41,46]. Further, the potential implications of poor management and reduced compliance are serious, with consistently negative effects for individuals with asthma being reported, including increased morbidity [47] and mortality [48,49].
Depression, which has negative effects on cognitive functioning, energy, and motivation [41], has been identified as one factor which may decrease the effectiveness of asthma self-management and compliance. A meta-analysis by DiMatteo et al [50] revealed that patients with a chronic disease and depression were three times more likely to be noncompliant with medical treatment than non-depressed patients. Bosley et al [51] found that individuals with asthma who were classified as 'noncompliant' (took less than 70% of their prescribed doses of inhaled medication) had significantly higher depression scores (measured on the HADS) than those who were compliant, and Cluley and Cochrane [11] reported that in their investigation of a group of 19 people with asthma who had been diagnosed with depression (as assessed by both the HADS and the Structured Clinical Interview for the DSM-III-R Patient Edition (SCID-P)), only one was adherent to their medication regime.
Depression might interfere with asthma treatment compliance and self-management via several pathways: firstly, depression-related hopelessness may lead to a patient seeing little point in taking their medication as instructed [52]; depression can also result in isolation from family and friends who could offer the support that has often been noted as important for compliance [52]; and finally, depression may also be associated with declines in areas of cognitive functioning (such as problem solving, complex task performance, concentration, attention span, and memory) that are vital for compliance with treatment recommendations [37,53-55].
Unfortunately, the correlational studies mentioned here cannot determine whether depression causes reduced compliance or vice-versa, whether there are mediating factors in the relationship, or whether the relationship between depression and compliance may be bi-directional [50]. DiMatteo et al [50] have also hypothesised the possibility of a 'feedback loop', in which depression leads to treatment non-compliance, non-compliance further exacerbates asthma, asthma exacerbations lead to increased depression, and so on, resulting in a cycle of ever-worsening outcomes for the individual.
Corticosteroid use and depression
Corticosteroid use has also been hypothesised as a link between asthma and depression [18,46,56]. The creation of more potent inhaled corticosteroids (ICS), together with more efficient delivery systems, has greatly increased the use of ICS in the last 20 years [18]. While these changes have had positive effects on overall health, they have also been related to adverse side effects [57].
Although early reports of a possible relationship between asthma medications and depression were rare, and often anecdotal, recent studies in this area seem to suggest a stronger link than first believed. For example, an investigation by Patten and Lavorato [58] found that corticosteroid use was significantly associated with a 'syndrome resembling major depression' (measured by the Composite International Diagnostic Interview Short-Form (CIDI-SF)), and in a study by Patten [59] of 73,402 community members, it was found that among individuals taking corticosteroids (current or within the past month), the prevalence of major depression (11.1%; also measured by the CIDI-SF) was significantly higher than for those who had not taken steroids (4.1%). Perceived health status was not a confounder in this study. In a smaller outpatient study (N = 50), Bonala et al [18] found high-dose ICS usage was associated with positive effects in relation to pulmonary functioning and 'physical' quality of life, but was inversely related to 'mental' quality of life.
There appear to be a number of gaps and problems in the literature to date on ICS and depression in asthma. For example, some studies seem to have overlooked the possibility that the illness for which the corticosteroid was being taken may have been responsible for the increased levels of depression, instead of the medication. Also, it is unclear if depression related to corticosteroid use is actually a common problem. As mentioned, earlier accounts of this relationship were infrequent, and it may be that as ICS have become more potent and prescribed, the problem has increased exponentially. Another possible explanation for increased recognition of depression in persons using ICS for asthma treatment may simply be that medical practitioners have become more adept in recognising depression.
A 'feedback loop' of depression and asthma?
A number of researchers have suggested that a so-called 'feedback loop' may exist between asthma and depression [42,45]. Lehrer et al [42] noted that negative emotion (such as depression) often experienced by people with asthma may be as much a result of having asthma, as it is a cause of it, and that this bidirectional association may lead to a continual cycle of asthma and depression, resulting in ever-worsening physical and mental health. DiMatteo et al [50] noted the possibility that non-compliance with medical treatment might also be a component in this cycle.
Clinical implications: a 'whole person' approach to treatment
As discussed above, the coexistence of asthma and depression has been linked to a number of negative effects on both the physical and mental health of the individual. These adverse effects have led several researchers to propose that the most successful treatment is likely to require an integrated treatment approach, using interventions to address the physical, psychological, and social consequences of asthma [45,60].
One suggested treatment combination has been to treat depression and asthma together using antidepressants with anticholinergic properties, such as tricyclic antidepressant medication [4,45,60]. However, some who have advocated the use of antidepressant medications have also warned that antidepressants alone may not be the entire answer for a chronic disease such as asthma, because people with asthma need to be able to "successfully grieve over physical losses, combat changes in self-esteem, and overcome the social isolation that illness can cause" [[60], p.296]. For these reasons, counselling and psychosocial aspects in treatment are likely to have a very important role in successful asthma treatment, and cognitive-behavioural therapy (CBT) and group and individual counselling are already being combined with asthma self-management information to try to improve health outcomes for asthma sufferers [61].
It is hoped that by treating depression in asthma, the negative effects of this coexistence can be minimised [33,51]. While treating depression may increase adherence and lead to more effective asthma self-management, decrease asthma symptoms, and possibly even decrease asthma-related mortality [11,44,61-63], at a minimum, treating depression is likely to dramatically improve the HRQOL of individuals with asthma [64].
To date, only one study is believed to have examined the impact of treating depression in asthma. Grover et al [65] recruited a sample of 10 asthma out-patients, with participants sequentially allotted to either the experimental group (CBT and standard pharmacotherapy for asthma) or control group (standard pharmacotherapy only). CBT in the experimental group consisted of 15 individual sessions involving asthma education, muscle relaxation techniques, behavioural techniques, cognitive restructuring, and coping skills. Following the full course of therapy and medication, the experimental group had significant decreases in asthma symptoms, anxiety, and depression (measured by the Beck Depression Index), and a significant increase in quality of life, while the control group did not show any significant changes. Although these results are promising, further investigation into the effects of employing combined treatment approaches is obviously necessary.
Methodological issues in the literature
A number of problematic methodological issues were observed in the studies examined in this review, many of which limited interpretations of the findings. Firstly, the majority of the reviewed studies were cross-sectional and retrospective in design, meaning that neither directionality nor causality could be reliably inferred from the results [22,28,37,42,50,66]. Instead, many researchers hypothesised causality and relationship direction in their own findings based on the conclusions of other studies.
The different measurement instruments used and names given to the constructs of depression examined in the studies, ('depression', 'depressive symptoms', 'major depression', 'a syndrome resembling major depression', etc) also made it difficult both to understand what was actually being measured and investigated, and to compare results and interpretations between studies. Comparatively few studies used standardised diagnostic instruments such as the DSM and SCID, or standardised clinical rating scales such as the Hamilton Rating Scale for Depression, the consistent use of which would make interpretation and cross-study comparisons more straightforward. The problems of unclear definitions of depression and lack of standardised assessment measures were also noted by Rodin and Voshart [cited in [67]] as issues hindering progress on research in the medically ill.
Because asthma and depression share a number of symptoms, there is also potential for the use of self-report measures in studies to result in inaccurate diagnoses of depression or depressive symptoms [23,37,68]. Sherwood Brown et al [23] noted that on some measures, secondary symptoms of asthma such as decreased sleep or fatigue may elevate scores on measures of depression, even if these symptoms are actually unrelated to psychological problems. This issue will require careful selection of instruments to measure depression.
Finally, most samples utilised in studies investigating asthma and depression have been drawn from in-patient or outpatient samples, with very few population or primary care studies conducted thus far. Although these studies have been instrumental in highlighting links between asthma and depression, the generalisability of these findings to the broader asthma population is not yet well understood [39].
Future research directions
There are many potential research avenues to consider in regards to the coexistence of asthma and depression. Firstly, further investigation of the various links considered in this review (especially those explored in studies that may have been limited by methodological issues) would serve to provide a clearer understanding of the relationships between asthma and depression, and the potential repercussions of this association.
Future research should also consider 'big picture' studies, in which a number of different variables such as medication compliance, asthma self-management, quality of life, and lung function might all be examined in one investigation. These studies are likely to be particularly vital in aiding our understanding of possible 'feedback loops' of asthma and depression, more fully exploring how asthma and depression coexist, and may be valuable in determining how best to reduce or eliminate the negative effects of this coexistence.
Researchers have been suggesting for some time that the most important next step in asthma and depression research is to investigate the effects of treating the depression of persons with asthma. However, as previously mentioned, only one small study appears to have attempted this so far [65]. The first step in this new research phase would be to develop effective combinations of pharmacological, psychological, and/or psychosocial interventions as an integrated treatment program for people with asthma and depression. The most sought after knowledge, however, would result from studies using these integrated programs to actually treat the depression of individuals with asthma, and assessing the effects of this treatment on variables such as the depression itself, compliance, self-management, HRQOL, pulmonary function, asthma symptom exacerbations, overall asthma severity, and asthma-related mortality. Large scale studies of this type would be complex to set up, expensive to carry out, and require long-term commitment to the research.
Although they are most likely to be targeted toward patients already suffering from both asthma and depression, integrated treatment programs may also be valuable from a prevention focus, working to inhibit the development of depression and the negative psychological and physical health effects that may follow. Large scale, long-term research also needs to be carried out to examine this important possibility.
While a great deal of investigation still needs to be carried out, the move into this new phase of asthma and depression research has great potential to result in more effective ways of caring for people living with these coexisting illnesses, and significantly improve the lives of many individuals.
Declarations
Ethical clearance was not required for this literature review. MO received a University of Adelaide Vacation Scholarship to conduct the literature review. The authors do not have any conflict of interests in relation to this study.
==== Refs
Vila G Nollet-Clemencon C de Blic J Mouren-Simeoni MC Scheinmann P Prevalence of DSM IV anxiety and affective disorders in a pediatric population of asthmatic children and adolescents J Affect Disord 2000 58 223 231 10.1016/S0165-0327(99)00110-X 10802131
Rubin NJ Severe asthma and depression Arch Fam Med 1993 2 433 440 10.1001/archfami.2.4.433 8130924
Verhaak PF Heijmans MJ Peters L Rijken M Chronic disease and mental disorder Soc Sci Med 2005 60 789 797 10.1016/j.socscimed.2004.06.012 15571896
Thompson WL Thompson TL Treating depression in asthmatic patients Psychosomatics 1984 25 809 812 6505127
Kovacs M Stauder A Szedmak S Severity of allergic complaints: the importance of depressed mood J Psychosom Res 2003 54 549 557 10.1016/S0022-3999(02)00477-4 12781309
Rosner F Moses Maimonides' treatise on asthma Thorax 1981 36 245 251 7025335
Dunbar F Mind and body: psychosomatic medicine. 1947 New York, Random House
Alexander F Psychosomatic medicine: its principles and applications. 1950 New York, Norton
Moran MG Stoudemire A Pulmonary and rheumatologic diseases. Psychological factors affecting medical conditions 1995 Washington, D.C., American Psychiatric Press. 141 151
French T Alexander F Psychogenic factors in bronchial asthma. 1941 Washington, D.C., National Research Council
Cluley S Cochrane GM Psychological disorder in asthma is associated with poor control and poor adherence to inhaled steroids Respir Med 2001 95 37 39 10.1053/rmed.2000.0968 11207015
Harrison BD Psychosocial aspects of asthma in adults Thorax 1998 53 519 525 9713455
Roberts C Mayer JD Henderson WRJ Asthma deaths in Washington State, 1980-1989: geographic and demographic distributions Ann Allergy Asthma Immunol 1996 76 20 26 8564624
Ford ES Mannino DM Homa DM Gwynn C Redd SC Moriarty DG Mokdad AH Self-reported asthma and health-related quality of life: findings from the behavioral risk factor surveillance system Chest 2003 123 119 127 10.1378/chest.123.1.119 12527612
American Thoracic Society Adult asthma http://www.atsqol.org/adultasthma.asp
Juniper EF Effect of asthma on quality of life Can Respir J 1998 5 Suppl A 77A 84A 9753523
Australian Centre for Asthma Monitoring Measuring the impact of asthma on quality of life in the Australian population 2004 Canberra, Australian Institute of Health and Welfare
Bonala SB Pina D Silverman BA Amara S Bassett CW Schneider AT Asthma severity, psychiatric morbidity, and quality of life: correlation with inhaled corticosteroid dose. Journal of Asthma 2003 40 691 699 10.1081/JAS-120023491 14580001
Stewart AL Greenfield S Hays RD Wells K Rogers WH Berry SD McGlynn EA Ware JEJ Functional status and well-being of patients with chronic conditions. Results from the Medical Outcomes Study Jama 1989 262 907 913 10.1001/jama.262.7.907 2754790
Wells KB Stewart A Hays RD Burnam MA Rogers W Daniels M Berry S Greenfield S Ware J The functioning and well-being of depressed patients. Results from the Medical Outcomes Study Jama 1989 262 914 919 10.1001/jama.262.7.914 2754791
Afari N Schmaling KB Barnhart S Buchwald D Psychiatric comorbidity and functional status in adult patients with asthma. Journal of Clinical Psychology in Medical Settings 2001 8 245 252 10.1023/A:1011912712262
Mancuso CA Rincon M McCulloch CE Charlson ME Self-efficacy, depressive symptoms, and patients' expectations predict outcomes in asthma Med Care 2001 39 1326 1338 10.1097/00005650-200112000-00008 11717574
Sherwood Brown E Khan DA Nejtek VA Rajan Thomas N Mahadi SF Depressive symptoms and functioning in asthmatic patients. Primary Care Psychiatry 2000 6 155 161 10.1185/135525700543389
Anderson KL The effect of chronic obstructive pulmonary disease on quality of life Res Nurs Health 1995 18 547 556 7480855
Lyons RA Lo SV Littlepage BN Comparative health status of patients with 11 common illnesses in Wales J Epidemiol Community Health 1994 48 388 390 7964339
Vamos M Kolbe J Psychological factors in severe chronic asthma Aust N Z J Psychiatry 1999 33 538 544 10.1046/j.1440-1614.1999.00591.x 10483849
Janson C Bjornsson E Hetta J Boman G Anxiety and depression in relation to respiratory symptoms and asthma Am J Respir Crit Care Med 1994 149 930 934 8143058
Rimington LD Davies DH Lowe D Pearson MG Relationship between anxiety, depression, and morbidity in adult asthmatic patients. Thorax 2001 56 266 271 10.1136/thorax.56.4.266 11254816
Chaney JM Mullins LL Uretsky DL Pace TM Werden D Hartman VL An experimental examination of learned helplessness in older adolescents and young adults with long-standing asthma J Pediatr Psychol 1999 24 259 270 10.1093/jpepsy/24.3.259 10379141
Badoux A Levy DA Psychologic symptoms in asthma and chronic urticaria Ann Allergy 1994 72 229 234 8129215
Centanni S Di Marco F Castagna F Boveri B Casanova F Piazzini A Psychological issues in the treatment of asthmatic patients Respir Med 2000 94 742 749 10.1053/rmed.1999.0766 10955748
Kessler RC McGonagle KA Zhao S Nelson CB Hughes M Eshleman S Wittchen HU Kendler KS Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey Arch Gen Psychiatry 1994 51 8 19 8279933
Gillaspy SR Hoff AL Mullins LL Van Pelt JC Chaney JM Psychological distress in high-risk youth with asthma J Pediatr Psychol 2002 27 363 371 10.1093/jpepsy/27.4.363 11986359
Mrazek DA Psychiatric complications of pediatric asthma Ann Allergy 1992 69 285 290 1416262
Fitzpatrick MF Engleman H Whyte KF Deary IJ Shapiro CM Douglas NJ Morbidity in nocturnal asthma: sleep quality and daytime cognitive performance Thorax 1991 46 569 573 1926025
Leigh D Marley E A psychiatric assessment of adult asthmatics: a statistical study J Psychosom Res 1956 1 128 136 10.1016/0022-3999(56)90014-9 13346680
Adams RJ Wilson DH Taylor AW Daly A Tursan d'Espaignet E Dal Grande E Ruffin RE Psychological factors and asthma quality of life: a population based study Thorax 2004 59 930 935 10.1136/thx.2003.010256 15516466
Janson-Bjerklie S Ferketich S Benner P Becker G Clinical markers of asthma severity and risk: importance of subjective as well as objective factors Heart Lung 1992 21 265 272 1592618
Goldney RD Ruffin R Fisher LJ Wilson DH Asthma symptoms associated with depression and lower quality of life: a population survey Med J Aust 2003 178 437 441 12720509
Ritz T Steptoe A Emotion and pulmonary function in asthma: reactivity in the field and relationship with laboratory induction of emotion Psychosom Med 2000 62 808 815 11139001
Lehrer PM Feldman J Giardino N Song HS Schmaling K Psychological aspects of asthma J Consult Clin Psychol 2002 70 691 711 10.1037//0022-006X.70.3.691 12090377
Lehrer PM Isenberg S Hochron SM Asthma and emotion: a review J Asthma 1993 30 5 21 8428858
Ritz T Claussen C Dahme B Experimentally induced emotions, facial muscle activity, and respiratory resistance in asthmatic and non-asthmatic individuals Br J Med Psychol 2001 74 167 182 10.1348/000711201160894
Krommydas GC Gourgoulianis KI Angelopoulos NV Kotrotsiou E Raftopoulos V Molyvdas PA Depression and pulmonary function in outpatients with asthma Respir Med 2004 98 220 224 10.1016/j.rmed.2003.09.018 15002757
Miller BD Wood BL Influence of specific emotional states on autonomic reactivity and pulmonary function in asthmatic children J Am Acad Child Adolesc Psychiatry 1997 36 669 677 10.1097/00004583-199705000-00018 9136502
Main J Moss-Morris R Booth R Kaptein AA Kolbe J The use of reliever medication in asthma: the role of negative mood and symptom reports. Journal of Asthma 2003 40 357 365 10.1081/JAS-120018635 12870831
Horn CR Clark TJ Cochrane GM Compliance with inhaled therapy and morbidity from asthma Respir Med 1990 84 67 70 2371425
Sears MR Rea HH Beaglehole R Gillies AJ Holst PE O'Donnell TV Rothwell RP Sutherland DC Asthma mortality in New Zealand: a two year national study N Z Med J 1985 98 271 275 2859567
British Thoracic Association Death from asthma in two regions of England. Br Med J (Clin Res Ed) 1982 285 1251 1255 6812831
DiMatteo MR Lepper HS Croghan TW Depression is a risk factor for noncompliance with medical treatment: meta analysis of the effects of anxiety and depression on patient adherence. Arch Intern Med 2000 160 2101 2107 10.1001/archinte.160.14.2101 10904452
Bosley CM Fosbury JA Cochrane GM The psychological factors associated with poor compliance with treatment in asthma Eur Respir J 1995 8 899 904 7589375
DiMatteo MR Enhancing patient adherence to medical recommendations Jama 1994 271 79, 83 10.1001/jama.271.1.79 8258895
Tarbuck AF Paykel ES Effects of major depression on the cognitive function of younger and older subjects Psychol Med 1995 25 285 295 7675916
Willner P Cognitive functioning in depression: a review of theory and research Psychol Med 1984 14 807 823 6545415
Sweeney JA Wetzler S Stokes P Kocsis J Cognitive functioning in depression J Clin Psychol 1989 45 836 842 2613891
O'Byrne PM Pedersen S Measuring efficacy and safety of different inhaled corticosteroid preparations J Allergy Clin Immunol 1998 102 879 886 9847425
Toogood JH Side effects of inhaled corticosteroids J Allergy Clin Immunol 1998 102 705 713 9819285
Patten SB Lavorato DH Medication use and major depressive syndrome in a community population. Comprehensive Psychiatry 2001 42 124 131 10.1053/comp.2001.21218 11244148
Patten SB Exogenous corticosteroids and major depression in the general population J Psychosom Res 2000 49 447 449 10.1016/S0022-3999(00)00187-2 11182439
Koenig HG Depression in the medically ill: a common and serious disorder Int J Psychiatry Med 2000 30 295 297 10.2190/P57Y-0PT5-FRB8-7QK7 11308033
Fleming SL Pagliari C Churchill R Shuldham CM McKean M Psychotherapeutic interventions for adults with asthma Cochrane Database Syst Rev 2004 CD002982 14974000
Coffman K Psychiatric issues in pulmonary disease Psychiatr Clin North Am 2002 25 89 127 10.1016/S0193-953X(03)00054-6 11912946
Galil N Depression and asthma in children Curr Opin Pediatr 2000 12 331 335 10.1097/00008480-200008000-00008 10943812
Jackson JL DeZee K Berbano E Can treating depression improve disease outcomes? Annals of Internal Medicine 2004 140 1054 1056 15197024
Grover N Kumaraiah V Prasadrao PS D'Souza G Cognitive behavioural intervention in bronchial asthma J Assoc Physicians India 2002 50 896 900 12126343
Lehrer PM Sargunaraj D Hochron S Psychological approaches to the treatment of asthma J Consult Clin Psychol 1992 60 639 643 10.1037/0022-006X.60.4.639 1506512
Cassem EH Depression and anxiety secondary to medical illness Psychiatr Clin North Am 1990 13 597 612 2281008
Miller BD Depression and asthma: a potentially lethal mixture J Allergy Clin Immunol 1987 80 481 486 10.1016/0091-6749(87)90080-7 3624703
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Cost Eff Resour AllocCost effectiveness and resource allocation : C/E1478-7547BioMed Central London 1478-7547-3-91618802110.1186/1478-7547-3-9ResearchTechnical efficiency of public district hospitals and health centres in Ghana: a pilot study Osei Daniel [email protected]'Almeida Selassi [email protected] Melvill O [email protected] Joses M [email protected] Ayayi Omar [email protected] Lenity H [email protected] Planning and Budget Unit, PPME, Ghana Health Service, Accra, Ghana2 WHO Country Office, Accra, Ghana3 World Health Organization, Regional Office for Africa, Brazzaville, Congo4 Department of Health Sciences, School of Public Health, Kenyatta University, Nairobi, Kenya2005 27 9 2005 3 9 9 16 5 2005 27 9 2005 Copyright © 2005 Osei et al; licensee BioMed Central Ltd.2005Osei 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 Government of Ghana has been implementing various health sector reforms (e.g. user fees in public health facilities, decentralization, sector-wide approaches to donor coordination) in a bid to improve efficiency in health care. However, to date, except for the pilot study reported in this paper, no attempt has been made to make an estimate of the efficiency of hospitals and/or health centres in Ghana. The objectives of this study, based on data collected in 2000, were: (i) to estimate the relative technical efficiency (TE) and scale efficiency (SE) of a sample of public hospitals and health centres in Ghana; and (ii) to demonstrate policy implications for health sector policy-makers.
Methods
The Data Envelopment Analysis (DEA) approach was used to estimate the efficiency of 17 district hospitals and 17 health centres. This was an exploratory study.
Results
Eight (47%) hospitals were technically inefficient, with an average TE score of 61% and a standard deviation (STD) of 12%. Ten (59%) hospitals were scale inefficient, manifesting an average SE of 81% (STD = 25%). Out of the 17 health centres, 3 (18%) were technically inefficient, with a mean TE score of 49% (STD = 27%). Eight health centres (47%) were scale inefficient, with an average SE score of 84% (STD = 16%).
Conclusion
This pilot study demonstrated to policy-makers the versatility of DEA in measuring inefficiencies among individual facilities and inputs. There is a need for the Planning and Budgeting Unit of the Ghana Health Services to continually monitor the productivity growth, allocative efficiency and technical efficiency of all its health facilities (hospitals and health centres) in the course of the implementation of health sector reforms.
==== Body
Background
The strategic health objectives of Vision 2020 in Ghana envisage: a significant reduction in the rates of infant, child and maternal mortality; effective control of the risk factors that expose individuals to major communicable diseases; increased access to health services, especially in rural areas; establishment of a health system effectively reoriented toward delivery of public health services; and effective and efficient management of the health system [1].
The Ministry of Health, following the thrust of Vision 2020, developed its current policy and strategy guidelines in 1995 in the Medium-Term Health Strategy (MTHS) document [2]. The five main objectives of the MTHS are: improving access to health services; improving quality of care; improving efficiency; fostering partnership between private and public health service-providers; and improving financing of health services.
Subsequently, the first [3] and second [4] Health Sector Five-Year Programme of Work were developed to enable the country attain the MTHS objectives. One of the five underlying objectives of the two programmes of work is "improved efficiency in health services delivery". Furthermore, the Ghana Poverty Reduction Strategy 2002–2004 [5] also highlights "enhancing efficiency in health service delivery" as one of the three priority health sector-related interventions. Thus, efficiency concerns are deeply embedded in the national Vision 2020, national poverty reduction strategy, health policy and programme of work.
In a bid to improve the efficiency of health services delivery, the Ministry of Health is implementing the following health sector reforms: separation of functions between the Ministry of Health (policy formulation, planning, donor coordination and resource mobilization) and the Ghana Health Services (responsible for service delivery); autonomy of tertiary hospitals; decentralized planning and budgeting systems, strengthening of financial management and performance monitoring system, and investing in overall management development capacity within the sector; sector-wide approach (SWAP); and strengthening of existing regulatory bodies and laws [6,7].
Since 1978, Data Envelopment Analysis (DEA) has been extensively used in the Americas [8-10], Western Europe [11-17] and Asia [18,19] to shed light on the efficiency of various aspects of national health systems. In Africa, the application of DEA in the health sector has been quite limited. So far, the approach has been applied to health facilities in only three countries, i.e. South Africa [20-22], Kenya [23,24] and Zambia [25]. Yet, the assessment of the efficiency ought to be more prevalent in low-income countries like Ghana in order to optimise health benefits from the available meagre health sector resources.
In Ghana, prior to the current study, no attempt had been made to estimate the efficiency of health care facilities, using either parametric (econometric) or non-parametric methods. The Planning Unit in the Ministry of Health (with support from the World Health Organization) decided to undertake a limited pilot study to demonstrate to policy-makers the potential usefulness of DEA in the pursuit of health sector efficiency objectives. Once policy-makers were adequately sensitised, hopefully, a national efficiency study would be conducted among all health centres and district, regional and tertiary hospitals.
The objectives of the exploratory study reported in this paper were: (i) to estimate the relative technical efficiency of a sample of public hospitals and health centres in Ghana; and (ii) to demonstrate policy implications for health sector policy-makers.
Methods
Study area
Ghana is situated on the west coast of Africa. It is divided into ten administrative regions (i.e. Upper East, Upper West, Northern, Brong Ahafo, Ashanti, Volta, Eastern, Greater Accra, Central and Western) and 110 districts. The organisation of health services more or less mimics the administrative structure.
The country's health services are organised at the following levels [2]:
a) Community: Delivered through outreach programmes, resident or itinerant herbalists, traditional birth attendants and/or retail drug peddlers.
b) Sub-district: A health centre services a geographical area with 15 000 to 30 000 population. It provides basic curative care, disease prevention services and maternity services (primary health care). A health centre constitutes an essential component of the close-to-client health services.
c) District: A district hospital provides support to sub-districts in disease prevention and control, health promotion and public health education; referral outpatient and inpatient care, training and supervision of health centres; maternity services, especially the management of complications and emergencies and surgical contraception.
d) Regional: A regional hospital provides specialised clinical and diagnostic care; management of high-risk pregnancies and complications of pregnancy; technical and logistical back up for epidemiological surveillance; and research and training.
e) Tertiary: At the apex of the referral system, there are two government-owned teaching hospitals that offer specialised services, undertake research, and provide undergraduate and postgraduate training in health and allied areas.
f) National (i.e. Ministry of Health headquarters): The national level is responsible for the development of national health policy and for providing strategic directions for service delivery as well as coordination and monitoring.
Ghana's population of 19.7 million is served by a total of 2189 health facilities, of which 952 are government owned, 181 are owned by religious organisations, 75 are quasi-government and 980 belong to the private sector. Out of the total number of health facilities, 2 are teaching hospitals, 9 regional hospitals, 91 district hospitals, 124 other hospitals, 558 health centres, 1085 clinics and 320 are maternity homes. All these health facilities are serviced by 1294 doctors, 29 dentists, 207 pharmacists and 326 medical assistants, along with other paramedical and support staff [7].
The country spends a total of US$ 252 million (4.2% of the GDP of US$ 6 billion) annually on health. About 53.5% of this expenditure is incurred by the government and 46.5% by the households through out-of-pocket expenses. The total per capita expenditure on health at an average exchange rate is US$ 11 [26].
The life expectancy at birth in Ghana is 57.4 years. The infant and under-5-years mortality rates per 1000 live births are 57 and 97 respectively [26]. The probability of dying (per 1000 live births) between ages 15 and 59 years is 303. The maternal mortality per 100 000 live births is 214. Nearly 72% of the population has access to improved sanitation, while 73% has access to an improved water supply source [27].
The question is whether the people of Ghana are deriving maximum health care benefits from the aforementioned investments in health sector, especially from hospitals and health centres which consume over 75% of both the recurrent and capital budgets of the Ministry of Health. The next sub-section presents a DEA conceptual framework, which is used to shed light on this issue.
DEA conceptual framework
In the production process, hospitals and health centres turn inputs (factors of production) into outputs (health services). We can divide the inputs into broad categories of labour, materials and capital, each of which will often include more narrow sub-divisions. Labour inputs include skilled health personnel (doctors, nurses, paramedics, support staff) and unskilled workers (drivers, watchmen, gardeners, ward attendants, cooks, etc.), as well as the entrepreneurial efforts of managers of health facilities. Materials include pharmaceuticals, non-pharmaceutical supplies and any other goods that health facilities require to produce health care. Capital includes buildings, medical equipment, vehicles and beds.
The relationship between inputs and the production process and resulting outputs is described in Figure 1. It is clear that hospitals and health centres employ multiple inputs to produce multiple outputs. We used DEA approach since it allows the measurement of relative efficiency when decision-making units (in this case hospitals/health centres) have multiple inputs and multiple outputs.
Figure 1 Relationship between inputs and the production process and resulting outputs.
DEA is a linear programming methodology for evaluating relative efficiency of each production unit among a set of fairly homogeneous decision-making units (DMUs), e.g. district hospitals, health centres, etc. It sketches a production possibilities frontier (data envelop or efficient frontier) using combinations of inputs and outputs from best performing health facilities. Health facilities that compose the "best practice frontier" are assigned an efficiency score of one (or 100%) and are deemed technically efficient compared to their peers. The efficiency of the health facilities below the efficiency frontier is measured in terms of their distance from the frontier. The inefficient health facilities are assigned a score between one and zero. The larger the score the more efficient a health facility is.
Since hospitals and health centres employ multiple inputs to produce multiple outputs, their individual technical efficiency can be defined as [28]:
The technically inefficient health facility uses more weighted inputs per weighted output, or produce less weighted output per weighted input than those health facilities on the "best practice frontier".
Algebraically, technical efficiency score of each hospital and health centre in the sample were obtained by solving the models (1) and (2) (See Table 8) [29].
Table 8 Model 1. DEA weights model, input-oriented, constant returns to scale (CRS) Model 2. DEA weights model, input-oriented, variable returns to scale (VRS)
Where:
yrj = the amount of output r produced by hospital or health centre j,
xij = the amount of input i used by hospital or health centre j,
ur = the weight given to output r, (r = 1,..., t and t is the number of outputs),
vi = the weight given to input i, (i = 1, ..., m and m is the number of inputs),
n = the number of hospitals or health centres,
j0 = the hospital or health centre under assessment.
We need to explain what we mean by constant returns to scale and variable returns to scale. Returns to scale refers to the changes in output as all inputs change by the same proportion. For instance, suppose that for a specific hospital (or health centre) j we start from an initial level of inputs (doctors = D, Other technical staff = T, Subordinate staff = S, Beds = B) and output (Q)
Qo = f(D,T,S,B)
and we increase all the factors by the same proportion φ. We will obviously obtain a new level of output Q*, higher than the original level Q0,
Q* = f(φD,φT,φS,φB)
If Q* increases by: (i) the same proportion φ as the inputs, we say that there are constant returns to scale (CRS); (ii) less than proportionally with the increase in inputs, we have decreasing returns to scale (DRS); (iii) more than proportionally with the increase in the inputs, we increasing returns to scale (IRS).
Those hospitals and health centres manifesting CRS can be said to be operating at their most productive scale sizes. In order to operate at the most productive scale size, a health facility displaying DRS should scale down both outputs and inputs. If a health facility is exhibiting IRS, it should expand both outputs and inputs in order to become scale efficient [8].
We have illustrated below how DEA works using hypothetical hospitals.
Illustration of the DEA analysis
Lets assume that a hypothetical country called Nkrumah has 9 district hospitals. Each hospital produces two outputs (i.e. outpatient department visits (OPVisits) and inpatient admissions (Admissions)) from a single input of technical staff. The number of staff employed, Opvisits, Admissions, ratios of Opvisits to staff, and Admissions to staff are contained in the Table 1.
Table 1 Illustration of DEA analysis using a hypothetical example of nine hospitals
DMUs OPVisits (A) Admissions (B) Staff (C) OPvisits/staff D = (A/C) Admissions/staff E = (B/C)
Anim 7020 5451 102 69 53
Akoa 20566 7610 92 224 83
Addai 25200 7148 143 176 50
Mensa 33568 7958 96 350 83
Kofi 17406 2429 95 183 26
Gyau 10573 8094 117 90 69
Anani 10500 8944 115 91 78
Amoi 20421 10969 117 175 94
Asamoa 20647 8619 46 449 187
The efficiency of each hospital in producing the two outputs were estimated by dividing each of their outputs by their input and seeing which hospital(s) have the highest ratios. The results are contained in the last two columns of Table 1. The higher the ratio of an output to input the more efficient a hospital is in producing that output. In this example, Asamoa hospital had the highest number of Opvisits per staff (449) and Admissions (187) for each member of technical staff employed.
By plotting Opvisits/staff against Admissions/staff for the nine hospitals we derive the production possibilities frontier graph contained in Figure 2.
Figure 2 Production possibilities frontier graph.
The diagram shows the efficiency frontier, which is the fundamental concept of DEA. The straight lines from Asamoa hospital to the Y axis (labelled Admissions/staff) and from Asamoa to the X axis (labelled Opvisits/staff) represent the efficient frontier. The efficiency frontier, derived from the most efficient hospital(s) (i.e. Asamoa hospital in our example) in the dataset/sample, represents a standard of technically best performance that can be achieved from available input and technology endowment. Consequently, it is used as a threshold against which to measure the performance of all other hospitals.
The efficiency frontier 'envelops' the inefficient hospitals within it and clearly shows the relative efficiency of each hospital. A hospital like Asamoa, which located on the frontier, is considered 100% technically efficient. Any hospital like Mensa, Amoi, Akoa, Anani, Kofi, Addai, Gyau and Anim that is below the production possibilities frontier is relatively less efficient and is given a technical efficiency rating of less than 100%.
Anim hospital, for instance, could become efficient if it increased its outputs, in the same proportions, while holding its input constant, i.e. assuming an output-orientated model/situation. Instead, it could become efficient by reducing its input while keeping its outputs the same, i.e. assuming an input-orientated scenario. Its technical efficiency is calculated by the ratio of its distance from the origin over the distance from the origin to the point of intersection on the production possibilities frontier or the efficiency frontier. This gives Anim hospital a technical efficiency score of 28.52%. Likewise, Mensa hospital is 77.90% as efficient as Asamoa hospital (i.e. the best practice hospital), Amoi hospital is 50.04%, Akoa hospital is 49.80%, Anani hospital is 41.51%, Kofi hospital is 40.82%, Addai hospital is 39.26%, and Gyau hospital is 36.92%. These scores were estimated assuming constant returns to scale (CRS).
However, often health services production process are not linear, and thus it may be more appropriate to assume variable returns to scale (VRS). Thus, we estimated the DEA model assuming VRS, the efficiency scores for various hospitals were as follows: Asamoa = 100%, Amoi = 100%, Mensa = 100%, Akoa = 50%, Anani = 48.54%, Kofi = 48.42%, Anim = 45.10%, Addai = 44.49% and Gyau = 39.32%. This finding implies that if Akoa, Anani, Kofi, Anim, Addai and Gyau hospitals were to operate efficiently, they are capable of producing their current output levels with 50%, 51.5%, 51.58%, 54.9%, 55.51% and 60.68% less inputs than they are currently using. Whereas in the CRS model only Asamoa hospital had a 100% efficiency score, in the VRS model three hospitals (Asamoa, Amoi, Mensa) achieved a pure technical efficiency score of 100%.
The impact of hospital scale/size on their technical efficiency was evaluated using a three-step process. First, the model was estimated assuming CRS. Second, the model was run assuming VRS. Third, scale efficiency was obtained by dividing each hospital's CRS technical efficiency score by its VRS technical efficiency score. Akoa and Asamoa hospitals has scale efficiency score of 100%, implying they had an optimal size. Gyau scored 94%, Addai scored 88%, Anani scored 86%, Kofi scored 84%, Mensa scored 78%, Anim scored 63% and Amoi scored 50%. These seven hospitals were scale inefficient since they were not operating at their most productive size for their observed input mix. It is important to mention that DEA is only an exploratory tool for efficiency measurement, and indicates directions for further investigations into how to improve/enhance efficiency.
Input and output orientation
Input Orientation for hospitals
In the hospital analysis the input orientation assumed that these facilities had limited control over the volume of their outputs. There was no linkage between staff earnings and output; thus, there was no incentive for inducing demand for health services. Otherwise, in Ghana hospital management has got greater control over the use of inputs. Thus, an input-oriented DEA model was used for hospital analysis.
Output orientation for health centres
On the other hand, output orientation was assumed for health centres. The management of health centres has no control over inputs, especially it's staffing. However, given their primary health care orientation, with a strong bias towards health promotion and disease prevention, they can influence a great number of people seeking, for example, antenatal and postnatal care, family planning services, birthing services, immunisations and health education, through their public health outreach work among communities. Thus, the output-oriented DEA model was used for the health centre analysis.
Strengths and weaknesses of DEA
Strengths of DEA
We chose to employ DEA approach to estimate technical efficiency of individual hospitals and health centres because of its unique strengthens: (i) it can handle multiple input and multiple output models/scenarios typical of hospitals and health centres; (ii) it does not require an assumption of a functional form relating inputs to output (as regression methods do); (iii) health facilities are directly compared against a peer or combination of peers; (iv) inputs and outputs can be very different units; (v) it does not require information on prices of inputs and outputs [22,30].
Weaknesses of DEA
Even though we chose to use DEA, we were fully aware that it has two main limitations. Firstly, it attributes any deviation from the "best practice frontier" to inefficiency, while some could be due to statistical noise, e.g. epidemics or measurement errors. Secondly, given that DEA is deterministic/nonparametric technique, it is difficult to conduct statistical tests of hypotheses concerning the inefficiency and the structure of the production function [22,31,32].
Variables
The hospital DEA model had a total of 7 variables, including 3 outputs and 4 inputs. The three outputs for each individual hospital were: (i) the number of maternal and child care (MCH) (i.e. antenatal care, postnatal care, family planning, tetanus toxoid, child immunisation and growth monitoring); (ii) the number of child deliveries; and (iii) the number of patients discharged (not including deaths). The four inputs included: (i) number of medical officers; (ii) the number of technical officers (including medical assistants, nurses and paramedical staff); (iii) the support or subordinate staff (including orderlies, ward assistants, cleaners, drivers, gardeners, watchmen, etc.); and (iv) the number of hospital beds.
On the other hand, the health centre DEA model was estimated with a total of 6 variables: 4 outputs and 2 inputs. The four outputs for each individual health centre were: (i) the number of child deliveries; (ii) the number of fully immunised children under the age of 5 years; (iii) the number of other maternal (i.e. antenatal care, postnatal care and family planning services) and childcare (nutritional/child growth monitoring) visits; and (iv) the number of outpatient curative visits. The two inputs were: (i) the number of technical staff (this included medical assistants, nurses and paramedical staff); and (ii) the number of support or subordinate staff (including cleaners, drivers, gardeners, watchmen and others).
The choice of inputs and outputs for the DEA analysis was guided in part by the previous DEA health care studies in the African Region and availability of data.
Data
The data used in this study are for 2000. In order to have a feel of the usefulness of DEA in the measurement technical efficiency of hospitals and health centres, the policy-makers instructed the Planning and Budget Unit (PBU) to draw a pilot sample of 21 hospitals and 21 health centres. PBU decided to use simple random sampling technique to draw the two samples. Data were collected from a random sample of 21 public health centres using a WHO African Regional Office (WHO/AFRO) efficiency questionnaire for primary health care facilities [32]. However, information on health personnel in four health centres was missing; thus, they were left out of the analysis. Data were also collected from a random sample of 21 district hospitals; however, information on inputs and outputs from only 17 hospitals was included in the analysis. Data on hospitals were collected using a WHO/AFRO efficiency questionnaire for hospitals [33]. The data were analysed using the DEAP software developed by Professor Tim Coelli [31].
Results
Hospitals analysis
In 2000, all 17 hospitals in the sample produced a total of 200 589 maternal and child health (MCH) visits, 24 152 deliveries and 69 361 discharges. Those outputs were produced employing a total of 55 medical officers/dentists, 1345 technical staff, 721 subordinate staff and 1543 beds. Table 2 presents the means and standard deviations for input and output variables of the 17 district hospitals.
Table 2 Means and standard deviations for public hospitals inputs and outputs
Variable Mean Standard deviation
Outputs: Maternal and child health care visits 11799 9516
Deliveries 1421 1186
Inpatient discharges 4080 2274
Inputs: Doctors/dentists 3 3
Technical staff (including nurses) 79 34
Subordinate staff 42 27
Beds 91 43
The VRS model technical and scale efficiency scores for individual hospitals are contained in Table 3. Of the 17 hospitals, 9 (53%) were technically efficient since they had a relative technical efficiency (TE) score of 100%. The remaining 8 (47%) had a TE score of less than 100%, which means that they were technically inefficient. The TE score among the latter facilities ranged from 74% in Atua hospital to 43% in Winneba hospital. This finding implies that Atua and Winneba hospitals could potentially reduce their current input endowments by 26% and 57% while leaving their output levels unchanged. The average TE score among the inefficient hospitals was 61% (standard deviation = 12%), which means that these hospitals could, on average, produce their current levels of output with 39% less inputs than they were currently using.
Table 3 Technical and scale efficiency scores for district hospitals
DMU (Hospitals) Technical efficiency (%) Scale efficiency (%)
Swendru 100 100
Half Asin 100 100
Turkwa 100 100
Kwesiminstmu 100 100
Yendi 100 100
Takoradi 100 100
St Francis Xavier 100 100
West End 100 91.6
Walewale 100 43.9
Atua 74.4 99.8
Tetteh Quarshie 73.6 99.9
Cape Coast 68.6 93.2
Akuse 66.3 43.9
Axim 57.4 50.9
Suhum 56.5 98.0
Akim 46.0 99.6
Winneba 42.7 93.9
Seven (41%) of the hospitals had a scale efficiency (SE) of 100%, which means that they had the most productive size for that particular input-output mix. The remaining 10 (59%) hospitals had a SE of less than 100% and as such they were scale inefficient. The average SE among the inefficient hospitals was 81% (standard deviation = 25%), meaning that, on average, the scale inefficient hospitals could reduce their size by 19% without affecting their current output levels.
All the seven scale-efficient hospitals displayed constant returns to scale (CRS), implying thereby that they were operating at their most productive scale sizes. Eight of the 10 scale-inefficient hospitals had increasing returns to scale (IRS) while one of the hospitals revealed decreasing returns to scale (DRS). In order to operate at the most productive scale size (MPSS), a hospital exhibiting DRS should scale down both its outputs and inputs. Similarly, if a hospital is displaying IRS, it should expand both its outputs and inputs.
Health centre analysis
The total output of all health centres in the sample combined was as follows: (i) 67 739 MCH visits; (ii) 4541 deliveries; (iii) 28 909 fully immunised children; and (iv) 81 665 outpatient curative visits. The total input endowment of all health centres consisted of 181 technical staff and 87 subordinate staff. Table 4 presents the means and standard deviations for input and output variables of 17 health centres.
Table 4 Means and standard deviations for public health centres inputs and outputs
Variables Mean Standard deviation
Outputs: Maternal and child health care visits 3985 2579
Deliveries 267 207
Fully-immunized children 1701 1526
OPD curative visits 4804 5475
Inputs: Medical assistants/nurses/other technical staff 11 5
Subordinate staff 5 3
The VRS technical and scale efficiency scores for individual health centres are given in Table 5. Out of the 17 health centres, 14 (82%) were technically efficient since they had a relative technical efficiency (TE) score of 100%. The remaining 3 (18%) had a TE score of less than 100% and thus were deemed to be technically inefficient. The TE score among the latter facilities varied from about 80% at Daboase and 42% at Tikobo to 27% at the Abomoso health centre. This means that Daboase, Tikobo and Abomoso could potentially produce 20%, 58% and 73% more outputs respectively using their current input endowment if they were to operate efficiently.
Table 5 Technical and scale efficiency scores for public health centres
DMU (Health centres) Technical efficiency (%) Scale efficiency (%)
Diare 100 100
Nafong 100 100
Anyingse 100 100
Anyinam 100 100
Akroso 100 100
Elubo 100 100
Adisadel 100 100
Nkwanyawum 100 100
Adukrom 100 100
Osino 100 98.3
Fanti Nyankomasia 100 90.1
Okrakwadwo 100 88.7
Ewim 100 69.5
Savelugu 100 67.6
Daboase 79.7 58.5
Tikobo 42.0 96.6
Abomoso 26.6 99.9
On the other hand, 9 (53%) of the health centres were scale efficient because they had a relative scale efficiency (SE) score of 100%. The remaining 8 health centres (47%) had a SE of less than 100%, and as such they were scale inefficient. The average SE among the inefficient health centres was 84% (standard deviation = 16%). This implies that, on average, the scale inefficient health centres could produce their current output levels with 17% less capacity than they were actually using.
All the 9 scale-efficient health centres exhibited constant returns to scale (CRS). Except for Abomoso, all the other 7 scale-inefficient health centres manifested decreasing returns to scale (DRS).
Discussion
Hospitals analysis
Key Findings
Forty-seven per cent of the hospitals in the sample were technically inefficient and 59% of them were scale inefficient. A similar study among 55 public hospitals in Kwazulu-Natal province in South Africa found 40% of the hospitals to be technically inefficient and 42% to be scale inefficient [20,22]. Another DEA analysis of 54 public hospitals in Kenya revealed that 26% of them were technically inefficient while about 30% were scale inefficient [23]. Masiye et al. [25] undertook DEA among 20 hospitals in Zambia and found 75% of them to be technically inefficient. Thus, the available evidence indicates that although there is significant technical inefficiency among health facilities in Ghana, Kenya, South Africa and Zambia, the magnitude of inefficiency does vary.
Table 6 shows the total output increases and/or input reductions needed to make inefficient district public hospitals efficient. The inefficient hospitals could be technically efficient if they were to increase their output levels by 25% more MCH visits, 12% more deliveries and 1% more discharges, while holding their current input endowment constant. Alternatively, the inefficient hospitals could become technically efficient if they were to reduce their current number of medical officers/dentists by 44%, technical staff by 22%, and subordinate staff by 28% and beds by 29% while holding the output constant.
Table 6 Total output increases and/or input reductions needed to make inefficient district public hospitals efficient
Variables Radial movement (A) Slack movement (B) Total Value (C = A+B)
Outputs: Maternal and child Health care visits 0 50845 50845
Deliveries 0 2865 2865
Inpatient discharges 0 808 808
Inputs: Doctors/dentists 13 11 24
Technical staff (including nurses) 292 0 292
Subordinate staff 173 27 200
Beds 397 51 448
Policy implications for hospitals
In regard to the excess resources, which were wasted and not utilized in the production of hospital outputs, decision-makers in the Ghana Ministry of Health have a number of policy options available to them. These are as follows:
a) Do nothing and continue with the wasteful situation as it exists. However, judging from the strategic and policy documents produced by the Ministry of Health, it is clear that this option is considered unacceptable by its policy-makers.
b) Option related to excess medical officers/dentists and other technical staff: In our opinion, given the need for strengthening health services at sub-district and community levels, it would not be rational to offer any category of technical staff the option of early retirement. Instead, excess medical officers/dentists and other technical staff should be transferred to health centres to provide primary health care. We believe that this would increase health coverage and quality of service provided at sub-district and community levels.
c) Options related to excess subordinate staff include: (i) Offering employees early retirement with severance pay plans (i.e. voluntary retirement); (ii) forced retrenchment with severance package; and (iii) transfer of excess labour force to under-staffed primary health care facilities.
d) Options related to excess beds and space include: (i) Convert the space excess beds occupy to provide outpatients secondary prevention services; (ii) rent excess beds and space to private medical practitioners if there is a demand for them; and (iii) sell excess beds and the space they occupy and use the money thus realised to improve the quality of hospital care.
e) Closure of some hospitals: Holding equity and political concerns constant, in principle, health policy-makers could opt to close down those hospitals with efficiency score below a certain threshold. However, in reality, members of parliament representing the concerned constituencies might be opposed to such an option due to potential political fallout.
f) Conversion of specific hospitals into health centres: This option would entail downsizing both the services provided and staff composition and their numbers. If this option were to be pursued, there would be need for working out details of the conversion process.
Implementation of any of the aforementioned options will need to be preceded by more detailed studies into the determinants of inefficiencies.
Health centres analysis
Key findings
In the Ghana pilot study reported in the paper, 18% of the health centres were technically inefficient and 47% were scale inefficient. A DEA study of 155 primary health care clinics in Kwazulu-Natal province in South Africa found 70% of them to be technically inefficient while 84% manifested some scale inefficiency [21]. A similar study of 32 public health centres in Kenya revealed that 56% of them were technically inefficient while 41% were scale inefficient [24].
Table 7 presents the total output increases and/or input reductions needed to make inefficient public health centres efficient. In order to become efficient, health centres will need to expand their: (i) maternal and child health visits by 9%; (ii) deliveries by 11%; (iii) fully-immunised children by 9%; and (iv) outpatient curative visits by 6%. If the excess inputs in district hospitals were to be transferred to primary health care facilities, health centres would potentially be able to increase their outputs by even larger magnitudes than those indicated above.
Table 7 Total output increases and/or input reductions needed to make inefficient public health centres efficient
Variable Radial movement (A) Slack movement (B) Total movement (C = A+B)
Outputs: Maternal and child health care visits 5306 1100 6406
Deliveries 507 0 507
Fully-immunized children 2645 74 2719
OPD curative visits 3451 1564 5015
Inputs: Medical assistants/nurses/other technical staff 0 7 7
Subordinate staff 0 1 1
Policy implications for health centres
Health centres provide affordable promotive, preventive and basic curative care in localities inhabited mainly by the poor. Their location makes them critically important in the ongoing efforts to scale up pro-poor cost-effective public health interventions geared at achieving the health-related Millennium Development Goals [34] and New Partnership for Africa's Development (NEPAD) health targets [35]. Thus, the importance of these close-to-client health facilities in all efforts to reduce the burden of disease and improve health conditions, especially in rural areas, cannot be overemphasised.
Health promotion strategies and methods may be crucial in inducing the necessary demand for services mentioned above in order to reduce technical inefficiencies in health centres [36]. Health promotion uses approaches/methods such as advocacy (including lobbying), health education, communication for behavioural change, social marketing, social mobilisation, information, education and communication (IEC), legislation and economic and environmental policies to reduce health risks involving health as well as non-health sectors (e.g. agriculture, education, housing, sanitation, trade, transport, water) [37,38].
Given their strategic position amongst communities and closeness to actual and potential clients, health centres make a vital contribution to the development, implementation, monitoring and evaluation of health-promoting initiatives. For example, through the combined use of the aforementioned health promotion strategies and approaches, health centre personnel (with some additional basic health-promotion training) can proactively motivate and persuade households in order to:
• support pregnant women to seek antenatal care, to give birth under the care of skilled birth attendants and seek postnatal care;
• get their children immunised against vaccine-preventable diseases;
• take their children to outpatient departments for integrated management of childhood illnesses (IMCI).
Apart from health promotion, once the Ghana National Health Insurance (GNHI) programme is fully implemented up to the community level, demand for health services is bound to increase due to reduction in financial barriers. The GNHI consists of Social Health Insurance Schemes (District Mutual Health Insurance Schemes and Private Mutual Health Insurance Schemes) and Private Commercial Health Insurance Schemes [39,40].
Limitations of the study
It could be argued that the objective function of health facilities is to maximise health gains from available resources. And the ideal output indicator would be the one that captures in a sensitive, valid, reliable and culturally acceptable manner changes in both the quantity and quality of the lives of those who interact with hospitals and health centres [20]. However, given the unavailability of data on either Disability-Adjusted Life Expectancy (DALE) [41] or Quality-Adjusted Life Years (QALY) [42,43] gained due to care in each of the facilities in the data set, we opted to use proxies that had been used in similar studies in the past [20-24].
It may be argued that there may be variation in the quality of care provided by different health facilities, e.g. facilities offering higher quality of care may require more personnel time and other inputs than those offering low quality of care. Given the fact that all the hospitals studied were district-level public hospitals, designed and resourced to provide a fairly similar level and mix of care, it is unlikely that there would be any significant variance in the quality of care across these facilities. The health centres studied were also fairly homogeneous in size and mix of services provided [2].
The analysis assumed that the case mix of a specific hospital and its Efficiency Reference Set (ERS) hospitals was similar. We were not able to verify whether that assumption was plausible. However, the fact that the study hospitals were all non-specialist first referral-level hospitals, the above assumption will most likely hold.
Drugs were largely supplied from the Central Medical Stores. However, some health facilities often used their cost-sharing funds to make supplementary acquisitions as and when needed. Unfortunately, data on drug expenditure were not forthcoming in most of the questionnaires; thus, it was decided to drop this variable from the analysis altogether.
Lastly, since the sample for health centres constituted only 3.7% of the total number of public health centres and hospitals formed about 22% of the public district hospitals, the results cannot be generalized to the entire population of health centres and hospitals in Ghana.
Suggestions for further research
In the light of the challenges of health financing, equity and efficiency (both allocative and technical) confronting the public health sector, there is an urgent need for:
• Conducting technical and allocative efficiency studies in all the public, private-for-profit and religious mission hospitals and health centres with a view to identifying inefficiencies in individual health facilities and their inputs;
• Conducting Malmquist Productivity Index analysis to monitor and evaluate the effects of different health care reforms on productivity growth, technical progress and efficiency change in health facilities over time [44].
Conclusion
Various governments in Africa have embarked on health sector reforms to improve the performance of their national health systems. Monitoring and evaluating the effects of those reforms on productivity growth, technical progress and efficiency change of fixed health facilities that consume the majority of the recurrent and development budgets of ministries of health is of paramount importance. Our study tried to contribute to establishing baseline technical and scale efficiency information that could be used in monitoring the efficiency effects of future policy changes. We have also briefly described how health promotion strategies and methods could be used to reduce inefficiencies in health centres.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DO, SD and MOG collected the data and participated in analysis and drafting of sections of the document. OM, LHK and DG participated in drafting sections of the manuscript. JMK did literature review and participated in the development of the conceptual framework, data analysis and writing of sections of the document. All the authors read and approved the final manuscript.
Acknowledgements
We are grateful to the Ministry of Health, Ghana, and the Ghana Health Services for authorising and facilitating the study. We are indebted to the two anonymous peer reviewers for their constructive comments and suggestions that helped to improve the quality of our manuscript. A.S. Kochar provided commendable editorial support. We owe profound gratitude to Sabbaoth El Mekodeshkum for multifaceted support, without which the manuscript would never have been completed.
Any mistakes remaining in the paper are those of the authors and should not be attributed to any of the persons acknowledged.
The article contains the views of the authors only and does not represent the opinions, decisions or stated policies of the Ghana Ministry of Health, the Ghana Health Services, the Kenyatta University or the World Health Organization.
==== Refs
Republic of Ghana The Ghana Vision 2020 Accra 1999
Republic of Ghana, Ministry of Health Medium-term health strategy towards Vision 2020 Accra 1999
Republic of Ghana, Ministry of Health Health sector 5-year programme of work 1997–2001 Accra 1999
Republic of Ghana, Ministry of Health Health sector 5-year programme of work 2002–2006 Accra 2001
Republic of Ghana, Ministry of Health Ghana Poverty Reduction Strategy 2002–2004: An agenda for growth and prosperity Accra 2002
Republic of Ghana, Ministryof Health Institutional Reform in the Health Sector Accra 1999
World Health Organization Regional Office for Africa WHO Country Cooperation Strategy: Ghana Brazzaville 2002
Chattopadhy S Ray CS Technical, scale, and size efficiency in nursing home care: a nonparametric analysis of Connecticut homes Health Economics 1996 5 363 373 8880173 10.1002/(SICI)1099-1050(199607)5:4<363::AID-HEC217>3.3.CO;2-T
Shroff HFE Gulledge TR Haynes KE Oneill MK Siting efficiency of long-term health care facilities Socio-Economic Planning Science 1998 32 25 43 10.1016/S0038-0121(97)00016-5
White KR Fache RN Ozcan YA Church ownership and hospital efficiency Hospital and Health Services Administration 1996 41 297 310
Hollingsworth B Parkin D The efficiency of the delivery of neonatal care in the UK Journal of Public Health Medicine 2001 23 47 50 11315693 10.1093/pubmed/23.1.47
Jacobs R Alternative methods to examine hospital efficiency: data envelopment analysis and stochastic frontier analysis Health Care Management Science 2001 4 103 115 11393739 10.1023/A:1011453526849
Ersoy K Kavuncubasi S Ozcan YA Harris JM II Technical efficiencies of Turkish hospitals: DEA approach Journal of Medical Systems 1997 21 67 74 9297615 10.1023/A:1022801222540
Zavras AI Tsakos G Economou C Kyriopoulos J Using DEA to evaluate efficiency and formulate policy within a Greek national primary health care network Journal of Medical Systems 2002 26 285 292 12118812 10.1023/A:1015860318972
Linna M Nordblad A Koivu M Technical and cost-efficiency of oral health care provision in Finnish health centres Social Science and Medicine 2002 56 343 353 12473319
Salinas-Jimenez J Smith P Data envelopment analysis applied to quality in primary health care Annals of Operational Research 1996 67 141 161 10.1007/BF02187027
Giuffrida A Gravelle H Measuring performance in primary care: econometric analysis and DEA Applied Economics 2001 33 163 175 10.1080/00036840150209183
Chang H Determinants of hospital efficiency: the case of central government-owned hospitals in Taiwan Omega International Journal of Management Science 1998 26 307 317 10.1016/S0305-0483(98)00014-0
Wan TTH Hsu N Feng R Ma A Pan S Chou M Technical efficiency of nursing units in a tertiary care hospital in Taiwan Journal of Medical Systems 2002 26 21 27 11778604 10.1023/A:1013086703159
Kirigia JM Lambo E Sambo LG Are public hospitals in Kwazulu-Natal Province of South Africa technically efficient? African Journal of Health Sciences 2000 7 25 32 17650022
Kirigia JM Sambo LG Scheel H Technical efficiency of public clinics in Kwazulu-Natal province of South Africa East African Medical Journal 2001 78 S1 S13 12002061
Zere EA Addison T McIntyre D Hospital efficiency in sub-Saharan Africa: Evidence from South Africa South African Journal of Economics 2000 69 336 358
Kirigia JM Emrouznejad A Sambo LG Measurement of technical efficiency of public hospitals in Kenya: using Data Envelopment Analysis Journal of Medical Systems 2002 26 39 45 11777310 10.1023/A:1013090804067
Kirigia JM Emrouznejad A Sambo LG Munguti N Liambila W Using Data Envelopment Analysis to measure the technical efficiency of public health centres in Kenya Journal of Medical Systems 2004 28 155 166 15195846 10.1023/B:JOMS.0000023298.31972.c9
Masiye F Ndulo M Roos P Odegaard K Seshamani V, Mwikisa CN, Odegaard K A comparative analysis of hospitals in Zambia: a pilot study on efficiency measurement and monitoring Zambia's Health Reforms Selected Papers 1995–2000 2002 7 Lund: Sweden 95 107
World Health Organization The World Health Report 2002 Geneva 2002
United Nations Development Programme Human Development Report 2003: Millennium Development Goals: A compact among nations to end human poverty 2003 New York: Oxford University Press
Charnes A Cooper WW Rhodes E Measuring efficiency of decision-making units European Journal of Operational Research 1978 2–6 429 444 10.1016/0377-2217(78)90138-8
Emrouznejad A Ali Emrouznejad Homepage: 1995–2001 2001 London: Warwick Business School
Commonwealth of Australia Data Envelopment Analysis: a technique for measuring the efficiency of government service delivery Melbourne 1997
Coelli TJ "A Guide to DEAP Version 2.1: A Data Envelopment Analysis (Computer) Program" CEPA Working Paper 96/8 1996 Department of Econometrics, University of New England, Armidale NSW Australia
World Health Organization, Regional Office for Africa Primary Health Care Facility Economics Efficiency Analysis Data Collection Instrument Brazzaville 2000
World Health Organization, Regional Office for Africa Hospitals Economic Efficiency Analysis Data Collection Instrument Brazzaville 2000
United Nations Organization Millennium Development Goals (MDG) New York 2000
New Partnership for Africa's Development (NEPAD) Human Development Programme: NEPAD Health Strategy Pretoria 2001
Ottawa Charter for Health Promotion, First International Conference on Health Promotion, Ottawa, 21 November 1986 – WHO/HPR/HEP/951
Egger G Spark R Lawson J Health promotion strategies and methods 1990 Sydney: McGraw-Hill Book Company
WHO/AFRO Health promotion: a strategy for the African Region Harare 2001
Republic of Ghana, Ministry of Health National health insurance policy framework Accra 2004
International Labour Organization Improving social protection for the poor: health insurance in Ghana Geneva 2005
World Health Organization The World Health Report 2000 – Improving performance of health systems Geneva 2000
Kirigia JM Health impacts of epidemiological environment change: measurement issues Environment and Development Economics 1996 1 359 367
Kirigia JM Cost-utility analysis of schistosomiasis intervention strategies in Kenya Environment and Development Economics 1998 3 319 346 10.1017/S1355770X98000187
Fare R Grosskopf S Norris M Zhang Z Productivity growth, technical progress, and efficiency change in industrialized countries The American Economic Review 1994 84 66 83
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Cost Eff Resour AllocCost effectiveness and resource allocation : C/E1478-7547BioMed Central London 1478-7547-3-91618802110.1186/1478-7547-3-9ResearchTechnical efficiency of public district hospitals and health centres in Ghana: a pilot study Osei Daniel [email protected]'Almeida Selassi [email protected] Melvill O [email protected] Joses M [email protected] Ayayi Omar [email protected] Lenity H [email protected] Planning and Budget Unit, PPME, Ghana Health Service, Accra, Ghana2 WHO Country Office, Accra, Ghana3 World Health Organization, Regional Office for Africa, Brazzaville, Congo4 Department of Health Sciences, School of Public Health, Kenyatta University, Nairobi, Kenya2005 27 9 2005 3 9 9 16 5 2005 27 9 2005 Copyright © 2005 Osei et al; licensee BioMed Central Ltd.2005Osei 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 Government of Ghana has been implementing various health sector reforms (e.g. user fees in public health facilities, decentralization, sector-wide approaches to donor coordination) in a bid to improve efficiency in health care. However, to date, except for the pilot study reported in this paper, no attempt has been made to make an estimate of the efficiency of hospitals and/or health centres in Ghana. The objectives of this study, based on data collected in 2000, were: (i) to estimate the relative technical efficiency (TE) and scale efficiency (SE) of a sample of public hospitals and health centres in Ghana; and (ii) to demonstrate policy implications for health sector policy-makers.
Methods
The Data Envelopment Analysis (DEA) approach was used to estimate the efficiency of 17 district hospitals and 17 health centres. This was an exploratory study.
Results
Eight (47%) hospitals were technically inefficient, with an average TE score of 61% and a standard deviation (STD) of 12%. Ten (59%) hospitals were scale inefficient, manifesting an average SE of 81% (STD = 25%). Out of the 17 health centres, 3 (18%) were technically inefficient, with a mean TE score of 49% (STD = 27%). Eight health centres (47%) were scale inefficient, with an average SE score of 84% (STD = 16%).
Conclusion
This pilot study demonstrated to policy-makers the versatility of DEA in measuring inefficiencies among individual facilities and inputs. There is a need for the Planning and Budgeting Unit of the Ghana Health Services to continually monitor the productivity growth, allocative efficiency and technical efficiency of all its health facilities (hospitals and health centres) in the course of the implementation of health sector reforms.
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Background
The strategic health objectives of Vision 2020 in Ghana envisage: a significant reduction in the rates of infant, child and maternal mortality; effective control of the risk factors that expose individuals to major communicable diseases; increased access to health services, especially in rural areas; establishment of a health system effectively reoriented toward delivery of public health services; and effective and efficient management of the health system [1].
The Ministry of Health, following the thrust of Vision 2020, developed its current policy and strategy guidelines in 1995 in the Medium-Term Health Strategy (MTHS) document [2]. The five main objectives of the MTHS are: improving access to health services; improving quality of care; improving efficiency; fostering partnership between private and public health service-providers; and improving financing of health services.
Subsequently, the first [3] and second [4] Health Sector Five-Year Programme of Work were developed to enable the country attain the MTHS objectives. One of the five underlying objectives of the two programmes of work is "improved efficiency in health services delivery". Furthermore, the Ghana Poverty Reduction Strategy 2002–2004 [5] also highlights "enhancing efficiency in health service delivery" as one of the three priority health sector-related interventions. Thus, efficiency concerns are deeply embedded in the national Vision 2020, national poverty reduction strategy, health policy and programme of work.
In a bid to improve the efficiency of health services delivery, the Ministry of Health is implementing the following health sector reforms: separation of functions between the Ministry of Health (policy formulation, planning, donor coordination and resource mobilization) and the Ghana Health Services (responsible for service delivery); autonomy of tertiary hospitals; decentralized planning and budgeting systems, strengthening of financial management and performance monitoring system, and investing in overall management development capacity within the sector; sector-wide approach (SWAP); and strengthening of existing regulatory bodies and laws [6,7].
Since 1978, Data Envelopment Analysis (DEA) has been extensively used in the Americas [8-10], Western Europe [11-17] and Asia [18,19] to shed light on the efficiency of various aspects of national health systems. In Africa, the application of DEA in the health sector has been quite limited. So far, the approach has been applied to health facilities in only three countries, i.e. South Africa [20-22], Kenya [23,24] and Zambia [25]. Yet, the assessment of the efficiency ought to be more prevalent in low-income countries like Ghana in order to optimise health benefits from the available meagre health sector resources.
In Ghana, prior to the current study, no attempt had been made to estimate the efficiency of health care facilities, using either parametric (econometric) or non-parametric methods. The Planning Unit in the Ministry of Health (with support from the World Health Organization) decided to undertake a limited pilot study to demonstrate to policy-makers the potential usefulness of DEA in the pursuit of health sector efficiency objectives. Once policy-makers were adequately sensitised, hopefully, a national efficiency study would be conducted among all health centres and district, regional and tertiary hospitals.
The objectives of the exploratory study reported in this paper were: (i) to estimate the relative technical efficiency of a sample of public hospitals and health centres in Ghana; and (ii) to demonstrate policy implications for health sector policy-makers.
Methods
Study area
Ghana is situated on the west coast of Africa. It is divided into ten administrative regions (i.e. Upper East, Upper West, Northern, Brong Ahafo, Ashanti, Volta, Eastern, Greater Accra, Central and Western) and 110 districts. The organisation of health services more or less mimics the administrative structure.
The country's health services are organised at the following levels [2]:
a) Community: Delivered through outreach programmes, resident or itinerant herbalists, traditional birth attendants and/or retail drug peddlers.
b) Sub-district: A health centre services a geographical area with 15 000 to 30 000 population. It provides basic curative care, disease prevention services and maternity services (primary health care). A health centre constitutes an essential component of the close-to-client health services.
c) District: A district hospital provides support to sub-districts in disease prevention and control, health promotion and public health education; referral outpatient and inpatient care, training and supervision of health centres; maternity services, especially the management of complications and emergencies and surgical contraception.
d) Regional: A regional hospital provides specialised clinical and diagnostic care; management of high-risk pregnancies and complications of pregnancy; technical and logistical back up for epidemiological surveillance; and research and training.
e) Tertiary: At the apex of the referral system, there are two government-owned teaching hospitals that offer specialised services, undertake research, and provide undergraduate and postgraduate training in health and allied areas.
f) National (i.e. Ministry of Health headquarters): The national level is responsible for the development of national health policy and for providing strategic directions for service delivery as well as coordination and monitoring.
Ghana's population of 19.7 million is served by a total of 2189 health facilities, of which 952 are government owned, 181 are owned by religious organisations, 75 are quasi-government and 980 belong to the private sector. Out of the total number of health facilities, 2 are teaching hospitals, 9 regional hospitals, 91 district hospitals, 124 other hospitals, 558 health centres, 1085 clinics and 320 are maternity homes. All these health facilities are serviced by 1294 doctors, 29 dentists, 207 pharmacists and 326 medical assistants, along with other paramedical and support staff [7].
The country spends a total of US$ 252 million (4.2% of the GDP of US$ 6 billion) annually on health. About 53.5% of this expenditure is incurred by the government and 46.5% by the households through out-of-pocket expenses. The total per capita expenditure on health at an average exchange rate is US$ 11 [26].
The life expectancy at birth in Ghana is 57.4 years. The infant and under-5-years mortality rates per 1000 live births are 57 and 97 respectively [26]. The probability of dying (per 1000 live births) between ages 15 and 59 years is 303. The maternal mortality per 100 000 live births is 214. Nearly 72% of the population has access to improved sanitation, while 73% has access to an improved water supply source [27].
The question is whether the people of Ghana are deriving maximum health care benefits from the aforementioned investments in health sector, especially from hospitals and health centres which consume over 75% of both the recurrent and capital budgets of the Ministry of Health. The next sub-section presents a DEA conceptual framework, which is used to shed light on this issue.
DEA conceptual framework
In the production process, hospitals and health centres turn inputs (factors of production) into outputs (health services). We can divide the inputs into broad categories of labour, materials and capital, each of which will often include more narrow sub-divisions. Labour inputs include skilled health personnel (doctors, nurses, paramedics, support staff) and unskilled workers (drivers, watchmen, gardeners, ward attendants, cooks, etc.), as well as the entrepreneurial efforts of managers of health facilities. Materials include pharmaceuticals, non-pharmaceutical supplies and any other goods that health facilities require to produce health care. Capital includes buildings, medical equipment, vehicles and beds.
The relationship between inputs and the production process and resulting outputs is described in Figure 1. It is clear that hospitals and health centres employ multiple inputs to produce multiple outputs. We used DEA approach since it allows the measurement of relative efficiency when decision-making units (in this case hospitals/health centres) have multiple inputs and multiple outputs.
Figure 1 Relationship between inputs and the production process and resulting outputs.
DEA is a linear programming methodology for evaluating relative efficiency of each production unit among a set of fairly homogeneous decision-making units (DMUs), e.g. district hospitals, health centres, etc. It sketches a production possibilities frontier (data envelop or efficient frontier) using combinations of inputs and outputs from best performing health facilities. Health facilities that compose the "best practice frontier" are assigned an efficiency score of one (or 100%) and are deemed technically efficient compared to their peers. The efficiency of the health facilities below the efficiency frontier is measured in terms of their distance from the frontier. The inefficient health facilities are assigned a score between one and zero. The larger the score the more efficient a health facility is.
Since hospitals and health centres employ multiple inputs to produce multiple outputs, their individual technical efficiency can be defined as [28]:
The technically inefficient health facility uses more weighted inputs per weighted output, or produce less weighted output per weighted input than those health facilities on the "best practice frontier".
Algebraically, technical efficiency score of each hospital and health centre in the sample were obtained by solving the models (1) and (2) (See Table 8) [29].
Table 8 Model 1. DEA weights model, input-oriented, constant returns to scale (CRS) Model 2. DEA weights model, input-oriented, variable returns to scale (VRS)
Where:
yrj = the amount of output r produced by hospital or health centre j,
xij = the amount of input i used by hospital or health centre j,
ur = the weight given to output r, (r = 1,..., t and t is the number of outputs),
vi = the weight given to input i, (i = 1, ..., m and m is the number of inputs),
n = the number of hospitals or health centres,
j0 = the hospital or health centre under assessment.
We need to explain what we mean by constant returns to scale and variable returns to scale. Returns to scale refers to the changes in output as all inputs change by the same proportion. For instance, suppose that for a specific hospital (or health centre) j we start from an initial level of inputs (doctors = D, Other technical staff = T, Subordinate staff = S, Beds = B) and output (Q)
Qo = f(D,T,S,B)
and we increase all the factors by the same proportion φ. We will obviously obtain a new level of output Q*, higher than the original level Q0,
Q* = f(φD,φT,φS,φB)
If Q* increases by: (i) the same proportion φ as the inputs, we say that there are constant returns to scale (CRS); (ii) less than proportionally with the increase in inputs, we have decreasing returns to scale (DRS); (iii) more than proportionally with the increase in the inputs, we increasing returns to scale (IRS).
Those hospitals and health centres manifesting CRS can be said to be operating at their most productive scale sizes. In order to operate at the most productive scale size, a health facility displaying DRS should scale down both outputs and inputs. If a health facility is exhibiting IRS, it should expand both outputs and inputs in order to become scale efficient [8].
We have illustrated below how DEA works using hypothetical hospitals.
Illustration of the DEA analysis
Lets assume that a hypothetical country called Nkrumah has 9 district hospitals. Each hospital produces two outputs (i.e. outpatient department visits (OPVisits) and inpatient admissions (Admissions)) from a single input of technical staff. The number of staff employed, Opvisits, Admissions, ratios of Opvisits to staff, and Admissions to staff are contained in the Table 1.
Table 1 Illustration of DEA analysis using a hypothetical example of nine hospitals
DMUs OPVisits (A) Admissions (B) Staff (C) OPvisits/staff D = (A/C) Admissions/staff E = (B/C)
Anim 7020 5451 102 69 53
Akoa 20566 7610 92 224 83
Addai 25200 7148 143 176 50
Mensa 33568 7958 96 350 83
Kofi 17406 2429 95 183 26
Gyau 10573 8094 117 90 69
Anani 10500 8944 115 91 78
Amoi 20421 10969 117 175 94
Asamoa 20647 8619 46 449 187
The efficiency of each hospital in producing the two outputs were estimated by dividing each of their outputs by their input and seeing which hospital(s) have the highest ratios. The results are contained in the last two columns of Table 1. The higher the ratio of an output to input the more efficient a hospital is in producing that output. In this example, Asamoa hospital had the highest number of Opvisits per staff (449) and Admissions (187) for each member of technical staff employed.
By plotting Opvisits/staff against Admissions/staff for the nine hospitals we derive the production possibilities frontier graph contained in Figure 2.
Figure 2 Production possibilities frontier graph.
The diagram shows the efficiency frontier, which is the fundamental concept of DEA. The straight lines from Asamoa hospital to the Y axis (labelled Admissions/staff) and from Asamoa to the X axis (labelled Opvisits/staff) represent the efficient frontier. The efficiency frontier, derived from the most efficient hospital(s) (i.e. Asamoa hospital in our example) in the dataset/sample, represents a standard of technically best performance that can be achieved from available input and technology endowment. Consequently, it is used as a threshold against which to measure the performance of all other hospitals.
The efficiency frontier 'envelops' the inefficient hospitals within it and clearly shows the relative efficiency of each hospital. A hospital like Asamoa, which located on the frontier, is considered 100% technically efficient. Any hospital like Mensa, Amoi, Akoa, Anani, Kofi, Addai, Gyau and Anim that is below the production possibilities frontier is relatively less efficient and is given a technical efficiency rating of less than 100%.
Anim hospital, for instance, could become efficient if it increased its outputs, in the same proportions, while holding its input constant, i.e. assuming an output-orientated model/situation. Instead, it could become efficient by reducing its input while keeping its outputs the same, i.e. assuming an input-orientated scenario. Its technical efficiency is calculated by the ratio of its distance from the origin over the distance from the origin to the point of intersection on the production possibilities frontier or the efficiency frontier. This gives Anim hospital a technical efficiency score of 28.52%. Likewise, Mensa hospital is 77.90% as efficient as Asamoa hospital (i.e. the best practice hospital), Amoi hospital is 50.04%, Akoa hospital is 49.80%, Anani hospital is 41.51%, Kofi hospital is 40.82%, Addai hospital is 39.26%, and Gyau hospital is 36.92%. These scores were estimated assuming constant returns to scale (CRS).
However, often health services production process are not linear, and thus it may be more appropriate to assume variable returns to scale (VRS). Thus, we estimated the DEA model assuming VRS, the efficiency scores for various hospitals were as follows: Asamoa = 100%, Amoi = 100%, Mensa = 100%, Akoa = 50%, Anani = 48.54%, Kofi = 48.42%, Anim = 45.10%, Addai = 44.49% and Gyau = 39.32%. This finding implies that if Akoa, Anani, Kofi, Anim, Addai and Gyau hospitals were to operate efficiently, they are capable of producing their current output levels with 50%, 51.5%, 51.58%, 54.9%, 55.51% and 60.68% less inputs than they are currently using. Whereas in the CRS model only Asamoa hospital had a 100% efficiency score, in the VRS model three hospitals (Asamoa, Amoi, Mensa) achieved a pure technical efficiency score of 100%.
The impact of hospital scale/size on their technical efficiency was evaluated using a three-step process. First, the model was estimated assuming CRS. Second, the model was run assuming VRS. Third, scale efficiency was obtained by dividing each hospital's CRS technical efficiency score by its VRS technical efficiency score. Akoa and Asamoa hospitals has scale efficiency score of 100%, implying they had an optimal size. Gyau scored 94%, Addai scored 88%, Anani scored 86%, Kofi scored 84%, Mensa scored 78%, Anim scored 63% and Amoi scored 50%. These seven hospitals were scale inefficient since they were not operating at their most productive size for their observed input mix. It is important to mention that DEA is only an exploratory tool for efficiency measurement, and indicates directions for further investigations into how to improve/enhance efficiency.
Input and output orientation
Input Orientation for hospitals
In the hospital analysis the input orientation assumed that these facilities had limited control over the volume of their outputs. There was no linkage between staff earnings and output; thus, there was no incentive for inducing demand for health services. Otherwise, in Ghana hospital management has got greater control over the use of inputs. Thus, an input-oriented DEA model was used for hospital analysis.
Output orientation for health centres
On the other hand, output orientation was assumed for health centres. The management of health centres has no control over inputs, especially it's staffing. However, given their primary health care orientation, with a strong bias towards health promotion and disease prevention, they can influence a great number of people seeking, for example, antenatal and postnatal care, family planning services, birthing services, immunisations and health education, through their public health outreach work among communities. Thus, the output-oriented DEA model was used for the health centre analysis.
Strengths and weaknesses of DEA
Strengths of DEA
We chose to employ DEA approach to estimate technical efficiency of individual hospitals and health centres because of its unique strengthens: (i) it can handle multiple input and multiple output models/scenarios typical of hospitals and health centres; (ii) it does not require an assumption of a functional form relating inputs to output (as regression methods do); (iii) health facilities are directly compared against a peer or combination of peers; (iv) inputs and outputs can be very different units; (v) it does not require information on prices of inputs and outputs [22,30].
Weaknesses of DEA
Even though we chose to use DEA, we were fully aware that it has two main limitations. Firstly, it attributes any deviation from the "best practice frontier" to inefficiency, while some could be due to statistical noise, e.g. epidemics or measurement errors. Secondly, given that DEA is deterministic/nonparametric technique, it is difficult to conduct statistical tests of hypotheses concerning the inefficiency and the structure of the production function [22,31,32].
Variables
The hospital DEA model had a total of 7 variables, including 3 outputs and 4 inputs. The three outputs for each individual hospital were: (i) the number of maternal and child care (MCH) (i.e. antenatal care, postnatal care, family planning, tetanus toxoid, child immunisation and growth monitoring); (ii) the number of child deliveries; and (iii) the number of patients discharged (not including deaths). The four inputs included: (i) number of medical officers; (ii) the number of technical officers (including medical assistants, nurses and paramedical staff); (iii) the support or subordinate staff (including orderlies, ward assistants, cleaners, drivers, gardeners, watchmen, etc.); and (iv) the number of hospital beds.
On the other hand, the health centre DEA model was estimated with a total of 6 variables: 4 outputs and 2 inputs. The four outputs for each individual health centre were: (i) the number of child deliveries; (ii) the number of fully immunised children under the age of 5 years; (iii) the number of other maternal (i.e. antenatal care, postnatal care and family planning services) and childcare (nutritional/child growth monitoring) visits; and (iv) the number of outpatient curative visits. The two inputs were: (i) the number of technical staff (this included medical assistants, nurses and paramedical staff); and (ii) the number of support or subordinate staff (including cleaners, drivers, gardeners, watchmen and others).
The choice of inputs and outputs for the DEA analysis was guided in part by the previous DEA health care studies in the African Region and availability of data.
Data
The data used in this study are for 2000. In order to have a feel of the usefulness of DEA in the measurement technical efficiency of hospitals and health centres, the policy-makers instructed the Planning and Budget Unit (PBU) to draw a pilot sample of 21 hospitals and 21 health centres. PBU decided to use simple random sampling technique to draw the two samples. Data were collected from a random sample of 21 public health centres using a WHO African Regional Office (WHO/AFRO) efficiency questionnaire for primary health care facilities [32]. However, information on health personnel in four health centres was missing; thus, they were left out of the analysis. Data were also collected from a random sample of 21 district hospitals; however, information on inputs and outputs from only 17 hospitals was included in the analysis. Data on hospitals were collected using a WHO/AFRO efficiency questionnaire for hospitals [33]. The data were analysed using the DEAP software developed by Professor Tim Coelli [31].
Results
Hospitals analysis
In 2000, all 17 hospitals in the sample produced a total of 200 589 maternal and child health (MCH) visits, 24 152 deliveries and 69 361 discharges. Those outputs were produced employing a total of 55 medical officers/dentists, 1345 technical staff, 721 subordinate staff and 1543 beds. Table 2 presents the means and standard deviations for input and output variables of the 17 district hospitals.
Table 2 Means and standard deviations for public hospitals inputs and outputs
Variable Mean Standard deviation
Outputs: Maternal and child health care visits 11799 9516
Deliveries 1421 1186
Inpatient discharges 4080 2274
Inputs: Doctors/dentists 3 3
Technical staff (including nurses) 79 34
Subordinate staff 42 27
Beds 91 43
The VRS model technical and scale efficiency scores for individual hospitals are contained in Table 3. Of the 17 hospitals, 9 (53%) were technically efficient since they had a relative technical efficiency (TE) score of 100%. The remaining 8 (47%) had a TE score of less than 100%, which means that they were technically inefficient. The TE score among the latter facilities ranged from 74% in Atua hospital to 43% in Winneba hospital. This finding implies that Atua and Winneba hospitals could potentially reduce their current input endowments by 26% and 57% while leaving their output levels unchanged. The average TE score among the inefficient hospitals was 61% (standard deviation = 12%), which means that these hospitals could, on average, produce their current levels of output with 39% less inputs than they were currently using.
Table 3 Technical and scale efficiency scores for district hospitals
DMU (Hospitals) Technical efficiency (%) Scale efficiency (%)
Swendru 100 100
Half Asin 100 100
Turkwa 100 100
Kwesiminstmu 100 100
Yendi 100 100
Takoradi 100 100
St Francis Xavier 100 100
West End 100 91.6
Walewale 100 43.9
Atua 74.4 99.8
Tetteh Quarshie 73.6 99.9
Cape Coast 68.6 93.2
Akuse 66.3 43.9
Axim 57.4 50.9
Suhum 56.5 98.0
Akim 46.0 99.6
Winneba 42.7 93.9
Seven (41%) of the hospitals had a scale efficiency (SE) of 100%, which means that they had the most productive size for that particular input-output mix. The remaining 10 (59%) hospitals had a SE of less than 100% and as such they were scale inefficient. The average SE among the inefficient hospitals was 81% (standard deviation = 25%), meaning that, on average, the scale inefficient hospitals could reduce their size by 19% without affecting their current output levels.
All the seven scale-efficient hospitals displayed constant returns to scale (CRS), implying thereby that they were operating at their most productive scale sizes. Eight of the 10 scale-inefficient hospitals had increasing returns to scale (IRS) while one of the hospitals revealed decreasing returns to scale (DRS). In order to operate at the most productive scale size (MPSS), a hospital exhibiting DRS should scale down both its outputs and inputs. Similarly, if a hospital is displaying IRS, it should expand both its outputs and inputs.
Health centre analysis
The total output of all health centres in the sample combined was as follows: (i) 67 739 MCH visits; (ii) 4541 deliveries; (iii) 28 909 fully immunised children; and (iv) 81 665 outpatient curative visits. The total input endowment of all health centres consisted of 181 technical staff and 87 subordinate staff. Table 4 presents the means and standard deviations for input and output variables of 17 health centres.
Table 4 Means and standard deviations for public health centres inputs and outputs
Variables Mean Standard deviation
Outputs: Maternal and child health care visits 3985 2579
Deliveries 267 207
Fully-immunized children 1701 1526
OPD curative visits 4804 5475
Inputs: Medical assistants/nurses/other technical staff 11 5
Subordinate staff 5 3
The VRS technical and scale efficiency scores for individual health centres are given in Table 5. Out of the 17 health centres, 14 (82%) were technically efficient since they had a relative technical efficiency (TE) score of 100%. The remaining 3 (18%) had a TE score of less than 100% and thus were deemed to be technically inefficient. The TE score among the latter facilities varied from about 80% at Daboase and 42% at Tikobo to 27% at the Abomoso health centre. This means that Daboase, Tikobo and Abomoso could potentially produce 20%, 58% and 73% more outputs respectively using their current input endowment if they were to operate efficiently.
Table 5 Technical and scale efficiency scores for public health centres
DMU (Health centres) Technical efficiency (%) Scale efficiency (%)
Diare 100 100
Nafong 100 100
Anyingse 100 100
Anyinam 100 100
Akroso 100 100
Elubo 100 100
Adisadel 100 100
Nkwanyawum 100 100
Adukrom 100 100
Osino 100 98.3
Fanti Nyankomasia 100 90.1
Okrakwadwo 100 88.7
Ewim 100 69.5
Savelugu 100 67.6
Daboase 79.7 58.5
Tikobo 42.0 96.6
Abomoso 26.6 99.9
On the other hand, 9 (53%) of the health centres were scale efficient because they had a relative scale efficiency (SE) score of 100%. The remaining 8 health centres (47%) had a SE of less than 100%, and as such they were scale inefficient. The average SE among the inefficient health centres was 84% (standard deviation = 16%). This implies that, on average, the scale inefficient health centres could produce their current output levels with 17% less capacity than they were actually using.
All the 9 scale-efficient health centres exhibited constant returns to scale (CRS). Except for Abomoso, all the other 7 scale-inefficient health centres manifested decreasing returns to scale (DRS).
Discussion
Hospitals analysis
Key Findings
Forty-seven per cent of the hospitals in the sample were technically inefficient and 59% of them were scale inefficient. A similar study among 55 public hospitals in Kwazulu-Natal province in South Africa found 40% of the hospitals to be technically inefficient and 42% to be scale inefficient [20,22]. Another DEA analysis of 54 public hospitals in Kenya revealed that 26% of them were technically inefficient while about 30% were scale inefficient [23]. Masiye et al. [25] undertook DEA among 20 hospitals in Zambia and found 75% of them to be technically inefficient. Thus, the available evidence indicates that although there is significant technical inefficiency among health facilities in Ghana, Kenya, South Africa and Zambia, the magnitude of inefficiency does vary.
Table 6 shows the total output increases and/or input reductions needed to make inefficient district public hospitals efficient. The inefficient hospitals could be technically efficient if they were to increase their output levels by 25% more MCH visits, 12% more deliveries and 1% more discharges, while holding their current input endowment constant. Alternatively, the inefficient hospitals could become technically efficient if they were to reduce their current number of medical officers/dentists by 44%, technical staff by 22%, and subordinate staff by 28% and beds by 29% while holding the output constant.
Table 6 Total output increases and/or input reductions needed to make inefficient district public hospitals efficient
Variables Radial movement (A) Slack movement (B) Total Value (C = A+B)
Outputs: Maternal and child Health care visits 0 50845 50845
Deliveries 0 2865 2865
Inpatient discharges 0 808 808
Inputs: Doctors/dentists 13 11 24
Technical staff (including nurses) 292 0 292
Subordinate staff 173 27 200
Beds 397 51 448
Policy implications for hospitals
In regard to the excess resources, which were wasted and not utilized in the production of hospital outputs, decision-makers in the Ghana Ministry of Health have a number of policy options available to them. These are as follows:
a) Do nothing and continue with the wasteful situation as it exists. However, judging from the strategic and policy documents produced by the Ministry of Health, it is clear that this option is considered unacceptable by its policy-makers.
b) Option related to excess medical officers/dentists and other technical staff: In our opinion, given the need for strengthening health services at sub-district and community levels, it would not be rational to offer any category of technical staff the option of early retirement. Instead, excess medical officers/dentists and other technical staff should be transferred to health centres to provide primary health care. We believe that this would increase health coverage and quality of service provided at sub-district and community levels.
c) Options related to excess subordinate staff include: (i) Offering employees early retirement with severance pay plans (i.e. voluntary retirement); (ii) forced retrenchment with severance package; and (iii) transfer of excess labour force to under-staffed primary health care facilities.
d) Options related to excess beds and space include: (i) Convert the space excess beds occupy to provide outpatients secondary prevention services; (ii) rent excess beds and space to private medical practitioners if there is a demand for them; and (iii) sell excess beds and the space they occupy and use the money thus realised to improve the quality of hospital care.
e) Closure of some hospitals: Holding equity and political concerns constant, in principle, health policy-makers could opt to close down those hospitals with efficiency score below a certain threshold. However, in reality, members of parliament representing the concerned constituencies might be opposed to such an option due to potential political fallout.
f) Conversion of specific hospitals into health centres: This option would entail downsizing both the services provided and staff composition and their numbers. If this option were to be pursued, there would be need for working out details of the conversion process.
Implementation of any of the aforementioned options will need to be preceded by more detailed studies into the determinants of inefficiencies.
Health centres analysis
Key findings
In the Ghana pilot study reported in the paper, 18% of the health centres were technically inefficient and 47% were scale inefficient. A DEA study of 155 primary health care clinics in Kwazulu-Natal province in South Africa found 70% of them to be technically inefficient while 84% manifested some scale inefficiency [21]. A similar study of 32 public health centres in Kenya revealed that 56% of them were technically inefficient while 41% were scale inefficient [24].
Table 7 presents the total output increases and/or input reductions needed to make inefficient public health centres efficient. In order to become efficient, health centres will need to expand their: (i) maternal and child health visits by 9%; (ii) deliveries by 11%; (iii) fully-immunised children by 9%; and (iv) outpatient curative visits by 6%. If the excess inputs in district hospitals were to be transferred to primary health care facilities, health centres would potentially be able to increase their outputs by even larger magnitudes than those indicated above.
Table 7 Total output increases and/or input reductions needed to make inefficient public health centres efficient
Variable Radial movement (A) Slack movement (B) Total movement (C = A+B)
Outputs: Maternal and child health care visits 5306 1100 6406
Deliveries 507 0 507
Fully-immunized children 2645 74 2719
OPD curative visits 3451 1564 5015
Inputs: Medical assistants/nurses/other technical staff 0 7 7
Subordinate staff 0 1 1
Policy implications for health centres
Health centres provide affordable promotive, preventive and basic curative care in localities inhabited mainly by the poor. Their location makes them critically important in the ongoing efforts to scale up pro-poor cost-effective public health interventions geared at achieving the health-related Millennium Development Goals [34] and New Partnership for Africa's Development (NEPAD) health targets [35]. Thus, the importance of these close-to-client health facilities in all efforts to reduce the burden of disease and improve health conditions, especially in rural areas, cannot be overemphasised.
Health promotion strategies and methods may be crucial in inducing the necessary demand for services mentioned above in order to reduce technical inefficiencies in health centres [36]. Health promotion uses approaches/methods such as advocacy (including lobbying), health education, communication for behavioural change, social marketing, social mobilisation, information, education and communication (IEC), legislation and economic and environmental policies to reduce health risks involving health as well as non-health sectors (e.g. agriculture, education, housing, sanitation, trade, transport, water) [37,38].
Given their strategic position amongst communities and closeness to actual and potential clients, health centres make a vital contribution to the development, implementation, monitoring and evaluation of health-promoting initiatives. For example, through the combined use of the aforementioned health promotion strategies and approaches, health centre personnel (with some additional basic health-promotion training) can proactively motivate and persuade households in order to:
• support pregnant women to seek antenatal care, to give birth under the care of skilled birth attendants and seek postnatal care;
• get their children immunised against vaccine-preventable diseases;
• take their children to outpatient departments for integrated management of childhood illnesses (IMCI).
Apart from health promotion, once the Ghana National Health Insurance (GNHI) programme is fully implemented up to the community level, demand for health services is bound to increase due to reduction in financial barriers. The GNHI consists of Social Health Insurance Schemes (District Mutual Health Insurance Schemes and Private Mutual Health Insurance Schemes) and Private Commercial Health Insurance Schemes [39,40].
Limitations of the study
It could be argued that the objective function of health facilities is to maximise health gains from available resources. And the ideal output indicator would be the one that captures in a sensitive, valid, reliable and culturally acceptable manner changes in both the quantity and quality of the lives of those who interact with hospitals and health centres [20]. However, given the unavailability of data on either Disability-Adjusted Life Expectancy (DALE) [41] or Quality-Adjusted Life Years (QALY) [42,43] gained due to care in each of the facilities in the data set, we opted to use proxies that had been used in similar studies in the past [20-24].
It may be argued that there may be variation in the quality of care provided by different health facilities, e.g. facilities offering higher quality of care may require more personnel time and other inputs than those offering low quality of care. Given the fact that all the hospitals studied were district-level public hospitals, designed and resourced to provide a fairly similar level and mix of care, it is unlikely that there would be any significant variance in the quality of care across these facilities. The health centres studied were also fairly homogeneous in size and mix of services provided [2].
The analysis assumed that the case mix of a specific hospital and its Efficiency Reference Set (ERS) hospitals was similar. We were not able to verify whether that assumption was plausible. However, the fact that the study hospitals were all non-specialist first referral-level hospitals, the above assumption will most likely hold.
Drugs were largely supplied from the Central Medical Stores. However, some health facilities often used their cost-sharing funds to make supplementary acquisitions as and when needed. Unfortunately, data on drug expenditure were not forthcoming in most of the questionnaires; thus, it was decided to drop this variable from the analysis altogether.
Lastly, since the sample for health centres constituted only 3.7% of the total number of public health centres and hospitals formed about 22% of the public district hospitals, the results cannot be generalized to the entire population of health centres and hospitals in Ghana.
Suggestions for further research
In the light of the challenges of health financing, equity and efficiency (both allocative and technical) confronting the public health sector, there is an urgent need for:
• Conducting technical and allocative efficiency studies in all the public, private-for-profit and religious mission hospitals and health centres with a view to identifying inefficiencies in individual health facilities and their inputs;
• Conducting Malmquist Productivity Index analysis to monitor and evaluate the effects of different health care reforms on productivity growth, technical progress and efficiency change in health facilities over time [44].
Conclusion
Various governments in Africa have embarked on health sector reforms to improve the performance of their national health systems. Monitoring and evaluating the effects of those reforms on productivity growth, technical progress and efficiency change of fixed health facilities that consume the majority of the recurrent and development budgets of ministries of health is of paramount importance. Our study tried to contribute to establishing baseline technical and scale efficiency information that could be used in monitoring the efficiency effects of future policy changes. We have also briefly described how health promotion strategies and methods could be used to reduce inefficiencies in health centres.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DO, SD and MOG collected the data and participated in analysis and drafting of sections of the document. OM, LHK and DG participated in drafting sections of the manuscript. JMK did literature review and participated in the development of the conceptual framework, data analysis and writing of sections of the document. All the authors read and approved the final manuscript.
Acknowledgements
We are grateful to the Ministry of Health, Ghana, and the Ghana Health Services for authorising and facilitating the study. We are indebted to the two anonymous peer reviewers for their constructive comments and suggestions that helped to improve the quality of our manuscript. A.S. Kochar provided commendable editorial support. We owe profound gratitude to Sabbaoth El Mekodeshkum for multifaceted support, without which the manuscript would never have been completed.
Any mistakes remaining in the paper are those of the authors and should not be attributed to any of the persons acknowledged.
The article contains the views of the authors only and does not represent the opinions, decisions or stated policies of the Ghana Ministry of Health, the Ghana Health Services, the Kenyatta University or the World Health Organization.
==== Refs
Republic of Ghana The Ghana Vision 2020 Accra 1999
Republic of Ghana, Ministry of Health Medium-term health strategy towards Vision 2020 Accra 1999
Republic of Ghana, Ministry of Health Health sector 5-year programme of work 1997–2001 Accra 1999
Republic of Ghana, Ministry of Health Health sector 5-year programme of work 2002–2006 Accra 2001
Republic of Ghana, Ministry of Health Ghana Poverty Reduction Strategy 2002–2004: An agenda for growth and prosperity Accra 2002
Republic of Ghana, Ministryof Health Institutional Reform in the Health Sector Accra 1999
World Health Organization Regional Office for Africa WHO Country Cooperation Strategy: Ghana Brazzaville 2002
Chattopadhy S Ray CS Technical, scale, and size efficiency in nursing home care: a nonparametric analysis of Connecticut homes Health Economics 1996 5 363 373 8880173 10.1002/(SICI)1099-1050(199607)5:4<363::AID-HEC217>3.3.CO;2-T
Shroff HFE Gulledge TR Haynes KE Oneill MK Siting efficiency of long-term health care facilities Socio-Economic Planning Science 1998 32 25 43 10.1016/S0038-0121(97)00016-5
White KR Fache RN Ozcan YA Church ownership and hospital efficiency Hospital and Health Services Administration 1996 41 297 310
Hollingsworth B Parkin D The efficiency of the delivery of neonatal care in the UK Journal of Public Health Medicine 2001 23 47 50 11315693 10.1093/pubmed/23.1.47
Jacobs R Alternative methods to examine hospital efficiency: data envelopment analysis and stochastic frontier analysis Health Care Management Science 2001 4 103 115 11393739 10.1023/A:1011453526849
Ersoy K Kavuncubasi S Ozcan YA Harris JM II Technical efficiencies of Turkish hospitals: DEA approach Journal of Medical Systems 1997 21 67 74 9297615 10.1023/A:1022801222540
Zavras AI Tsakos G Economou C Kyriopoulos J Using DEA to evaluate efficiency and formulate policy within a Greek national primary health care network Journal of Medical Systems 2002 26 285 292 12118812 10.1023/A:1015860318972
Linna M Nordblad A Koivu M Technical and cost-efficiency of oral health care provision in Finnish health centres Social Science and Medicine 2002 56 343 353 12473319
Salinas-Jimenez J Smith P Data envelopment analysis applied to quality in primary health care Annals of Operational Research 1996 67 141 161 10.1007/BF02187027
Giuffrida A Gravelle H Measuring performance in primary care: econometric analysis and DEA Applied Economics 2001 33 163 175 10.1080/00036840150209183
Chang H Determinants of hospital efficiency: the case of central government-owned hospitals in Taiwan Omega International Journal of Management Science 1998 26 307 317 10.1016/S0305-0483(98)00014-0
Wan TTH Hsu N Feng R Ma A Pan S Chou M Technical efficiency of nursing units in a tertiary care hospital in Taiwan Journal of Medical Systems 2002 26 21 27 11778604 10.1023/A:1013086703159
Kirigia JM Lambo E Sambo LG Are public hospitals in Kwazulu-Natal Province of South Africa technically efficient? African Journal of Health Sciences 2000 7 25 32 17650022
Kirigia JM Sambo LG Scheel H Technical efficiency of public clinics in Kwazulu-Natal province of South Africa East African Medical Journal 2001 78 S1 S13 12002061
Zere EA Addison T McIntyre D Hospital efficiency in sub-Saharan Africa: Evidence from South Africa South African Journal of Economics 2000 69 336 358
Kirigia JM Emrouznejad A Sambo LG Measurement of technical efficiency of public hospitals in Kenya: using Data Envelopment Analysis Journal of Medical Systems 2002 26 39 45 11777310 10.1023/A:1013090804067
Kirigia JM Emrouznejad A Sambo LG Munguti N Liambila W Using Data Envelopment Analysis to measure the technical efficiency of public health centres in Kenya Journal of Medical Systems 2004 28 155 166 15195846 10.1023/B:JOMS.0000023298.31972.c9
Masiye F Ndulo M Roos P Odegaard K Seshamani V, Mwikisa CN, Odegaard K A comparative analysis of hospitals in Zambia: a pilot study on efficiency measurement and monitoring Zambia's Health Reforms Selected Papers 1995–2000 2002 7 Lund: Sweden 95 107
World Health Organization The World Health Report 2002 Geneva 2002
United Nations Development Programme Human Development Report 2003: Millennium Development Goals: A compact among nations to end human poverty 2003 New York: Oxford University Press
Charnes A Cooper WW Rhodes E Measuring efficiency of decision-making units European Journal of Operational Research 1978 2–6 429 444 10.1016/0377-2217(78)90138-8
Emrouznejad A Ali Emrouznejad Homepage: 1995–2001 2001 London: Warwick Business School
Commonwealth of Australia Data Envelopment Analysis: a technique for measuring the efficiency of government service delivery Melbourne 1997
Coelli TJ "A Guide to DEAP Version 2.1: A Data Envelopment Analysis (Computer) Program" CEPA Working Paper 96/8 1996 Department of Econometrics, University of New England, Armidale NSW Australia
World Health Organization, Regional Office for Africa Primary Health Care Facility Economics Efficiency Analysis Data Collection Instrument Brazzaville 2000
World Health Organization, Regional Office for Africa Hospitals Economic Efficiency Analysis Data Collection Instrument Brazzaville 2000
United Nations Organization Millennium Development Goals (MDG) New York 2000
New Partnership for Africa's Development (NEPAD) Human Development Programme: NEPAD Health Strategy Pretoria 2001
Ottawa Charter for Health Promotion, First International Conference on Health Promotion, Ottawa, 21 November 1986 – WHO/HPR/HEP/951
Egger G Spark R Lawson J Health promotion strategies and methods 1990 Sydney: McGraw-Hill Book Company
WHO/AFRO Health promotion: a strategy for the African Region Harare 2001
Republic of Ghana, Ministry of Health National health insurance policy framework Accra 2004
International Labour Organization Improving social protection for the poor: health insurance in Ghana Geneva 2005
World Health Organization The World Health Report 2000 – Improving performance of health systems Geneva 2000
Kirigia JM Health impacts of epidemiological environment change: measurement issues Environment and Development Economics 1996 1 359 367
Kirigia JM Cost-utility analysis of schistosomiasis intervention strategies in Kenya Environment and Development Economics 1998 3 319 346 10.1017/S1355770X98000187
Fare R Grosskopf S Norris M Zhang Z Productivity growth, technical progress, and efficiency change in industrialized countries The American Economic Review 1994 84 66 83
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Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-3-501612902710.1186/1477-7525-3-50ResearchHealth related quality of life in sickle cell patients: The PiSCES project McClish Donna K [email protected] Lynne T [email protected] Viktor E [email protected] John D [email protected] Imoigele P [email protected] James L [email protected] Susan D [email protected] Wally R [email protected] Department of Biostatistics, Virginia Commonwealth University, Richmond, VA, USA2 Division of Quality Health Care, Department of Medicine, Virginia Commonwealth University, Richmond, VA, USA3 Department of Health Evaluation Sciences, University of Virginia, Charlottesville, VA, USA4 Division of Hematology/Oncology, Department of Medicine, Virginia Commonwealth University, Richmond, VA, USA5 Department of Emergency Medicine, Virginia Commonwealth University, Richmond, VA, USA6 Department of Psychiatry, Virginia Commonwealth University, Richmond, VA, USA7 Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA2005 29 8 2005 3 50 50 14 4 2005 29 8 2005 Copyright © 2005 McClish et al; licensee BioMed Central Ltd.2005McClish 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
Sickle cell disease (SCD) is a chronic disease associated with high degrees of morbidity and increased mortality. Health-related quality of life (HRQOL) among adults with sickle cell disease has not been widely reported.
Methods
We administered the Medical Outcomes Study 36-item Short-Form to 308 patients in the Pain in Sickle Cell Epidemiology Study (PiSCES) to assess HRQOL. Scales included physical function, physical and emotional role function, bodily pain, vitality, social function, mental health, and general health. We compared scores with national norms using t-tests, and with three chronic disease cohorts: asthma, cystic fibrosis and hemodialysis patients using analysis of variance and Dunnett's test for comparison with a control. We also assessed whether SCD specific variables (genotype, pain, crisis and utilization) were independently predictive of SF-36 subscales, controlling for socio-demographic variables using regression.
Results
Patients with SCD scored significantly worse than national norms on all subscales except mental health. Patients with SCD had lower HRQOL than cystic fibrosis patients except for mental health. Scores were similar for physical function, role function and mental health as compared to asthma patients, but worse for bodily pain, vitality, social function and general health subscales. Compared to dialysis patients, sickle cell disease patients scored similarly on physical role and emotional role function, social functioning and mental health, worse on bodily pain, general health and vitality and better on physical functioning. Surprisingly, genotype did not influence HRQOL except for vitality. However, scores significantly decreased as pain levels increased.
Conclusion
SCD patients experience health related quality of life worse than the general population, and in general, their scores were most similar to patients undergoing hemodialysis. Practitioners should regard their HRQOL as severely compromised. Interventions in SCD should consider improvements in health related quality of life as important outcomes.
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Background
Functional status and health-related quality of life (HRQOL) may be impaired in sickle cell disease (SCD) due to morbid events, such as stroke, or other organ system failures. The Cooperative Study of Sickle Cell Disease (CSSD) found that morbid events such as strokes that impaired function often preceded death in childhood [1-3] Until recent decades, SCD was associated with chronic childhood pain, organ failure and death in very early adulthood. Treatment advances have now transformed SCD into a chronic disease suffered by children and adults. Frequently, patients surviving until adulthood experience significant organ system damage that may include stroke, pulmonary failure and pulmonary hypertension, renal failure, congestive heart failure, leg ulcers, and osteonecrosis of the femoral or humeral heads [2].
Children and adolescents with SCD report poor HRQOL in qualitative studies using focus groups [4], and fare worse in their HRQOL compared to controls on health surveys [5] or on assessments of general physical, motor and independent daily functioning [6,7]. Despite the considerable evidence in children for reduced HRQOL in SCD, few studies have evaluated the impact of this disease on health related quality of life in adults [8-11].
The impact of this disease on HRQOL for adults may be even greater than for children. Quality of life is deteriorated by episodic, debilitating pain associated with substantial analgesic use, frequent hospitalization for pain episodes, and ultimately organ failure. Although SCD related pain can often be managed by analgesics and opioids, adults with SCD may be under-treated because clinicians suspect drug dependence in this population. This provider bias may lead to reluctance by patients to seek medical attention [12,13]. Further, HRQOL may be over-estimated by providers that do not regularly care for patients with SCD due to lack of understanding of the severity of the painful crises and the potential impact on function. Therefore, assessing the quality of life among a US adult SCD population and providing comparisons with HRQOL reported among comparable adults with other chronic diseases may be an important method for providing health care practitioners who care for these patients a more objective perspective on the impact and severity of this disease. In addition, quality of life in SCD is important to describe because it amplifies the ability to identify patients for whom potentially dangerous but potent interventions such as hydroxyurea or bone marrow transplantation is justified at an early stage.
Methods
Study description
The Pain in Sickle Cell Epidemology Study (PiSCES) is a longitudinal cohort study of over 300 adult patients with SCD designed to understand the relationship between pain and response to pain. The emphasis is on potentially mutable etiologic, and non-biologic variables. The PiSCES methods have been described in detail elsewhere [14]. Briefly, we enrolled 308 adult patients with SCD from July 2002 through August 2004. Baseline information, laboratory data and daily pain diary data were collected. Baseline data was collected at the time of enrollment using a self-administered questionnaire which included questions on demographics, health related quality of life, and other information including medical history and medication use. In addition to the survey information, blood was obtained for genotyping and urine specimens were collected to assess renal function.
As part of the PiSCES study, patients filled out daily diaries for up to 6 months [14]. The diary was modeled after the one used in the Multicenter Study of Hydroxyurea [15]. Among other things, the diary asked patients to report about the previous 24 hours: the worst sickle cell pain intensity, on a scale from 0 (none) to 9 (unbearable), whether or not they were in a sickle cell crisis, and whether they had gone for an unscheduled physician visit, ED visit or were hospitalized due to sickle cell pain.
Patient population
Patients were solicited for enrollment from across Virginia, but focused on the Richmond and Tidewater areas of Virginia. Patients aged 16 years and older were eligible for enrollment Patients identified as potentially eligible for the study were invited and scheduled for an enrollment visit, at which time informed consent was obtained. The study, along with recruitment methods, was approved by the VCU IRB.
Interested patients were then screened using the Mini Mental Status Examination [16] to assure competency (excluded if score less than 27) and the ability to provide informed consent. Patents were compensated ten dollars for the initial visit, when blood and urine specimens are obtained, and the baseline survey was completed.
To assess the relative HRQOL in SCD patients, comparison groups from published reports representing three different cohorts of patients with chronic diseases including asthma [17], cystic fibrosis [18] and hemodialysis [19] patients were included. These comparison groups were selected to be similar in age and gender to the PiSCES cohort. The asthma sample consisted of 301 patients whose mean age was 38 and 56% of whom were female. The hemodialysis sample consisted of 1000 prevalent cases with a mean age of 58 and of whom 50% were female. Data regarding cystic fibrosis came from 223 adolescents and adult subjects participating in a validation of the quality of life instrument with a mean age of 25, and 54% female.
Analytic variables
The Medical Outcome Study 36 item Short Form (SF-36) [20] is a generic measure of health related to functional status and well being. The survey is not disease- or age-specific and has been validated across a wide variety of age, race and disease groups, including many chronic diseases [20-22]. The SF-36 has high test-retest reliability, has been shown to predict a number of poor outcomes [23], has been compared with biological markers for their sensitivity to change in severity of chronic illness [24], and has been used as outcomes in clinical trials of chronic illness [25].
The SF-36 is multidimensional with subscales representing eight of the most important dimensions of HRQOL: physical function, physical role functions, emotional role functioning, bodily pain, vitality, general health, mental health and social function. Subscales are measured on a scale from 0 – 100 (with 0 being the worst and 100 the best score). Values are available for specific age and gender population subgroups for the US and other populations. In addition to the chronic disease samples used for comparison, the normal values for age matched males and females from the general US population are provided and compared with our study population.
Sickle cell genotype (Sβ+ Thalasemia, Sßo Thalasemia, SC and SS) is a known predictor of mortality and disease severity. The CSSCD evaluated the natural history of 3578 patients ranging in age from newborns to age 66. Hospital utilization due to pain varied according to genotype (SS = 0.8 episodes/pt/yr., Sßo Thalasemia = 1.0 episodes/pt/yr., SC and Sß+ Thalasemia = 0.4 episodes/pt/yr) [1]. Genotype was also a predictor of the age at death [2]. Further, among patients over the age of 20, the hospital utilization rate due to pain was correlated with mortality over the years [2]. For this study, genotype was obtained either directly from the blood specimen obtained from the patient at enrollment or from the patient's medical record. Since there were few patients with Sβ+ Thalasemia and Sßo Thalasemia, for purposes of analyses, two groups were defined. The more severe genotype grouping included SS and Sßo Thalasemia, the less severe group included SC and Sβ+ Thalasemia,
We used three calculated variables from the diary for this study. Mean daily pain was calculated as the sum of the pain intensity for all diary days, divided by the total number of days the diary was completed. The percentage of days for which a crisis was marked on the diary was calculated as 100*the number of days with crisis marked, divided by the total number of days the diary was completed. Percentage of days on which there was utilization was constructed similarly, with utilization consisting of either an unscheduled clinic visit, an ED visit or an overnight hospitalization. Since these latter two variable was very skewed, with many having no crisis or utilization, for analysis the diary variables were divided into 3 categories of roughly equal size (coded 1, 2, 3): Percent of days with self-reported crises: 0, 0.1–10, 10+; Percent of days with utilization: 0, 0.1–3, 3+.
Statistical methods
Means and standard deviations are presented. Comparison values were created from MOS national norms data by using a weighted average of age-gender specific values, with the weights equal to the proportion of the PiSCES sample in that age group. Subscales for the PiSCES cohort were compared to national norms with a t-test. Analysis of variance was used as an overall test for equality of each subscale across chronic disease cohorts. When the overall F test was significant, Dunnett's test was used to compare each of the chronic diseases to the mean for the PiSCES cohort. To determine whether SCD-specific variables were independently predictive of HRQOL, we used multiple linear regression, controlling for socio-demographic variables (age, gender, education). SCD-specific variables included genotype, mean pain, and percent days of diary days reporting crisis and utilization. These analyses were limited to patients who had returned at least 30 diaries. This reduced the sample from 308 to 226. Three additional subjects were excluded from regression analyses because they lacked information on genotype. Analysis used SAS 8.2 for UNIX.
Results
Table 1 describes the PiSCES cohort. The mean age was 33, and ranged from 16 to 64. There were more women than men in the study (60.4% vs 39.6), 48.6% attended college. Only 21.8% of subjects were currently married.
Table 1 Demographic description of PiSCES cohort
Variable Frequency (percent)
Gender
Male 122 (39.6)
Female 186 (60.4)
Education
<High school 41 (13.4)
High School grad 116 (37.9)
Some college 110 (35.9)
College Grad 39 (12.7)
Age group
16–24 79 (25.6)
25–34 92 (29.9)
35–44 82 (26.9)
45–54 42 (13.6)
55–64 12 (3.9)
Marital Status
Married 67 (21.8)
Never married 198 (64.5)
Divorced/separated/widowed 42 (13.7)
Genotype
SS 206 (66.9)
Sßo Thalasemia 8 (2.6)
SC 75 (4.3)
Sβ+ Thalasemia 10 (3.2)
Unknown 9 (2.9)
Table 2 has means and standard deviations for the 8 SF-36 subscales for the PiSCES cohort, separately for men and women, along with the age-adjusted national norms by gender. When the gender stratified PiSCES cohort was compared with national norms, values were significantly lower for all subscales (P < 0.0001) with one exception – the mental health scale was not significantly different from the national norm (men: p = 0.670, women: p = 0.102).
Table 2 SF-36 – PiSCES cohort vs National Norms (Mean ± standard deviation)
Male Female
PiSCES Norm PiSCES Norm
Physical Function 66.4 ± 24.1 92.3 ± 15.5 59.9 ± 25.1 87.4 ± 19.7
Role-Physical 40.1 ± 38.7 90.5 ± 24.0 38.6 ± 39.9 83.8 ± 31.5
Bodily Pain 50.8 ± 28.6 79.6 ± 21.1 45.2 ± 26.0 77.3 ± 22.1
General Health 42.7 ± 22.3 77.1 ± 17.3 37.0 ± 21.7 73.9 ± 19.0
Vitality 50.4 ± 22.5 64.9 ± 19.2 37.6 ± 21.0 59.2 ± 20.6
Social Function 62.3 ± 27.6 86.6 ± 19.9 62.4 ± 24.8 82.8 ± 22.1
Role-emotional 62.7 ± 43.1 85.0 ± 29.4 54.7 ± 42.8 80.7 ± 33.1
Mental Health 75.3 ± 20.7 76.4 ± 16.8 69.2 ± 20.0 72.8 ± 18.3
P < 0.0001 comparing PiSCES to SF-36 for all subscales, except MH, female: p = 0.102 and male: p = 0.670;
Table 3 has means and standard deviations of the 8 SF-36 subscales for the PiSCES cohort along with cohorts of patients with three other chronic diseases – asthma, cystic fibrosis and hemodialysis. There were significant differences (P < 0.0001) amongst cohorts for all subscales except mental health (p = 0.0582), which was marginal. Patients with SCD (PiSCES cohort) reported significantly worse HRQOL on all subscales (p < 0.05) except mental health as compared to adolescents and adults with cystic fibrosis. They had similar reported quality of life as asthma patients regarding physical function and role function (both physical and emotional), and mental health, but scored lower for the bodily pain, vitality, social function and general health subscales. When compared to patients on hemodialysis, SCD patients reported similar low scores for physical and emotional role function and social function. They also did not differ on the mental health subscale (p > 0.15). SCD patients had lower scores for the pain, vitality and general health subscales (P < 0.01), but reported a higher score for the physical function subscale compared with the hemodialysis cohort.
Table 3 SF-36: Comparison of PiSCES sample with other chronic disease cohorts (mean ± standard deviation)
PiSCES Hemo-Dialysis Cystic Fibrosis Asthma ANOVA F value†
N = 308 N = 1000 N = 223 N = 241
Physical Function 62.4 ± 24.9 44.3 ± 27.8* 76.3 ± 24.0* 63.2 ± 21.4 121.3
Role-Physical 39.2 ± 39.4 39.7 ± 40.4 72.9 ± 38.4* 38.7 ± 39.9 74.7
Bodily Pain 47.4 ± 27.2 60.4 ± 29.1* 82.2 ± 21.3* 67.2 ± 23.2* 49.5
General Health 39.2 ± 22.1 50.0 ± 22.4* 43.4 ± 23.7* 57.9 ± 19.0* 37.9
Vitality 42.7 ± 22.5 46.5 ± 22.3* 58.4 ± 23.1* 48.2 ± 20.8* 23.3
Social Function 63.5 ± 25.2 66.0 ± 29.9 80.4 ± 23.8* 72.1 ± 22.2* 21.3
Role-emotional 57.8 ± 43.1 58.2 ± 42.7 77.0 ± 36.9* 63.3 ± 41.5 13.1
Mental Health 71.6 ± 20.4 69.7 ± 21.6 73.7 ± 18.1 70.7 ± 18.4 2.56
†Numerator degrees of freedom are 3, denominator degrees of freedom are N-4;
*p < 0.0001 compared to PiSCES cohort
Multiple regressions were performed to look at the relationship of SCD specific variables (genotype, mean pain, percent of diary days subjects reported crisis and percent of diary days subjects reported utilization) and SF-36 subscales. Socio-demographic variables (age, gender, number of years of education) were included in the models as covariates. Results are in Table 4. For each subscale, mean SCD pain was highly predictive (p < 0.0001 for all subscales except p = 0.0396 for mental health). The more SCD pain a subject experienced, the worse the reported quality of life. One unit increase in pain (on a 0–9 scale) was associated with an approximate decrease of 1.4 (mental health) to 6 (both role functions) units on an SF-36 subscale. Percentage of days with crisis was an independent predictor of bodily pain (p = 0.0109), with an approximate 6 point decrease in bodily pain score for each increase in crisis category. Genotype was also an independent predictor of vitality (p = 0.0161), with SS/Sßo Thalasemia being associated with better vitality. No other variables were independent predictors of SF-36 subscales.
Table 4 Results of regression of SCD-specific variables on SF-36 subscales, controlling for socio-demographic variables1 (regression coefficients ± standard error)
Genotype2 Mean pain Proportion Days with Crisis3 Proportion Days with Utilization4
Physical Function -5.01 ± 3.30 -4.55 ± 0.72** -0.42 ± 2.12 3.51 ± 1.97
Role-Physical -1.62 ± 5.73 -6.12 ± 1.27** -0.91 ± 3.70 2.75 ± 3.39
Bodily Pain 0.14 ± 3.57 -4.41 ± 0.79** -5.93 ± 2.31* -0.59 ± 2.13
General Health 3.52 ± 3.19 -3.34 ± 0.71** -1.31 ± 2.09 -1.44 ± 1.92
Vitality 6.43 ± 3.27* -3.54 ± 0.72** 0.55 ± 2.10 3.06 ± 1.94
Social Function 3.16 ± 3.77 -4.27 ± 0.82** -3.28 ± 2.44 1.93 ± 2.25
Role-emotional 7.06 ± 6.27 -5.81 ± 1.39** 2.30 ± 4.09 2.12 ± 3.71
Mental Health 4.23 ± 3.14 -1.44 ± 0.69* -1.53 ± 2.02 0.35 ± 1.87
1controlling for age, gender, years of education
2SS and Sßo Thalasemia vs SC and Sß+ Thalasemia
3Percent of days with crises: 0, 0.1–10,10+ (coded 1,2,3)
4Percent of days with utilization: 0, 0.1–3, 3+ (coded 1,2,3)
*p ≤ 0.05; **p < 0.0001
Discussion
In general, SCD patients experience a poor health related quality of life. Except for mental health, the SF-36 subscale values were considerably lower than norms of the general US population. They reported a HRQOL that was equal to or poorer than patients with other significant chronic conditions in many domains. Similar to patients with SCD, until somewhat recently, patients with cystic fibrosis rarely lived until adulthood, marking this as a disease with significant sequelae and high mortality. It is interesting, then, to see that quality of life of adult survivors of this chronic disease, even though impaired, was comparable to national norms, and was generally far superior than that reported by adults with SCD. Of the three chronic conditions selected for comparison, the PiSCES cohort had HRQOL patterns most similar to that of patients undergoing chronic hemodialysis.
That SCD patients did not report poorer mental health and well being than the general US population is consistent with findings for many medical conditions. In 1978 Brickman, et al [26] presented a famous result showing that lottery winners are not much happier than paraplegics. Since that time many studies have confirmed similar results, that while people who are sick may report their health as being worse than the general population, they appear to have similar well-being. Not only was it true of the asthma, dialysis and cystic fibrosis cohorts presented here, but similar results have been shown for people with other chronic diseases both in the US and other countries [21,22,27].
It has been suggested that the fact that many people with chronic diseases report good psychological well being could be a result of increased social support, lack of other stressors, or a "response shift" associated with the managing their chronic disease [27]. The "response shift" could be a result of a scale recalibration, a change in the patient's values, or a reconceptualization of their mental health and well-being [28,29] in order to accommodate their illness. Riis et al [30] dispute the idea of scale recalibration, proposing instead that people have adapted to their illness or situation.
Heady and Wearing [31] propose that there is a baseline level of mood or well-being that people have to which they return after events cause them to move from that baseline. This is supported by a twin study indicating that most variation in well-being is due to variations in genetics, not life circumstances [32]. That would suggest that, while perhaps people with SCD may temporarily report poorer well-being associated with high levels of pain or other disease sequalae, most often they would report a baseline well-being similar to that of others.
Despite the similar level of well-being in SCD patients compared with both norms and patients with other chronic diseases, patients with SCD experience significant decrements in other important aspects of HRQOL. This is supported in a study of adult SCD patients in the UK, where Anie et al, found their population also had much lower HRQOL scores than general UK population norms. Further, the patients in this study had reported HRQOL similar to patients with arthritis due to hereditary haemochromatisis, another chronic disease [9]. Patients in the PiSCES cohort reported somewhat lower general health and higher mental health scores than Anie et al found. This may result in part from differences in the two cohorts, including their relative access to health care in the two settings.
Surprisingly, HRQOL was not associated with genotype except for the vitality subscale. However there was a strong association with reduced HRQOL and pain levels, and, for a few subscales, there was a trend with increasing levels of utilization. The relationship between genotype severity and HRQOL may have been mediated by these variables, particularly pain. Anie et al [9] also found a relationship between pain and some subscales of the SF-36 (physical and social functions, mental and general health) but found no significant association with utilization measures. Whether the more severe manifestations of the disease cause the poorer quality of life, or patients who report poorer quality of life suffer more and use more health care cannot be determined from this study.
It is unclear why the direction of the statistically significant association between vitality for SS/Sßo Thalasemia vs SC/Sß+ Thalasemia is opposite of what would be expected, with the more severe genotype being associated with better vitality. Even the nonsignificant relationships between genotype and the other HRQOL subscales in these regressions were in the same direction, except for physical functioning. Similarly, increased utilization tended to be associated, albeit nonstatistically with better HRQOL scores for most subscales These counter-intuitive findings should be explored by other SCD researchers.
There are several limitations of this study. First, this study enrolled patients from only one state, so may not be representative of the entire US SCD population. When comparing to populations with chronic disease, the gender and age distributions were not ideally matched. In particular, the PiSCES cohort, at a mean age of 33, is significantly younger than the dialysis comparison group However, since SF-36 subscale scores tend to decrease with age, the fact that these younger patients with SCD had worse HRQOL scores on some subscales than an older hemodialysis cohort is even more alarming
Conclusion
Practitioners caring for adult SCD patients should regard their quality of life as severely compromised, with scores that are most similar to hemodialysis patients in our comparison of other chronic diseases. Although reducing mortality is of paramount importance among SCD patients, future interventions should consider improving health related quality of life as a clinical endpoint.
Authors' contributions
DKM participated in the design and coordination of the study, performed the statistical analysis, interpreted results and drafted the manuscript. IPA, SDR, JDR participated in the coordination of the study, and helped to edit the manuscript. WRS, VEB, JLL, LTP conceived of the study, participated in its design and coordination, and helped to edit the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by a grant from NHLBI: 1R01HL64122-01A1
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Platt OS Thorington BD Brambilla DJ Milner PF Rosse WF Vichinsky E Kinney TR Pain in sickle cell disease: Rates and risk factors N Engl J Med 1991 325 11 16 1710777
Platt OS Brambilla DJ Milner PF Rosse WF Milner PF Castro O Steinberg MH Klug PP Mortality in sickle cell disease: Life expectancy and risk factors for early death N Engl J Med 1994 330 1639 1644 7993409 10.1056/NEJM199406093302303
Bonds DR Three decades of innovation in the management of sickle cell disease: the road to understanding the sickle cell disease clinical phenotype Blood Rev 2005 19 99 110 15603913 10.1016/j.blre.2004.04.002
Thomas VJ Taylor LM The psychosocial experience of people with sickle cell disease and its impact on quality of life: Qualitative findings from focus groups Br J Health Psychol 2002 7 345 363 12614505 10.1348/135910702760213724
Fuggle P Shand PA Gill LJ Davies SC Pain, quality of life, and coping in sickle cell disease Arch Dis Child 1996 75 199 203 8976657
Kater AP Heijboer H Peters M Vogels T Prins MH Heymans HS Quality of life in children with sickle cell disease in Amsterdam area Ned Tijdschr Geneeskd 1999 143 2049 2053 10560546
Stegenga KA Ward-Smith P Hinds PS Routhieaux JA Woods GM Quality of life among children with sickle cell disease receiving chronic transfusion therapy J Pediatr Oncol Nurs 2004 21 207 213 15490865 10.1177/1043454204265841
Strickland OL Jackson G Gilead M McGuire DB Quarles S Use of focus groups for pain and quality of life assessment in adults with sickle cell disease J Natl Black Nurses Assoc 2001 12 36 43 11902019
Anie KA Steptoe A Bevan D Sickle cell disease: Pain, coping and quality of life in a study of adults in the UK Br J Health Psychol 2002 7 331 344 12614504 10.1348/135910702760213715
Thomas VJ Taylor LM The psychosocial experience of people with sickle cell disease and its impact on quality of life: Qualitative findings from focus groups Br J Health Psychol 2002 7 345 363 12614505 10.1348/135910702760213724
Ramsey LT Woods KF Callahan LA Mensay GA Barbeau P Gutin B Quality of life improvement for patients with sickle cell disease Am J Hematol 2001 66 155 156 11421301 10.1002/1096-8652(200102)66:2<155::AID-AJH1038>3.0.CO;2-D
Maxwell K Streetly A Bevan D Experience of hospital care and treatment seeking for pain from sickle cell disease: qualitative study BMJ 1999 318 1585 1590 10364116
Elander J Lusher J Bevan D Telfer P Pain management and symptoms of substance dependence among patients with sickle cell disease Soc Sci Med 2003 57 1683 1696 12948577 10.1016/S0277-9536(02)00553-1
Smith WR Bovbjerg VE Penberthy LT McClish DK Levenson JL Roberts JD Roseff SD Aisidu IP Understanding pain and improving management of sickle cell disease: the PiSCES Study J Natl Med Assoc 2005 97 183 192 15712781
Carache S Terrin ML Moore RD Dover GJ McMahon RP Barton FB Waclawiw M Eckert SV the Investigators of the Multicenter Study of Hydroyurea Design of the multicenter study of hydroxyrea in sickle cell anemia Controlled Clinical Trials 1995 16 432 446 8925656 10.1016/S0197-2456(95)00098-4
Folstein MF Folstein SE McHugh PR "Mini-mental state". A practical guide for grading the cognitive state of patients for the clinician J Psychiatr Res 1975 12 189 98 1202204 10.1016/0022-3956(75)90026-6
Lee TA Hollingworth W Sullivan Comparison of directly elicited preferences to preferences derived from the SF-36 in adults with asthma Med Decis Making 2003 23 323 334 12926582 10.1177/0272989X03256009
Gee L Abbott J Conway SP Etherington C Webb AK Validation of the SF-36 for the assessment of quality of life in adolescents and adults with cystic fibrosis J Cyst Fibros 2002 1 137 145 15463820 10.1016/S1569-1993(02)00079-6
DeOreo PB Hemodialysis patient-assessed functional health status predicts continued survival, hospitalization and dialysis attendance compliance Am J Kidney Dis 1997 30 204 212 9261030
Ware JE Snow KK Kosinski M Gandek B SF-36 Health Survey Manual & Interpretation Guide 1993 Boston MA. Health Institute
Alonso J Ferrer M Gandek B Ware JE JrAaronson NK Mosconi P Rasmussen NK Bullinger M Fukuhara S Kaasa S Leplege A the IQOLA Project Group Health-related quality of life associated with chronic conditions in eight countries: results from the International Quality of Life Assessment (IQOLA) Project Qual Life Res 2004 13 283 298 15085901 10.1023/B:QURE.0000018472.46236.05
Rijken M van Kerkhof M Dekker J Schellevis FG Comorbidity of chronic diseases: effects of disease pairs on physical and mental functioning Qual Life Res 2005 14 45 55 15789940 10.1007/s11136-004-0616-2
Wachtel T Piette J Mor V Stein M Fleishman J Carpenter C Quality of life in persons with human immunodeficiency virus infection: measurement by the Medical Outcomes Study instrument Ann Intern Med 1992 116 129 137 1727616
Ware JE JrKemp JP Buchner DA Singer AE Nolop KB Goss TF The responsiveness of disease-specific and generic health measures to changes in the severity of asthma among adults Qual Life Res 1998 7 235 244 9584554
Kosinski M Keller SD Hatoum HT Kong SX Ware JE Jr The SF-36 Health Survey as a generic outcome measure in clinical trials of patients with osteoarthritis and rheumatoid arthritis: tests of data quality, scaling assumptions and score reliability Med Care 1999 37 MS10 22 10335740 10.1097/00005650-199905001-00002
Brickman P Coates D Janoff-Bulman R Lottery winners and accident victims: Is happiness relative? J Pers Soc Psychol 1978 36 917 927 690806 10.1037//0022-3514.36.8.917
Schlenk EA Erlen JA Dunbar-Jacob J McDowell J Engberg S Serinka SM Rohay JM Bernier MH Health related quality of life in chronic disorders: a comparison across studies using the SF-36 Qual Life Res 1998 7 57 65 9481151 10.1023/A:1008836922089
Sprangers MAG Schwarts CE Integrating response shift into health-related quality of life research: a theoretical model Soc Sci Med 1999 48 1507 1515 10400253 10.1016/S0277-9536(99)00045-3
Rapkin BD Schwartz CR Towards a theoretical model of quality-of-life appraisal: Implications of findings from studies of response shift HQLO 2004 2 14 15023229 10.1186/1477-7525-2-14
Riis J Loewenstien G Baron J Fepson C Fagerlin A Ubel PA Ignorance of hedonic adaptation for hemodialyis: A study using ecological momentary assessment J Exp Psychol: Gen 2005 134 3 9 15702959 10.1037/0096-3445.134.1.3
Heady B Wearing A Understanding happiness: A theory of subjective well being 1992 Melboune, Vicotira Australia: Longman Cheshire 1360101
Lykken D Tellegen A Happiness is a stochastic phenomenon Psychological Science 1996 7 186 189
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Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-3-531614454610.1186/1477-7525-3-53ResearchEngagement of patients in religious and spiritual practices: Confirmatory results with the SpREUK-P 1.1 questionnaire as a tool of quality of life research Büssing Arndt [email protected] Peter F [email protected] Thomas [email protected] Department of Medical Theory and Complementary Medicine, University Witten/Herdecke, Gerhard-Kienle-Weg 4, 58313 Herdecke, Germany2 Krebsforschung Herdecke, Department of Applied Immunology, Heinrichstraße 67, 44805 Bochum, Germany2005 6 9 2005 3 53 53 20 7 2005 6 9 2005 Copyright © 2005 Büssing et al; licensee BioMed Central Ltd.2005Büssing et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Quality of life is a multidimensional construct composed of functional, physical, emotional, social and spiritual well-being. In order to examine how patients with severe diseases view the impact of spirituality and religiosity on their health and how they cope with illness, we have developed the SpREUK questionnaire. We deliberately avoided the intermingling of attitudes, convictions and practices, and thus addressed the distinct forms and frequencies of spiritual/religious practices in an additional manual, the SpREUK-P questionnaire.
Methods
The SpREUK-P was designed to differentiate spiritual, religious, existentialistic and philosophical practices. It was tested in a sample of 354 German subjects (71% women; 49.0 ± 12.5 years). Half of them were healthy controls, while among the patients cancer was diagnosed in 54%, multiple sclerosis in 22%, and other chronic diseases in 23%. Reliability and factor analysis of the inventory were performed according to the standard procedures.
Results
We confirmed the structure and consistency of the previously described 18-item SpREUK-P manual and improved the quality of the current construct by adding several new items. The new 25-item SpREUK-P 1.1 (Cronbach's alpha = 0.8517) has the following scales: (1) conventional religious practice (CRP), (2) existentialistic practice (ExP), (3) unconventional spiritual practice (USP), (4) nature/environment-oriented practice (NoP), and (5) humanistic practice (HuP). Among the tested individuals, the highest engagement scores were found for HuP and NoP, while the lowest were found for the USP. Women had significantly higher scores for ExP than male patients. With respect to age, the engagement in CRP increases with increasing age, while the engagement in a HuP decreased. Individuals with a Christian orientation and with a religious and spiritual attitude had the highest engagement scores for CRP, while the engagement in an USP was high with respect to a spiritual attitude. Variance analyses confirmed that the SpR attitude and religious affiliation are the main relevant covariates for CRP and ExP, while for the USP the SpR attitude and the educational level are of significance, but not religious affiliation. Patients with multiple sclerosis overall had the lowest engagement scores for all five forms of SpR practice, while it is remarkable that cancer patients had lower scores for HuP and USP than healthy subjects.
Conclusion
The current re-evaluation of the SpREUK-P questionnaire (Version 1.1) indicates that it is a reliable, valid measure of five distinct forms of spiritual, religious and philosophical practice that may be especially useful for assessing the role of spirituality and religiosity in health related research. An advantage of our instruments is the clear-cut differentiation between convictions and attitudes on the one hand, and the expression of these attitudes in a concrete engagement on the other hand.
QuestionnairesReligion and MedicineSpirituality and Religionreligious practicescopingchronic disease, cancermultiple sclerosis
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Background
Quality of life is a multidimensional construct composed of functional, physical, emotional, social and (newly introduced) spiritual well-being [1,2]. In breast cancer patients, Levine and Targ [3] found significant correlations of spirituality and spiritual well-being with functional well-being, while items pertaining to meaning and peace tended to correlate significantly with physical well-being. However, "spiritual well-being" itself is also a multidimensional construct. Murray et al. [4] described the signs of spiritual well-being as "inner peace and harmony; having hope, goals and ambitions; social life and place in community retained; feeling of uniqueness and individuality; dignity; feeling valued; coping with and sharing emotions; ability to communicate with truth and honesty; being able to practice religion; finding meaning". Thus, it is obvious that spiritual well-being is inextricably intertwined with the physical, social and emotional needs of patients.
Within the last years scientific research approved several connections between religion, spirituality, and health. Several studies indicate that religious involvement and spirituality are associated with better recovery from illness, greater longevity, coping skills, health-related quality of life, less anxiety and less depression (reviewed by [5-11]). As mentioned by Koenig [12], the findings are particularly strong in patients with severe or chronic diseases who have stressful psychological and social changes, as well as existential struggles related to meaning and purpose. Surveys suggest that most patients regard their spiritual health and physical health as equally important [5].
However, the measurability of a religious and/or spiritual attitude respectively towards engagement in distinct forms of a spiritual and religious practice is somewhat difficult. Even though the constructs Religiosity and Spirituality may not be identical, they were interchangeable to a certain degree in their origins. Nowadays, it is well established practice to divide Religiosity into the three sub-constructs intrinsic, extrinsic, and quest religiosity [13-16]. In contrast, the construct Spirituality is commonly divided into the following sub-constructs: Cognitive Orientation Towards Spirituality, Experiential/Phenomenological Dimension of Spirituality, Existential Well-Being, Paranormal Beliefs, and Religiousness [17].
Due to their close contextual and cultural coherence, several inventories designed to measure spirituality ask for specific and locally valid religious beliefs and practices (i.e. church attendance and praying) and/or assume a belief in God [17-23]. But this may be inappropriate for patients with different religious, cultural or philosophical backgrounds, or atheist or agnostic patients who may still be spiritually oriented, but are not addressed with regard to their distinct forms of practice in the inventories. A useful tool which does not assume a specific belief in God is the Functional Assessment of Chronic Illness Therapy – Spiritual Well-Being (FACIT-Sp) [1,2]; however, it differentiates only two factors, i.e. "faith", and "meaning and peace".
Another new inventory is our SpREUK questionnaire, which asks for basic SpR attitudes and convictions (i.e. Search for meaningful support; Positive interpretation of disease; Trust in external guidance; Support in relations with the External through SpR; Stabilization of the inner condition through SpR). In order to examine how patients with severe diseases view the impact of spirituality and religiosity (SpR) on their health and how they cope with illness, we previously performed several studies with this new inventory [24-29]. We found that patients with both a religious and spiritual attitude had significantly higher values in the sub-scales dealing with the search for meaningful support, and the stabilizing effects of SpR than patients without such attitudes, while patients with a non-spiritual religious attitude had lower perception of the beneficial effects of their SpR and had significantly lower scores in the search for meaningful support sub-scale [24-26]. However, no significant differences were found in the SpR attitude groups with regard to the meaning of disease. Reflecting on meaning and sense of life and positive interpretation of disease obviously can have an impact on how patients change their further life, and thus on distinct forms and engagement frequencies of their SpR practice. Depending on the strength of SpR beliefs and convictions some may feel love and concern for others or have a sense of connectedness, but those who are religious or spiritual only literally might not show any concern for others and are self-centered.
Following these results, it is important to analyze the forms of religious or spiritual involvement of the patients, and to connect their engagement with the SpR attitudes and their convictions how this may have an effect on the course of disease. As the original SpREUK does neither ask for distinct forms nor frequency of SpR practices, these topics are addressed in the newly developped SpEUK-P manual [26,27]. To account for the fact of an institutional religion declines in Europe [30], and the alternative use of various existing esoteric and religious resources, we intended to ask for both, the conventional forms of SpR (i.e. praying, service attendance, recitation of distinct texts, reading distinct books, meditation etc.), and a more reflecting or philosophical practice and nature/environment-oriented practice.
In this paper, we aimed to examine the statical properties of this new SpREUK-P module and how it interacts with the given SpREUK-scores.
Methods
Procedure and subjects
All individuals were informed of the purpose of the study, were assured of confidentiality, and gave informed consent to participate. The patients were recruited consecutively in the cancer service, the multiple sclerosis service, and two internal medical units of the Communal Hospital in Herdecke (West-Germany). The healthy subjects were recruited among the medical staff of the Community Hospitals in Herdecke and Berlin, staff of an ambulant out-patient care unit in Essen, attendants of a meeting on "Spirituality and Medicine" in Berlin, a Caritas congress in Kevealer, and a meeting of contemporary Christian songwriters in Trier. All subjects completed the questionnaires (SpREUK 1.1 and SpREUK-P) by themselves. Demographic information is provided in Table 1.
Table 1 Demographic data and SpREUK-P scores of 354 subjects
CRP ExP USP HuP NoP
% (45.5 ± 27.6) (60.9 ± 18.3) (32.0 ± 24.0) (74.4 ± 20.0) (69.9 ± 18.7)
sex * (*)
female 71 46.6 ± 27.3 62.4 ± 18.1 31.8 ± 24.9 74.7 ± 19.8 71.0 ± 18.3
male 29 42.5 ± 29.0 56.9 ± 18.2 32.1 ± 22.2 73.4 ± 19.9 67.1 ± 19.5
age * *
< 30 years 4 34.0 ± 32.8 57.4 ± 21.2 21.4 ± 19.1 74.5 ± 23.1 61.1 ± 22.9
30–49 years 54 41.5 ± 27.1 60.6 ± 18.3 32.5 ± 24.8 75.8 ± 18.6 69.0 ± 18.1
50–69 years 35 51.8 ± 27.2 62.8 ± 17.7 33.4 ± 23.9 74.8 ± 19.1 71.4 ± 19.7
> 70 years 6 50.7 ± 25.8 55.9 ± 20.4 25.4 ± 19.1 60.5 ± 28.4 74.2 ± 14.3
marital status * **
married 59 47.8 ± 27.1 58.3 ± 17.5 29.1 ± 21.3 74.0 ± 21.2 68.5 ± 18.6
living with partner 13 37.6 ± 23.6 65.6 ± 16.3 32.9 ± 24.9 78.7 ± 16.7 70.4 ± 18.9
divorced 9 48.6 ± 26.8 72.2 ± 18.1 38.9 ± 28.7 74.1 ± 18.6 78.1 ± 19.7
alone 16 38.2 ± 30.6 59.4 ± 22.0 34.1 ± 27.3 73.1 ± 19.3 67.6 ± 19.3
widowed 3 57.4 ± 30.5 56.1 ± 11.3 26.6 ± 21.9 71.3 ± 19.1 71.5 ± 13.7
education1 ** ** ** (*)
level 1 14 44.3 ± 26.1 52.3 ± 18.6 20.1 ± 19.7 67.4 ± 22.8 66.4 ± 22.7
level 2 24 38.2 ± 24.6 57.5 ± 17.4 24.3 ± 17.6 74.1 ± 20.0 70.9 ± 17.7
level 3 40 52.3 ± 28.8 63.5 ± 17.5 38.6 ± 26.4 76.0 ± 18.8 69.2 ± 18.0
other 13 36.8 ± 24.8 67.0 ± 17.6 34.3 ± 21.6 76.5 ± 19.6 75.0 ± 18.1
disease (*) * ** ** *
Healthy 50 47.3 ± 28.8 62.3 ± 16.6 36.5 ± 25.3 78.4 ± 15.8 70.5 ± 19.0
Chronic diseases 12 41.6 ± 27.2 64.7 ± 20.9 35,5 ± 26.6 78.4 ± 22.0 73.9 ± 17.3
Multiple Sclerosis 11 36.2 ± 28.3 51.9 ± 17.9 16.2 ± 15.3 68.3 ± 27.8 60.3 ± 20.7
Cancer 26 46.4 ± 25.5 60.1 ± 19.5 28.6 ± 20.5 68.0 ± 20.4 72.2 ± 16.3
religious affiliation ** *
Christian 84 50.4 ± 26.2 61.0 ± 17.5 31.8 ± 23.1 74.1 ± 19.9 70.0 ± 19.1
Others2 3 38.3 ± 32.6 73.8 ± 18.9 46.0 ± 35.7 72.2 ± 15.5 76.6 ± 13.3
None 13 16.0 ± 15.4 55.8 ± 20.8 29.4 ± 27.0 77.1 ± 20.6 67.6 ± 17.5
spiritual attitude ** ** ** * **
R+S+ 47 57.3 ± 24.3 66.8 ± 17.0 42.4 ± 22.2 76.5 ± 18.2 72.8 ± 16.8
R+S- 25 51.3 ± 25.2 55.3 ± 15.5 19.6 ± 15.6 70.5 ± 22.2 67.6 ± 18.0
R-S+ 12 22.9 ± 18.7 65.5 ± 16.1 42.2 ± 26.7 78.0 ± 16.4 75.5 ± 14.7
R-S- 17 18.8 ± 16.2 47.6 ± 18.1 12.2 ± 15.3 71.1 ± 21.8 61.9 ± 19.6
1 Increasing educational level (based on German school system): 1 = secondary education (Hauptschule), 2 = secondary education (junior high; Realschule), 3 = high school education (Gymnasium).
2 Islam, Buddhism, "Christengemeinschaft" and some not-specified confessions. Scores are significantly different (** p < 0.01; * p < 0.05; (*) 0.05 < p < 0.10; ANOVA).
Deviations of >15% from the mean were highlighted.
The sample contained 354 subjects of whom 71% were women. The mean age was 49.0 ± 12.5 years. Half of the subjects were healthy, while among the patients cancer was diagnosed in 54%, multiple sclerosis in 22%, and other chronic diseases in 23% (i.e. Hepatitis C, liver cirrhosis, inflammatory bowel disease, severe hypertension etc.). Patients in final stages of their disease were not enrolled.
Measures
The SpREUK-P was designed to differentiate spiritual, religious, existentialistic, and philosophical practices. The items were developed with the patients' input (cancer service of the Herdecke Community Hospital) and experts' statements (physicians, therapists, and priest working with patients) [26,27].
According to a previously conducted pilot study on the reliability and factorial validity on the original 18-item SpREUK-P 1.0 version [26], the following scales were derived: (1) conventional religious practice (CRP), (2) nature-oriented practice (NoP), (3) existentialistic practice (ExP), (4) unconventional spiritual practice (USP), and (5) humanistic practice (HuP). As some of the scales had only a few items, eight new questions (No. 19–26) were added, in particular to strengthen the HuP construct. In total, our item pool therefore consisted of 26 items. All items were scored on a 4-point scale (0 – never; 1 – seldom; 2 – often; 3 – regularly). The SpREUK-P scores are referred to a 100% level (4 "regularly " = 100%), which reflects the degree of an engagement in the distinct forms of a SpR practice ("engagement scores").
Statistical analysis
Reliability and factor analysis of the new inventory were performed according to the standard procedures. In order to eliminate items from the item pool that were not contributing to the questionnaire reliability, the reliability of the scale and distinct sub-scales was evaluated with internal consistency coefficients, which reflect the degree to which all items on a particular scale measure a single (unidimensional) concept. To combine several items with similar content, we relied on the technique of factor analysis, which examines the correlations among a set of variables, in order to achieve a set of more general "factors." VARIMAX-factor analysis was repeated rotating different numbers of items in order to arrive at a convergent solution embodying both the simplest structure and the most coherent.
Differences in the SpREUK scores were tested using ANOVA. We judged p < 0.05 significant, and 0,05 < p < 0.10 as a trend. To test the impact of several variables on the SpREUK sub-scales, we performed analysis of univariate variance (ANOVA).
All statistical analyses were performed with SPSS for Windows 10.0.
Results
Reliability
Reliability analysis revealed that item "Church attendance" (P2) had a poor corrected item-total correlation (Table 2) and thus should have been eliminated. However, as this item is of major conceptual importance (also in other questionnaires), we decided not to eliminate it.
Table 2 Mean values of the items from SpREUK-P 1.1 and reliability parameters
Factors and Items Mean value (Score 0–3) SD Factor load Item difficulty Corrected Item-Total correlation Alpha if Item deleted (α = 0.8517)
Conventional Religious Practice (α = 0.8642)
P2 go to church: 1.30 0.94 0.794 0.44 0.116 0.858
P20 participate in religious events: 0.87 0.98 0.779 0.29 0.266 0.851
P19 religious symbols are important in private area 1.37 1.03 0.772 0.46 0.414 0.846
P1 pray: 1.66 0.99 0.692 0.55 0.499 0.843
Existentialistic Practice (α = 0.7797)
P13 work on my self-realization: 1.65 0.80 0.782 0.55 0.318 0.849
P14 work on my spiritual development: 2.01 0.75 0.667 0.67 0.483 0.844
P11 try to get insight (also into myself): 2.07 0.75 0.606 0.69 0.682 0.838
P15 try to achieve a higher level of consciousness: 1.23 0.91 0.568 0.41 0.531 0.842
P10 reflect upon the meaning of life: 2.08 0.75 0.520 0.70 0.460 0.845
P16 try to convey positive values & convictions to others: 1.91 0.77 0.436 0.64 0.432 0.846
Unconventional Spiritual Practice (α = 0.7535)
P7 work on a body-mind discipline: 0.92 1.04 0.768 0.31 0.450 0.844
P4 meditate: 0.91 0.93 0.723 0.31 0.471 0.844
P8 perform distinct rituals: 0.99 1.02 0.682 0.33 0.577 0.839
P6 read religious/spiritual books: 1.24 1.15 0.595 0.41 0.371 0.826
P5 recite distinct (i.e. holy) texts: 0.71 0.86 0.534 0.24 0.483 0.844
Humanistic Practice (α = 0.7907)
P23 try to consider the needs of others: 2.31 0.56 0.774 0.77 0.319 0.849
P22 try to help others: 2.41 0.58 0.746 0.80 0.355 0.848
P24 thoughts are with those in need: 1.98 0.70 0.717 0.66 0.370 0.848
P25 try to do good: 2.30 0.58 0.650 0.77 0.302 0.849
P26 feel connected with others: 2.26 0.66 0.574 0.75 0.340 0.848
P3 try make an effort for other people: 2.21 0.70 0.445 0.74 0.499 0.844
Nature-oriented Practice (α = 0.5853)
P17 try to be aware of how I treat the world around: 2.42 0.60 0.725 0.80 0.396 0.847
P18 try to have a healing effect on environment: 1.85 0.82 0.616 0.62 0.519 0.843
P9 turn to nature: 2.03 0.81 0.551 0.68 0.244 0.851
Guardian Angel
P21x belief in (my) Guardian Angel: 1.79 1.03 0.584 0.60 0.377 0.848
As shown in Table 2, the new construct had a good quality (Cronbach's alpha = 0.8517). The item difficulty (1.65 [mean value]/3) is 0.55. With the exception of item P17 ("I try to be aware of the way I treat the world around me"; item difficulty = 0.81), all values are in the acceptable range from 0.2 to 0.8.
Factor analysis
Factor analysis revealed a Kaiser-Mayer-Olkin value of 0.79, which as a measures for the degree of common variance, indicates that the item-pool seems to be suitable for a factorial validation. In addition, Barlett's test for non-sphericity was highly significant (p < 0,001).
Primary factor analysis of item pool pointed to a 7-factor solution, which would explain 60.4% of variance. However, due to a low item number in the tentative subscales 6 and 7 (with 2 items each), we favored the more appropriate 6-factor solution, which explains 56.2% of variance and is provided in Table 2.
The 4-item sub-scale CRP had an alpha of 0.8642, the 6-item sub-scale ExP had an alpha of 0.7797, sub-scale USP with its 5 items had an alpha of 0.7535, and the sub-scale HuP with its 6 items had a Cronbach's alpha of 0.7907, while the 3-item sub-scale NoP had an alpha of 0.5853. As the item P21x ("Guardian angel") made up a factor on its own – even in a tentative 5-factor solution -, we decided to use it as a marker item until the construct is revised for this topic. Thus, the internal consistency of the item pool was sufficiently high.
Analysis of the "side-loadings" of the item pool (only values > 0.35 were take into account) revealed that the marker item P21x ("guardian angel") together with items P11 ("get insight") and P10 ("reflect upon the meaning of life") from the ExP sub-scale and item P5 ("recite distinct texts") from the USP sub-scale would load to a tentative sub-scale 7, while item P21x would load only on the NoP sub-scale 5 (0.368). However, this solution would decrease the quality of the other respective sub-scales and thus was rejected. Item P15 ("higher level of consciousness") from the ExP sub-scale also loads on the NoP sub-scale (0.388).
Relation between SpREUK-P scores and demographic variables
The highest engagement scores were found for HuP and NoP, while the lowest were found for the USP. Means and standard deviations for study variables are provided in Table 1.
Women had significantly higher scores for ExP than male patients, and in trend also for NoP.
With respect to age, the engagement in CRP increases with increasing age, while the engagement in a HuP decreases with increasing age. Although not significant, it is remarkable that the lowest scores for USP were found in subjects < 30 years and > 70 years of age.
With respect to the marriage status, subjects living alone or with a partner but not married had the lowest scores for CRP, whilst widowed patients had the highest engagement scores. This is in agreement with our previous findings that these individuals rely on "external guidance" [25], but not the patients living with an unmarried partner. Divorced subjects had the highest engagement scores for ExP (and, although not significant, for NoP) which is again in accordance with our previous findings that they are highly in search of meaningful support [25].
Engagement in USP, CRP and ExP depended on the educational level: Patients with higher educational level had significantly higher scores than those with a lower level. Only the NoP did not depend on educational level, while for HuP we observed only a trend.
However, patients with MS overall had the lowest engagement scores, while patients with cancer or other chronic diseases did not differ in regard of the engagement in NoP, ExP and CRP. But it is somehow remarkable, that in contrast to patients with other chronic diseases, the cancer patients had lower scores for HuP and USP than healthy subjects.
It is not surprising that individuals without any religious affiliation had the lowest scores for the engagement in CRP, while their engagement in USP was similar to that of Christian subjects. The few individuals with religious affiliation other than Christian had the highest scores for USP and ExP, but due to a too small investigation group, this statement can be valued only as a hint. There were no significant differences between these three groups with respect to an engagement in NoP and HuP.
Since nominational affiliation is not necessarily identical with religiosity or spirituality, we asked whether the patients would describe themselves as religious or spiritual [24-29]. 47 % of the 354 subjects analyzed herein reported themselves as both religious and spiritual (R+S+); 25% as religious, but not spiritual (R+S-); 17% as neither religious nor spiritual (R-S-), while 12% claimed that they were spiritual, but not religious (R-S+). Thus, the numbers of patients with denominational affiliation and self-reported spiritual/religious attitudes are somewhat similar.
However, R+S+ subjects had the highest engagement score for CRP (even higher than that of R+S-). The lower score of subjects with a R-S+ attitude was comparable with that of R-S-. The engagement in an USP was high with respect to a spiritual attitude (R+S+ and R-S+), while the lowest scores were found for R-S- and R+S- subjects. It is remarkable that R-S- subjects had the lowest engagement scores for all five forms of a SpR practice.
At present the item P21x ("belief in (my) Guardian Angel"), which was a factor on its own, should be regarded just as a marker item. We found significant differences in this item between the SpR attitude groups (F = 7.649; p < 0.0001). R+S+ had the highest belief score (2.06 ± 0.932), followed by R+S- (1.75 ± 0.98), while R-S+ and R-S- had less faith in their Guardian Angel (1.27 ± 1.16 resp. 1.14 ± 1.04). The highest belief scores were found in widowed individuals (2.33 ± 0.58) and those > 70 years of age (2.25 ± 0.96), while the lowest score was found in subjects < 30 years (1.13 ± 0.83). Women and men did not significantly differ in their belief scores.
Analyses of variance
Next we tested the impact of several variables on the SpREUK P sub-scales, such as age, sex, marital status, educational level, religious affiliation, SpR attitude, disease and duration of disease. Using the method of univariate analyses of variance we identified several sources of variability (Table 3):
Table 3 Univariate variance analyses
variables Levene's test * F-value p-value
(1) conventional religious practice SpR attitude
age 0.000 45.165
1.350 0.000
n.s.
religious affiliation
educational level 0.007 23.192
1.396 0.000
n.s.
(2) existentialistic practice SpR attitude
age 0.000 15.064
0.921 0.000
n.s.
religious affiliation
educational level 0.028 3.557
2.768 0.030
0.040
gender
marital status
gender * marital status n.s. 4.390
2.276
2.325 0.037
0.029
0.056
(3) unconventional spiritual practice SpR attitude
age 0.000 29.344
0.982 0.000
n.s.
religious affiliation
educational level 0.003 0.417
4.429 n.s.
0.005
disease
duration of disease 0.001 2.555
1.228 0.042
n.s.
(4) humanistic practice disease
duration of disease disease
* duration of disease 0.053 2.047
1.711
3.188 0.091
n.s.
0.002
(5) nature-oriented practice SpR attitude
age 0.000 4.058
0.302 0.008
n.s.
disease duration of disease n.s. 2.484
1.088 0.046
n.s.
In this table, only significant results were given.
*Levene's test for equality of variances was significant and thus the level of significance for the variance analyses should be p < 0.01
• SpR attitude is an important covariate for four of the distinct forms of practice (CRP, NoP, ExP and USP), but not for the HuP.
• Religious affiliation is an important covariate for CRP and ExP, but not for USP.
• Educational level is a covariate for USP and ExP.
• Gender andMarital status are covariates only for ExP.
• Age is not a relevant covariate for any of the five forms of SpR practice.
• Disease itself has an impact on NoP (and a minor impact on HuP and USP), while the duration of disease has no impact on the forms of SpR practice. However, disease and its duration are of relevance for an engagement in HuP.
Correlation between engagement in the different forms of SpR practice and SpR attitude
Bivariate correlation between the five forms of a SpR practice revealed a strong correlation between NoP and ExP, and between ExP and USP, while CRP did not correlate with NoP or HuP, regardless of their SpR attitude (Table 4). For individuals with a spiritual attitude (R+S+ and R-S+), their ExP is strongly associated with USP and NoP (Table 4), while the ExP of R+S- individuals correlates only with NoP, but not with USP. Moreover, for those with a spiritual attitude (R+S+ and R-S+), their USP correlated well with CRP. However, the ExP of in R-S- individuals significantly correlated with a CRP.
Table 4 Pearson correlation between SpREUK-P sub-scales with respect to SpR attitude
CRP ExP USP HuP NoP
all individuals
conventional religious practice 1.000 .230 ** .371 ** .027 .066
existentialistic practice 1.000 .490 ** .316 ** .512 **
unconventional spiritual practice 1.000 .199 ** .302 **
humanistic practice 1.000 .220 **
nature-oriented practice 1.000
R+S+ individuals
conventional religious practice 1.000 .019 .324 ** -.047 -.030
existentialistic practice 1.000 .429 ** .278 ** .505 **
unconventional spiritual practice 1.000 .216** .307 **
humanistic practice 1.000 .284 **
nature-oriented practice 1.000
R+S- individuals
conventional religious practice 1.000 .179 .255 * .119 .000
existentialistic practice 1.000 .165 .276 * .450 **
unconventional spiritual practice 1.000 .070 .090
humanistic practice 1.000 .'157
nature-oriented practice 1.000
R-S+ individuals
conventional religious practice 1.000 .056 .400 ** -.167 .166
existentialistic practice 1.000 .465 ** .311 ** .581 **
unconventional spiritual practice 1.000 .055 .216
humanistic practice 1.000 .329 *
nature-oriented practice 1.000
R-S- individuals
conventional religious practice 1.000 .410 ** .227 .083 -.030
existentialistic practice 1.000 .268 * .313 * .396 **
unconventional spiritual practice 1.000 .164 .153
humanistic practice 1.000 .015
nature-oriented practice 1.000
Bivariate correlations are statistically significant with * p < 0.05 and ** p < 0.01 (2-tailed significance)
As shown in Table 5, Pearson's correlation between the SpR practice and the distinct SpR measures of the SpREUK manual revealed strong correlations:
Table 5 Pearson correlation between SpREUK sub-scales and SpR practice
Search Meaning Message Disease Trust Guidance Support External Support Internal
SpREUK-P engagement scores
conventional religious practice .381 ** .093 .659 ** .472 ** .367**
existentialistic practice .510 ** .465 ** .361 ** .487 ** .433 **
unconventional spiritual practice .551 ** .385 ** .330 ** .560 ** .479 **
humanistic practice .243 ** .154 ** .014 .130 * .112
nature-oriented practice .322 ** .284 ** .190 ** .314 ** .367 **
(Guardian Angel) .327 ** .271 ** .421 ** .179 * .361 **
Bivariate correlations are statistically significant with * p < 0.05 and ** p < 0.01 (2-tailed significance)
• CRP correlates with "Trust in external guidance" and "Support in relations with the External life through SpR", but not with "Positive interpretation of disease".
• ExP correlates well with "Search for meaningful support", "Support in relations with the External life through SpR" and "Positive interpretation of disease".
• USP correlates well with "Support in relations with the External life through SpR" and "Search for meaningful support".
• HuP did not correlate at all with "Trust in external guidance" or "Support in relations with the Internal life through SpR", but marginally with both "Support through SpR" sub-scales.
• With respect to NoP, we found moderate correlations with all five SpREUK subscales.
The item "Belief in (my) Guardian Angel" which was used only as a preliminary marker item correlated moderately with ExP (r = 0.301) and NoP (r = 0.290), and with the SpREUK scales "Search for meaningful support" (r = 0.327) and "Support of the Internality through SpR" (r = 0.361), but somewhat higher with "Trust in external Guidance" (r = 0.421). Surprisingly, also individuals without any religious affiliations reported that they often/frequently believed in their Guardian Angel (36%, resp. 46% of R-S+ and 27% of R-S-), in contrast to 62% of the Christians (69% R+S+ and 62% of R+S-), and 86% of those with non-Christian affiliations.
Discussion
We have confirmed the structure and consistency of the previously described SpREUK-P manual [26,27], which is an integral part of the SpREUK construct [24-28], and improved the quality of the current construct by adding several new items. Apart from conventional religious and unconventional spiritual practices, three other distinct forms of engagement were of relevance to the patients with life-threatening diseases, i.e. existentialistic practice, humanistic practice, and nature/environment-oriented practice. The latter three topics are obviously more philosophical forms of SpR.
When confronted with a life-threatening disease, more existentialistic or self-centered issues become relevant to the patients. Existentialistic philosophers (such as Søren Kierkegaard and Jean-Paul Sartre) emphasized the universal struggle to find meaning in life, to live by moral standards, and to come to an understanding of suffering and death [31,32]. To them, life might be without inherent meaning (existential atheists) or it might be without a meaning we can understand (existential theists). Consequently, since man is ultimately alone, one is free to pick and choose one's own values, and to create one's own suitable religious patchwork. In accordance with these views we found strong correlations between ExP and the SpREUK scales "Search for meaningful support" and "Positive interpretation of disease". Moreover, the ExP engagement score was much higher compared to the more formalized practices, i.e. CRP and USP. The highest engagement scores were found for HuP and NoP. One could speculate that these forms reflect a higher level of "insight", but it is also true that less effort is needed to turn to others (HuP) and nature (NoP) than to reflect on yourself (ExP); and their social desirability is much higher. One the other hand, within recent decades, ecological issues and people's appreciation of nature ("earth connection") have gained much attention, and thus higher agreement levels are not surprising. Engagement in an ExP is significantly dependent on the SpR attitude (low engagement level were found for R-S- individuals and those without any religious affiliation); gender and marital status are also relevant variables. One may speculate that divorced individuals, who have the highest ExP engagement level, reflect more on themselves because of the process of divorce ("liberation", "self-realization").
A more self-centered attitude is also measured in a scale of Holland's Spiritual Beliefs Inventory (SBI-15R-D) [19,33]: The underlying attitude of a social support through a religious faith community can be described as "What will others do for me?" In our HuP and NoP scales, the question is "What can I do for others, for nature and environment?" These contrasting views are highly affected by the state of "insight" an individual has developed.
The items of the scale HuP are related to the views of Secular Humanism [34,35] and the Philosophical resp. Christian Humanism [36]. Secular Humanism is an atheistic and naturalistic philosophy promoting humanity as the measure of all things, and roots in the rationalism of the 18th Century and the free-thought movement of the 19th Century. Secular Humanists reject the concept of a personal creator God, and regard man as fully responsible for the future of the world, its political systems, its ecology, etc. [34,35]. Thus, it is not surprising that the scale HuP neither correlates to CRP nor to the SpREUK scale "Trust in External Guidance". In fact, it correlates somewhat better with ExP and the SpREUK scale "Search for meaningful support". Consequently, the lowest HuP engagement levels were found for individuals lacking a spiritual attitude (R+S- and R-S-). Low engagement levels were also found with respect to higher age, and in patients with cancer and MS. The impact of disease and its duration on HuP remains to be explained in further studies.
To our surprise, one of the most accepted topics defining conventional religious practice, "going to church" resp. "service attendance", had a low engagement score among the German individuals tested. The same is true for the participation in religious events, while praying seems to be much more attractive. The items from the USP scale had low engagement scores too, even meditation, which is highly valued in several other questionnaires.
The presumption that both scales do not measure what they are intended to do can be rejected both from a statistical but also from a contextual point of view, because individuals with a Christian affiliation had significantly higher engagement scores for CRP than those with other religious affiliations or none, while individuals with non-Christian affiliations had the highest scores for USP. Moreover, a religious attitude (R+S+ and R+S-) was associated with significantly higher mean levels for CRP than subjects with a spiritual attitude (R-S+ and R-S+), while in contrast a spiritual attitude (R+S+ and R-S+) was associated with higher levels for USP. In this context it is worth mentioning that an R-S- attitude was associated with the lowest engagement scores for all five forms of a SpR practice. Variance analyses confirmed that SpR attitude and religious affiliation are the main relevant covariate for CRP, while for USP, the SpR attitude and the educational level are of significance, but not religious affiliation.
The level of engagement in CRP also depends on the professional background of the tested subjects. We found that the engagement score was very high in attendants of a Christian Caritas meeting (mainly priests, chaplains, Christian social workers etc.) and in composers of Contemporary Christian Songs (mean values 79.9 ± 20.3 resp. 77.0 ± 13.1); high scores were found also in attendants of a meeting on "Spirituality and Health" (56.9 ± 33.3), while the lowest CRP score was found in hospital staff (32.7 ± 20.0). The engagement score of catholic nurses caring for out-patients (45.7 ± 25.6) was similar to the overall mean level (47.1 ± 28.8). Details of this investigation will be presented elsewhere.
Using the German version of Holland's Spiritual Beliefs Inventory (SBI-15R-D), Albani et al. found that higher religiosity is observed for women, older people, people with lower education, former West Germans vs. former East Germans, and people stating a religious affiliation [33]. These findings are only in part congruent with ours: The higher CRP engagement score in women was not statistically significant, while lower age and lower educational level were associated with significantly lower CRP engagement scores. However, we can confirm higher scores for patients with a Christian affiliation and a religious attitude (R+S+ and R+S-). Using the SpREUK 1.1 inventory, we have found that women with cancer have significantly higher scores for "Search for meaningful support", "Interpretation of disease" and "Support in relations with the external life through SpR", but not for "Trust in external Guidance"[24] Similar, cancer patients with a lower educational level had significantly lower scores for "Search for meaningful support" and "Interpretation of disease", though again not for "Trust in external Guidance" [24]. Thus, it is obvious that the condensed 10-item scale of the shortened SB-15-R [19], which measures mainly religious beliefs and convictions dealing with the support through God and faith, represents only a distinct aspect of religiosity.
In fact, "religiosity" is already multidimensional construct. Batson et al. described a three-dimensional model of religiosity: Means or external, End or internal, and Quest [14, 37]. Intrinsic religiosity identifies religion as an end in itself. Strong personal convictions, beliefs and values are what matter, while the social aspects of religion are not that important. In contrast, the motifs of extrinsic religiosity are based on social or external values and beliefs; religion is used to gain social standing and endorsement. The Quest orientation is founded on a willingness to question complex ideas. The persons are open to the exploration of existential questions and they are open for new information and doubts. Thus, as we have to assume a complex interconnection of various existing views, attitudes and concepts, an oversimplification ("two scales are enough") of SpR concerns in QoL research is not appropriate.
Conclusion
Our scales are in congruence with external factors influencing the distinct forms and frequency of a patients SpR engagement. The SpREUK questionnaire with its SpREUK-P manual thus could be of value in measuring SpR attitudes and engagement of patients coping with life-threatening illness, and in the measurement of distinct aspects of QoL.
An advantage of our instruments is the clear-cut differentiation between convictions and attitudes on the one hand, and the expression of these attitudes in concrete engagement on the other. A second advantage is the differentiation of five distinct forms of spiritual, religious and philosophical practice. Finally the fact that the validation was performed in a sample with at least two different types of life-changing diseases (cancer and MS, and other chronic diseases) and a healthy control group is advantageous for interpreting the results.
In future studies we will emphasize the correlation our scales with conventional QoL instruments. Nevertheless, evaluation of the SpREUK-P questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR practices. The focus of a larger study is to enroll patients from the highly secular Eastern Europe, and to run longitudinal studies with cancer, multiple sclerosis patients, but also cardiac failure and spinal cord damage.
The conventional SpREUK (Version 1.1) and its SpREUK-P manual (Version 1.1) are currently available in English and German.
Authors' contributions
AB conceived the study, designed and developed the questionnaire, performed statistical analysis and drafted the manuscript. TO participated to conceive and design the study assisted in statistical analysis and helped to draft the manuscript. PFM participated in the design and development of the questionnaire. All authors read and approved the final manuscript.
Acknowledgements
We are grateful to our patients and to Dr. Cristina Stumpf and Dr. Mette Käder for their cooperation in recruiting them, and to Hugh Featherstone Blyth for his grammatical support.
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Brady MJ Peterman AH Fitchett G Mo M Cella D A case for including spirituality in quality of life measurement in oncology Psychooncology 1999 8 417 428 10559801 10.1002/(SICI)1099-1611(199909/10)8:5<417::AID-PON398>3.3.CO;2-W
Peterman AH Fitchett G Brady MJ Hernandez L Cella D Measuring spiritual well-being in people with cancer: the functional assessment of chronic illness therapy–Spiritual Well-being Scale (FACIT-Sp) Ann Behav Med 2002 24 49 58 12008794 10.1207/S15324796ABM2401_06
Levine EG Targ E Spiritual correlates of functional well-being in women with breast cancer Integr Cancer Ther 2002 1 166 174 14664742 10.1177/1534735402001002008
Murray SA Kendall M Boyd K Worth A Benton TF Exploring the spiritual needs of people dying of lung cancer or heart failure: a prospective qualitative interview study of patients and their carers Palliat Med 2004 18 39 45 14982206 10.1191/0269216304pm837oa
Mueller PS Plevak DJ Rummans TA Religious involvement, spirituality, and medicine: implications for clinical practice Mayo Clin Proc 2001 76 1225 1235 11761504
Sloan RP Bagiella E Powell T Religion, spirituality, and medicine Lancet 1999 353 664 667 10030348 10.1016/S0140-6736(98)07376-0
Sloan RP Bagiella E Claims about religious involvement and health outcomes Ann Behav Med 2002 24 14 21 12008790 10.1207/S15324796ABM2401_03
McCullough ME Hoyt WT Larson DB Koenig HG Thoresen C Religious involvement and mortality: a meta-analytic review Health Psychol 2000 19 211 222 10868765 10.1037/0278-6133.19.3.211
Luskin F Review of the effect of spiritual and religious factors on mortality and morbidity with a focus on cardiovascular and pulmonary disease J Cardiopulm Rehabil 2000 20 8 15 10680093 10.1097/00008483-200001000-00002
Levine EG Targ E Spiritual correlates of functional well-being in women with breast cancer Integr Cancer Ther 2002 1 166 174 14664742 10.1177/1534735402001002008
Seeman TE Dubin LF Seeman M Religiosity/spirituality and health. A critical review of the evidence for biological pathways Am Psychol 2003 58 53 63 12674818 10.1037/0003-066X.58.1.53
Koenig HG Religion, spirituality, and medicine: research findings and implications for clinical practice South Med J 2004 97 1194 1200 15646757 10.1097/01.SMJ.0000146489.21837.CE
Allport GW Ross JM Personal religious orientation and prejudice J Pers Soc Psychol 1967 5 432 443 6051769 10.1037/0022-3514.5.4.432
Batson CD Schoenrade PA Measuring religion as Quest: Validity concerns J Scien Study Religion 1991 30 416 429
Maltby J Lewis CA Measuring Intrinsic and Extrinsic Orientation Toward Religion: Amendments for its use among religious and non-religious samples Personal Indiv Differences 1996 21 937 946 10.1016/S0191-8869(96)00154-7
Maltby J Day L Amending a measure of the Quest Religious Orientation: Applicability of the scale's use among religious and non-religious persons Personal Indiv Differences 1998 25 517 522 10.1016/S0191-8869(98)00078-6
Bufford RK Paloutzian RF Ellison CW Norms for the spiritual well-being scale J Psychol Theol 1991 19 56 70
Paloutzian RF Ellison CW Peplau LA, Perlman D Loneliness, spiritual well-being and the quality of life Loneliness: A sourcebook of current theory, research and therapy 1982 Wiley-Interscience 224 237
Holland JC Kash KM Passik S Gronert MK Sison A Lederberg M A brief spiritual beliefs inventory for use in quality of life research in life-threatening illness Psychooncology 1998 7 460 469 9885087 10.1002/(SICI)1099-1611(199811/12)7:6<460::AID-PON328>3.0.CO;2-R
Underwood LG Teresi JA The Daily Spiritual Experience Scale: Development, Theoretical Description, Reliability, Exploratory Factor Analysis, and Preliminary Construct Validity Using Health-Related Data Ann Behav Med 2002 24 22 33 12008791 10.1207/S15324796ABM2401_04
Plante TG Boccaccini M The Santa Clara Strength of Religious Faith Questionnaire Pastoral Psychology 1997 45 375 387
Plante TG Vallaeys C Sherman AC Wallston KA The development of a brief version of the Santa Clara Strength of Religious Faith Questionnaire Pastoral Psychology 2002 50 359 368 10.1023/A:1014413720710
Sherman AC Plante TG Simonton S Adams DC Harbison C Burris SK A multidimensional measure of religious involvement for cancer patients: the Duke Religious Index Support Care Cancer 2000 8 102 109 10739356 10.1007/s005200050023
Bussing A Ostermann T Matthiessen PF Search for meaningful support and the meaning of illness in German cancer patients Anticancer Res 2005 25 1449 1455 15865104
Bussing A Ostermann T Matthiessen PF Role of religion and spirituality in medical patients: confirmatory results with the SpREUK questionnaire Health Qual Life Outcomes 2005 3 10 15705195 10.1186/1477-7525-3-10
Bussing A Ostermann T Matthiessen PF Spirituelle Bedürfnisse krebskranker Menschen – Einstellung und Praxis Deutsche Zeitschrift für Onkologie 2005 37 13 22 10.1055/s-2005-862520
Bussing A Ostermann T Patzek M Caritas und ihre neuen Dimensionen: Spiritualität und Krankheit Caritas plus Qualität hat einen Namen 2004 Kevelaer: Butzon and Bercker 110 133
Ostermann T Bussing A Matthiessen PF [Pilot study for the development of a questionnaire for the measuring of the patients' attitude towards spirituality and religiosity and their coping with disease(SpREUK)] Forsch Komplementarmed Klass Naturheilkd 2004 11 346 353 15604625 10.1159/000082816
Bussing A Ostermann T Matthiessen PF Role of Religion and Spirituality in Medical patients in Germany Journal of Religion and Health 2005 44 321 340 10.1007/s10943-005-5468-8
Jagodzinski W Dobbelaere K Der Wandel kirchlicher Religiosität in Westeuropa Kölner Zeitschrift für Soziologie und Sozialpsychologie 1993 68 91
Kaufmann W Existentialism: From Dostoevsky to Sartre Plume Books 1975
Sartre J-P Being and Nothingness 1943 Washington Square Press
Albani C Bailer H Geyer M Brähler E Grulke N Erfassung religiöser und spiritueller Einstellungen – Psychometrische Überprüfung der deutschen Version des „System of Belief Inventory" (SBI-15R-D) von Holland et al. an einer repräsentativen Bevölkerungsstichprobe Psychother Psych Med 2002 52 306 313
Kurtz P A Secular Humanist Declaration 1980 New York: Prometheus Books
Kurtz P The Humanist Alternative 1973 New York: Prometheus Books
Holland J Religious Humanism: The Past We Inherit; The Future We Create Humanism today 1998 12
Batson CD Schoenrade P Ventis WL Religion and the individual: A social-psychological perspective 1993 New York: Oxford University Press
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Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-3-591618803410.1186/1477-7525-3-59ResearchThe effect of patient characteristics on variability in pain and function over two years in early knee osteoarthritis Paradowski Przemyslaw T [email protected] Martin [email protected] L Stefan [email protected] Ewa M [email protected] Department of Orthopedics, Lund University, Lund University Hospital, SE-221 85 Lund, Sweden2 Department of Reconstructive Surgery and Artrhroscopy of the Knee Joint, Medical University, Drewnowska 75, 91-002 Lodz, Poland2005 27 9 2005 3 59 59 23 3 2005 27 9 2005 Copyright © 2005 Paradowski et al; licensee BioMed Central Ltd.2005Paradowski 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
Large variations in pain and function are seen over time in subjects at risk for and with radiographic knee osteoarthritis (OA). We hypothesized that this variation may be related not only to knee OA but also to patient characteristics. The objective of this study was to investigate the influence of age, gender, and body mass index (BMI) on clinically relevant change in pain and function over two years in subjects at high risk for or with knee OA.
Methods
We assessed 143 individuals (16% women, mean age 50 years [range 27–83]) twice; 14 and 16 years after isolated meniscectomy. Subjects completed one disease-specific questionnaire, the Knee injury and Osteoarthritis Outcome Score (KOOS) and one generic measure, the SF-36. Individuals with a BMI between 25 and 29.9 were considered overweight, while individuals with a BMI of 30 or more were considered obese.
Results
Subjects aged 46–56 (the middle tertile) were more likely to change (≥10 points on a 0–100 scale) in the KOOS subscale Activities of Daily Living (ADL) than younger subjects (odds ratio [OR] 4.5, 95% confidence interval [95% CI] 1.5–13.0). Essentially the same result was obtained after adjusting for baseline values. Overweight or obesity was a risk factor for clinically relevant change for knee pain (OR 2.4, 95% CI 1.0 – 5.8, OR 4.0, 95% CI 1.2 – 13.6) and obesity for change in ADL (OR 4.3, 95% CI 1.2 – 15.4). The results did not remain significant when adjusted for the respective baseline value. Being symptomatic was strongly associated with increased variation in pain and function while presence or absence of radiographic changes did not influence change over two years in this cohort.
Conclusion
In a population highly enriched in early-stage and established knee OA, symptomatic, middle-aged, and overweight or obese subjects were more likely to vary in their knee function and pain over two years. The natural course of knee pain and function may be associated with subject characteristics such as age and BMI.
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Background
The osteoarthritis (OA) disease process begins much earlier than radiographic changes can be detected on plain radiographs. Individuals with such incipient OA may represent an attractive target group for future therapy aimed at slowing or stopping the further progression of OA. Individuals that have undergone meniscectomy constitute a high risk group for development of knee OA, and may represent a suitable group for studies on OA progression, as well as clinical trials in OA [1-3]. In a previous study, we investigated the natural variation in symptoms in a cohort of patients who had undergone meniscectomy 14–16 years earlier, and found that one in three patients reported a clinically relevant change in pain and function over two years [4]. Variability of symptoms in either direction is of interest to understand the natural course of knee OA. We hypothesized that the natural variation of symptoms over time may not only depend on factors related to the knee (such as the presence of joint pathology), but also on factors not directly related to the knee, e.g. patient characteristics such as age and weight. The objective of this study was to investigate the influence of age, gender, and body mass index (BMI) on clinically significant variation in pain and function over two years in subjects who had undergone meniscectomy several years before. We used a knee-specific instrument as the primary outcome measure and a generic questionnaire as a secondary tool.
Methods
Patients
Approval was obtained from the Research Ethics Committee at the Faculty of Medicine, Lund University, Sweden. All patients recruited were identified by searching the surgical records at the Department of Orthopedics, Lund University Hospital, Sweden [4]. A total of 552 subjects had undergone meniscectomy between 1983 and 1985. Inclusion and exclusion criteria allowed us to identify 264 patients who, in 1998, were sent self-administered questionnaires that assessed their knee-specific symptoms, knee function, and general health status. Of those, 211 replied (response rate 80%). In 2000, follow-up questionnaires were distributed to 200 subjects (eleven subjects were excluded because of death or newly-discovered co-morbidity). Replies were received from 146 individuals (73%). Three patients completed only one of two different questionnaires. The remaining 143 patients (84% men) formed the study group. Neither the baseline scores nor patient characteristics for the non-responders differed from the subjects completing the questionnaires at both time points. Self-reported weight and height was obtained from 141 of the patients at the second evaluation. Individuals with a BMI between 25 and 29.9 were considered overweight, while individuals with a BMI of 30 or more were considered obese. The subjects' mean age at the first follow-up was 50 (range 27–83) years. Assessments were carried out with a median interval of 2.3 (range 2.3 to 3.0) years.
Disease-specific questionnaire
The Knee injury and Osteoarthritis Outcome Score (KOOS) Swedish version LK 1.0 was used [5]. KOOS is a 42-item self-administered knee-specific questionnaire based on the WOMAC Osteoarthritis Index [6]. KOOS was developed to be used for short- and long-term follow-up studies of knee injuries, and it comprises five subscales: Pain, other Symptoms, Activities of Daily Living (ADL), Sports and Recreation Function, and knee-related Quality of Life. A separate score ranging from 0–100, where 100 represents the best result, is calculated for each subscale. The questionnaire and scoring manual are available from the website . The KOOS is valid and reliable in follow-up of meniscectomy [5], anterior cruciate ligament reconstruction [7] and total knee replacement [8]. The patients completed the KOOS questionnaire answering questions on their operated knee.
Generic questionnaire
The 36-item Short Form (SF-36) of the Medical Outcome Study, Swedish version was used [9]. The SF-36 is a self-administered, generic measurement tool which has been previously validated for use in the general populations [10] as well as in selected populations with knee OA [11]. Responses to 35 of 36 questions are aggregated into eight dimensions: Physical Function, Role-Physical, Role-Emotional, Bodily Pain, Social Functioning, Mental Health, Vitality, and perception of General Health. Responses vary from dichotomous (yes/no) to a six-point verbal rating scale. Results are calculated and presented as a profile of scores of each of eight dimensions.
Clinically important variation
The minimal perceptible clinical improvement (MPCI) represents the difference on the measurement scale associated with the smallest change in the health status that could be detected by the patient. Since the KOOS questionnaire contains the full and original version of the WOMAC index and WOMAC scores can easily be calculated, we used the MPCI as described for the WOMAC subscales pain, physical function, and stiffness [12]. According to this standard a level of 10 points or more on a 0–100 scale was established as a cut-off representing a clinically significant difference. We applied the same cut-off for the generic SF-36 outcome measure as for the KOOS.
Definition of a symptomatic index knee
Since there is no agreement upon a cut-off with regard to 'symptomatic' in this context, we used a previously applied definition based on the patient's self-report from the KOOS questionnaire [2]. This operational definition aimed at identifying individuals symptomatic enough to possibly seek medical care. The definition required that the value of the KOOS subscale QOL and two out of the four additional subscales should be equal to or less than the score obtained as follows: At least 50% of the questions within the subscale were answered with at least one step decrease from the best response (indicating no pain/best possible function etc.) on a 5-point Likert scale. After conversion to a 0–100 worst to best scale the cut-offs were as follows: Pain ≤ 86.1, Symptoms ≤ 85.7, ADL ≤ 86.8, Sport/Rec ≤ 85.0 and QOL ≤ 87.5.
Radiographic examination
One-hundred and thirty-three (93%) of the subjects had undergone radiographic knee examination at the second assessment. The radiographic technique and the grading of radiographs have been detailed [3]. The cut-off for radiographic OA corresponded to grade 2 or worse on the Kellgren and Lawrence scale [13].
Statistics
Logistic regression models, both unadjusted and adjusted for baseline values, were used to evaluate the association of each study variable with clinically relevant change. The logistic regression model included age divided into tertiles, gender, and body mass index (categorized). The odds ratio (OR) estimates with 95% confidence intervals (95% CIs), and results from the likelihood ratio test, expressed as P-values, were based on the models. We considered a P-value of 0.05 or less significant, and all tests were two-sided (SPSS for Windows release 12.0.1, SPSS Inc.). No prior sample size determination was made due to the observational character of the present study. However, in a binomial post hoc power calculation, using prevalence estimates and numbers from the existing dataset (n = 143, clinical relevant change occurring in 8 of 50 subjects in the reference category, and at a significance level of 0.05), we would have 80% power to detect an OR of 3.16
Results
Age
There was an association of age 46–56 (middle tertile) and clinically relevant change (≥10 points on a 0–100 scale) for the KOOS subscale ADL (OR 4.5, 95% CI 1.5 – 13.0) when unadjusted for baseline (figure 1, table 2), but otherwise there were no significant associations detected with respect to age. The association between the intermediate age tertile and ADL remained significant after adjusting for baseline values, i.e., the subjects' ADL values from 1998 (table 2).
Figure 1 Variability in the subscale Activities of Daily Living of the Knee injury and Osteoarthritis Outcome Score (KOOS) over two years. Subjects are divided into three groups (tertiles) according to their age. Each line represents one individual visualizing the difference between the score in 1998 (presented as zero, left endpoint of line) and in 2000 (right endpoint of the same line).
Table 2 The effect of demographic characteristics on clinically relevant variation in function over two years
KOOS Activities of Daily Living SF-36 Physical Function
Characteristic Prevalence of clinically relevant change Unadjusted for baseline values Adjusted for baseline values Prevalence of clinically relevant change Unadjusted for baseline values Adjusted for baseline value
N % OR OR 95% CI N % OR OR 95% CI
Age
<46† 6/49 12 21/49 43
46–56 19/47 40 4.5 3.8 1.2–12.4 24/45 53 1.2 0.9 0.3–2.4
>56 13/47 28 2.5 1.8 0.5–6.2 23/46 50 1.3 0.5 0.2–1.4
Gender
men† 32/120 27 54/118 46
women 6/23 26 1.2 0.7 0.2–2.3 14/22 64 2.4 1.9 0.6–6.1
BMI (kg/m2)
<25† 9/61 15 21/60 35
25–29.9 22/64 34 1.4 2.0 0.5–3.9 39/63 62 3.2 2.4 1.0–5.7
≥30 7/16 44 1.4 1.8 0.3–6.3 6/15 40 1.2 0.1 0.2–0.9
KOOS = Knee injury and Osteoarthritis Outcome Score, OR = odds ratio, 95% CI = 95% confidence interval, BMI = body mass index.
* All risk factors listed are entered simultaneously in each model.
† Reference category.
Gender
No significant associations between gender and clinically relevant change for pain or function were detected (tables 1, 2).
Table 1 The effect of demographic characteristics on clinically relevant variation in pain over two years*
KOOS Pain SF-36 Bodily Pain
Characteristic Prevalence of clinically relevant change Unadjusted for baseline values Adjusted for baseline values Prevalence of clinically relevant change Unadjusted for baseline values Adjusted for baseline value
N % OR OR 95% CI N % OR OR 95% CI
Age
<46† 11/49 22 27/49 55
46–56 17/47 36 1.5 1.1 0.4–3.1 32/47 68 1.2 1.1 0.4–2.8
>56 12/47 26 1.0 0.9 0.3–2.4 34/47 72 1.9 1.7 0.7–4.5
Gender
men† 35/120 29 78/120 65
women 5/23 22 1.2 0.5 0.2–1.8 15/23 65 1.2 1.1 0.4–3.2
BMI (kg/m2)
<25† 10/61 16 34/61 56
25–29.9 22/64 34 2.4 2.0 0.8–4.9 47/64 73 2.3 1.9 0.8–4.3
≥30 7/16 44 4.0 1.8 0.4–7.1 11/16 69 1.4 0.6 0.2–2.5
KOOS = Knee injury and Osteoarthritis Outcome Score, OR = odds ratio, 95% CI = 95% confidence interval, BMI = body mass index.
* All risk factors listed are entered simultaneously in each model.
† Reference category.
Body mass index
In a model unadjusted for baseline, overweight or obesity was a risk factor for clinical variation in the knee-specific subscale Pain (OR 2.4, 95% CI 1.0 – 5.8, OR 4.0, 95% CI 1.2 – 13.6), and obesity for change in ADL of the KOOS questionnaire (OR 4.3, 95% CI 1.2 – 15.4). However, when the baseline values were included in the model, the associations did not remain significant (tables 1, 2). For pain in general and Physical Function (SF-36), overweight was associated with clinically relevant change (OR 2.2, 95% CI 1.0 – 4.8, OR 3.2, 95% CI 1.5 – 6.9) unadjusted for baseline. The association remained for SF-36 Physical Function after adjusting for baseline values, but not for SF-36 Bodily Pain (tables 1, 2).
Radiographic knee OA
Fifty-eight of the 133 x-rayed subjects (44%) had radiographic tibiofemoral or patellofemoral OA in their index knee. In separate analyses (adjusted for age, gender, BMI, and unadjusted and adjusted for baseline values, respectively), we also evaluated the effect of radiographic knee OA on clinically relevant change. We found no association between the presence of radiographic OA (K/L grade ≥ 2) and clinical variation, neither in the knee-specific KOOS nor the generic SF-36 subscales (p ≥ 0.09).
Symptomatic knee
In all the present analyses, the baseline values were highly significant, where lower (worse) score was associated with clinically relevant change (p ≤ 0.001).
According to our definition of a 'symptomatic' knee based on the KOOS questionnaire, there were 61 individuals who were symptomatic at entry and 66 who fulfilled the same criteria at the second assessment. In a model adjusted for age, gender, and BMI, the likelihood of clinically relevant variation over time for subjects who were symptomatic at baseline was significantly higher for all the outcomes than subjects defined as asymptomatic: KOOS Pain (OR 5.8, 95% CI 2.4 – 14.1), KOOS ADL (OR 4.5, 95% CI 1.9. – 11.0), SF-36 Bodily Pain (OR 3.0, 95% CI 1.3 – 6.8), and SF-36 Physical Function (OR 3.8, 95% CI 1.7 – 8.5).
Discussion
The present study indicates that being knee-symptomatic, middle-aged, and overweight may predispose for variation in pain and function over two years. These factors are therefore relevant to take into account when deciding patient inclusion and exclusion criteria, outcome measures, and the number needed in clinical trials in subjects with early-stage knee OA.
The Bristol 'OA 500' study is one of few studies on the natural variation of symptoms in knee OA [14]. In this cohort, several baseline variables including age, gender, and BMI were analyzed as possible predictors of change in a combined index including change in pain, change in index joints, and global change. Neither age, gender, nor BMI were shown to influence the variation over eight years [14]. In comparison with our study subjects, the Bristol 'OA 500' cohort was on average 10 years older, held more women (69 vs. 16%), and included subjects with OA not only of the knee but also of the hip and hand. The mean BMI or presence of previous knee injuries was not reported.
OA is usually studied in the elderly. However, it is well recognized that OA may develop during middle-age or earlier [15,16]. The age range of 27 to 83 for our cohort allowed us to study the variation in symptoms in different age categories. We found an increased variability in knee-related function in patients aged 46–56, compared with the younger age group. Younger subjects scored well at entry, and in general their outcome remained good over time. These age-related findings may reflect the developmental phases of OA and correspond to an increase in the report of knee disability seen in the population during middle-age [16,17]. With increased age there may also be large changes in lifestyle due to early retirement or other alterations of the psychosocial situation that may affect self-reported symptoms and knee function. The oldest subgroup (aged >56) seem to form a more stable group than the aged 46–56, but low subject numbers limit interpretation of the results.
In analytic models unadjusted for baseline, overweight or obesity was a predictor of clinically relevant variation in outcome over two years. However, when adjusting for baseline scores, these associations did not remain significant. This is likely an effect of the strong association between high BMI and being symptomatic. In a closely related study we found no evidence that subjects had become sedentary due to symptoms and then obese (as cause and effect) [3].
Having a symptomatic knee, in comparison with having a non-symptomatic knee, was strongly associated with large variability over two years. We cannot exclude the contribution of a ceiling effect, i.e., an individual with KOOS Pain ≥ 91 cannot improve by 10 points. There was no floor effect possible, as no study subjects had a score of 10 or worse. The finding of symptoms being a strong predictor of change has less importance for future design in OA trials since a common inclusion criterion is at least moderate pain and functional limitations to be able to detect a clinically relevant improvement from the intervention applied.
We found no significant influence of radiographic status on variation in pain and function in this cohort, consistent with the well-known discordance between radiographic status and pain in population-based studies of OA [18,19].
We arbitrarily applied the same cut-off for clinically relevant change for the generic SF-36 as for the knee-specific measure KOOS. This serves as an important limitation in interpreting the SF-36 results. The pain and function subscales of the KOOS and SF-36 measures hold different number of items, and the different items may have different numbers of response options. A change in response of one step, e.g. from mild to moderate, will have greater impact on the final score if the scale has fewer items, or fewer response options. For the KOOS subscale Pain, a one step change in response option will result in a score change of 2.8 points. For the SF-36 subscale Bodily Pain the corresponding change in response would yield a score change of 6–20 points (depending on the item/response option changed), explaining the larger proportion of patients changing on the SF-36, compared with the KOOS.
Other limitations of our study include low subject numbers and not taking into consideration several factors in that may be important to the outcome studied, most importantly medication, co-morbidities, educational level, coping, and mood status. Also, the use of self-estimates of weight and length from the second assessment and the low proportion of women serve as limitations. Meniscectomy is more frequently performed in men [20,21], the reasons for which are not clear. We were unable to evaluate the possible influence of "regression to the mean" phenomenon as we only had data from two time points. Strengths of our study include the use of validated self-reported questionnaires which help avoid investigator bias, and the high follow-up rate of 73%.
Conclusion
In conclusion, we report that among a group highly enriched in early-stage and established knee OA, symptoms at baseline, middle age, and overweight or obesity are factors related to clinically relevant change in pain or knee function over two years. Variation over time in knee symptoms related to OA may thus also be dependent on factors not directly related to the knee.
Authors' contributions
EMR and LSL planned the study and collected the data. ME identified the patient cohort, collected the data, and performed the statistical analysis. PTP participated in the statistical analysis and drafted the manuscript together with ME (ME and PTP contributed equally to this study). EMR and LSL revised the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by grants from the Articulum Fellowship Europe, the Swedish Rheumatism Association, The Swedish National Center for Research in Sports, The Swedish Research Council, The King Gustaf V 80-year Birthday Fund, Zoega Foundation for Medical Research, Kock Foundations and Lund University Medical Faculty and Region Skane.
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Roos H Lauren M Adalberth T Roos EM Jonsson K Lohmander LS Knee osteoarthritis after meniscectomy: prevalence of radiographic changes after twenty-one years, compared with matched controls Arthritis Rheum 1998 41 687 693 9550478 10.1002/1529-0131(199804)41:4<687::AID-ART16>3.0.CO;2-2
Englund M Roos EM Lohmander LS Impact of type of meniscal tear on radiographic and symptomatic knee osteoarthritis: a sixteen-year followup of meniscectomy with matched controls Arthritis Rheum 2003 48 2178 2187 12905471 10.1002/art.11088
Englund M Lohmander LS Risk factors for symptomatic knee osteoarthritis fifteen to twenty-two years after meniscectomy Arthritis Rheum 2004 50 2811 2819 15457449 10.1002/art.20489
Paradowski PT Englund M Roos EM Lohmander LS Similar group mean scores, but large individual variations, in patient-relevant outcomes over 2 years in meniscectomized subjects with and without radiographic knee osteoarthritis Health Qual Life Outcomes 2004 2 38 15279676 10.1186/1477-7525-2-38
Roos EM Roos HP Ekdahl C Lohmander LS Knee injury and Osteoarthritis Outcome Score (KOOS)--validation of a Swedish version Scand J Med Sci Sports 1998 8 439 448 9863983
Bellamy N Buchanan WW Goldsmith CH Campbell J Stitt LW Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee J Rheumatol 1988 15 1833 1840 3068365
Roos EM Roos HP Lohmander LS Ekdahl C Beynnon BD Knee Injury and Osteoarthritis Outcome Score (KOOS)--development of a self-administered outcome measure J Orthop Sports Phys Ther 1998 28 88 96 9699158
Roos EM Toksvig-Larsen S Knee injury and Osteoarthritis Outcome Score (KOOS) - validation and comparison to the WOMAC in total knee replacement Health Qual Life Outcomes 2003 1 17 12801417 10.1186/1477-7525-1-17
Sullivan M Karlsson J Ware JEJ The Swedish SF-36 Health Survey--I. Evaluation of data quality, scaling assumptions, reliability and construct validity across general populations in Sweden Soc Sci Med 1995 41 1349 1358 8560302 10.1016/0277-9536(95)00125-Q
Ware JEJ Sherbourne CD The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection Med Care 1992 30 473 483 1593914
Hawker G Melfi C Paul J Green R Bombardier C Comparison of a generic (SF-36) and a disease specific (WOMAC) (Western Ontario and McMaster Universities Osteoarthritis Index) instrument in the measurement of outcomes after knee replacement surgery J Rheumatol 1995 22 1193 1196 7674255
Ehrich EW Davies GM Watson DJ Bolognese JA Seidenberg BC Bellamy N Minimal perceptible clinical improvement with the Western Ontario and McMaster Universities osteoarthritis index questionnaire and global assessments in patients with osteoarthritis J Rheumatol 2000 27 2635 2641 11093446
Kellgren J Lawrence J Radiological assessment of osteoarthritis Ann Rheum Dis 1957 16 494 502 13498604
Dieppe P Cushnaghan J Tucker M Browning S Shepstone L The Bristol 'OA500 study': progression and impact of the disease after 8 years Osteoarthritis Cartilage 2000 8 63 68 10772234 10.1053/joca.1999.0272
Petersson IF Boegard T Saxne T Silman AJ Svensson B Radiographic osteoarthritis of the knee classified by the Ahlback and Kellgren & Lawrence systems for the tibiofemoral joint in people aged 35-54 years with chronic knee pain Ann Rheum Dis 1997 56 493 496 9306873
Hart DJ Doyle DV Spector TD Incidence and risk factors for radiographic knee osteoarthritis in middle-aged women: the Chingford Study Arthritis Rheum 1999 42 17 24 9920009 10.1002/1529-0131(199901)42:1<17::AID-ANR2>3.0.CO;2-E
Roos EM Paradowski PT Bergman S Lundius A Lohmander LS Knee specific outcomes related to age and gender in a population-based cohort Arthritis Rheum 2004 50 S666
Lethbridge-Cejku M Scott WWJ Reichle R Ettinger WH Zonderman A Costa P Plato CC Tobin JD Hochberg MC Association of radiographic features of osteoarthritis of the knee with knee pain: data from the Baltimore Longitudinal Study of Aging Arthritis Care Res 1995 8 182 188 7654803
Hannan MT Felson DT Pincus T Analysis of the discordance between radiographic changes and knee pain in osteoarthritis of the knee J Rheumatol 2000 27 1513 1517 10852280
Chatain F Robinson AH Adeleine P Chambat P Neyret P The natural history of the knee following arthroscopic medial meniscectomy Knee Surg Sports Traumatol Arthrosc 2001 9 15 18 11269578 10.1007/s001670000146
Englund M Roos EM Roos HP Lohmander LS Patient-relevant outcomes fourteen years after meniscectomy: influence of type of meniscal tear and size of resection Rheumatology (Oxford) 2001 40 631 639 11426019 10.1093/rheumatology/40.6.631
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Int J Behav Nutr Phys ActThe International Journal of Behavioral Nutrition and Physical Activity1479-5868BioMed Central London 1479-5868-2-121615940410.1186/1479-5868-2-12ResearchGender differences in perceived environmental correlates of physical activity Bengoechea Enrique Garcia [email protected] John C [email protected] Kerry R [email protected] Alberta Centre for Active Living, Faculty of Physical Education and Recreation E-424 Van Vliet, University of Alberta, Edmonton, Alberta, Canada2 Faculty of Physical Education and Recreation, E-424 Van Vliet, University of Alberta, Edmonton, Alberta, Canada3 Department of Health and Sport Studies, University of Iowa, Iowa City, USA2005 13 9 2005 2 12 12 15 4 2005 13 9 2005 Copyright © 2005 Bengoechea et al; licensee BioMed Central Ltd.2005Bengoechea 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
Limited research has been conducted on gender differences in perceived environmental correlates of physical activity (PA). The purpose of this study was to explore the potential role of gender in the link between perceived environment and PA.
Methods
Using a telephone-administered survey, data was collected on leisure time physical activity (LTPA), perceptions of the neighbourhood environment, and self-efficacy in a representative sample of 1209 adults from the province of Alberta, Canada. LTPA was regressed on ten measures of perceived neighbourhood environment and self-efficacy in a series of logistic regressions.
Results
Women were more likely than men to perceive their neighbourhood as unsafe to go for walks at night (χ2 = 67.46, p < 0.001) and to report seeing people being active in their neighbourhood (χ2 = 6.73, p < 0.01). Conversely, women were less likely to perceive easy access to places for PA (χ2 = 11.50, p < 0.01) and availability of places to buy things within easy walking distance from home (χ2 = 4.30, p < 0.05). Adjusting for age, education, income, and place of residence, access to places for PA (OR = 2.49) and interesting things to look at in the neighbourhood (OR = 1.94), were associated with higher levels of LTPA in men. Access to places for PA (OR = 2.63) and reporting seeing people being active (OR = 1.50) were associated with increased LTPA among women. After controlling for sociodemographic variables and self-efficacy, the presence of shops and places to buy things within easy walking distance from home (OR = 1.73), interesting things to look at in the neighbourhood (OR = 1.65), and access to places for PA (OR = 1.82) were associated with higher levels of LTPA in men. Among women, no significant relationships were observed between perceived environment and LTPA after adjusting for self-efficacy.
Conclusion
The results provide additional support for the use of models in which gender is treated as a potential moderator of the link between the perceived environment and PA. Further, the results suggest the possibility of differential interventions to increase PA based on factors associated with gender.
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Background
A growing body of scientific evidence has brought to public attention the negative effects of physical inactivity on health and the benefits of a physically active lifestyle [1,2]. Despite the well-documented physical, psychological, and social benefits of regular physical activity (PA) [3,4], physical inactivity remains pervasive. It is estimated that upwards of two-thirds of the industrialized world does not achieve minimum PA guidelines [5,2]. Physical inactivity thus constitutes a major public health concern [2] with related social and economic costs [6,7].
In an effort to solve the PA participation problem, research in the past two decades has employed theoretical perspectives to better understand the factors that enhance and detract from PA participation. In particular, social cognitive models that emphasize the interaction of intrapersonal factors, micro-environmental influences and PA behavior, have gained empirical support. This has allowed researchers to identify cognitive (e.g., efficacy beliefs), affective, and social influences on the individual's choice to be active and to aid in the development of interventions to increase PA levels [8]. Despite being identified as contributing toward behavior change, such individually-focused factors have generally been found to account for a modest proportion of the variance in PA behavior.
In recent years, it has been suggested that wider use of ecological models, which emphasize PA as being the result of multiple influences (i.e., intrapersonal, interpersonal, environmental, organizational and policy levels), hold great promise for addressing the PA participation problem [8-10]. The foregoing is underscored by significant changes observed over the past 50 years in the North American environment which promote decreased PA at the level of the individual. For example, in the United States, while daily vehicle miles traveled per person increased by 224% between 1950 and 2000, the proportion of trips to work by walking decreased 71% between 1960 and 2000 [11]. Transport systems and urban design (e.g., number and quality of pathways and cycleways, availability of buses and bus stops) play an important role in supporting or discouraging PA [12]. Urban sprawl increases distances between differential land use so that roads are more available than cycling paths or sidewalks [13-15].
Consistent with an ecological perspective [9,16], researchers have attempted to document and understand how different aspects of the physical environment may influence the extent to which individuals are more or less physically active [17-19]. Distribution and quality of local sport and recreational facilities, community clubs and churches, as well as features of the physical environment have all been shown to be associated with PA participation [20,21].
Researchers have begun to use models that allow selected demographic and personal characteristics such as socioeconomic status [22,23], gender [24-26] and weight status [27] to act as potential moderators of the effect of the perceived environment rather than as confounding variables. This is also consistent with an ecological perspective, which posits that there are interactions among levels in the system linking individuals with their environments [9,16].
Understanding the role of gender in the relationship between the perceived environment and PA participation is of particular importance since women typically exhibit lower levels of PA than their male counterparts [24,28]. Research that has attempted to elucidate gender differences in PA determinants has also revealed that women may face different barriers (e.g., lack of time due to multiple roles, perceptions of safety, and environmental access) than men, which can limit their PA participation [29,30]. For instance, in a survey of 3,032 U.S. adults [16], psychological factors such as mastery, exercise self-efficacy, and perceived control over health explained educational differences in PA among women.
Despite the emerging interest in the role of the environment and PA participation, few studies have systematically explored differences between women and men in terms of correlates of PA in general, and environmental correlates in particular. Among the exceptions is a recent study [24] that prospectively examined associations of changes in perceptions of local environmental attributes with changes in neighbourhood walking. Results revealed contrasting findings for men and women who reported traffic as less of a problem, with men being 61% less likely to increase walking. Women, on the other hand, were 76% more likely to have done so. Another finding was that men reporting positive changes in aesthetics and convenience were twice as likely to increase their walking. Women who reported positive changes in convenience were more than twice as likely to have increased their walking. In a related study [25], neighborhood walking was associated with high ratings of "aesthetics", "convenience" of facilities and "access" to facilities in men, and with high ratings of "convenience" in women. "Access", on the other hand, was negatively associated with neighborhood walking in women. Finally, in one recent study examining the role of gender on the link between PA and the perceived environment [26], perceptions of safety during day time and convenience (e.g., having shops within walking distance) were positively associated with walking among women. Having a park/open space within walking distance was the only environmental dimension associated with walking in men. Furthermore, there was evidence of substantial differences in perceptions of the environment between genders for both low and high walking groups.
These studies contribute to the literature that documents significant cross-sectional associations of perceived environmental attributes with PA [31]. However, more studies are needed to further elucidate relevant gender differences in PA participation [24]. In particular, little is known yet about how gender may interact with perceptions of the environment in order to influence PA participation. For instance, in relation to PA, it is likely self-efficacy beliefs vary by gender [16] and thus may account for much of any gender differences in perceived environment. Furthermore, no studies have been published on how perceptions of neighbourhood environment relate to PA in Canada. Consequently, the purposes of this study were to determine: a) if gender differences in perceptions of the physical environment exist; b) how these differences may have an impact on leisure time physical activity (LTPA) patterns of individuals in a representative sample of adults from the Province of Alberta; and c) if these gender differences are explained by PA self-efficacy.
Methods
Design and Sample
Participants included a representative sample of 1,209 adults (Males = 600; Females = 609) aged 18 years and over residing in the Province of Alberta, Canada. Data were collected by telephone interview between October 29, 2002, and December 1, 2002. Three separate subsamples represented Edmonton, Calgary, and the rest of the province. A random-digit dialling approach ensured that respondents had an equal chance of being contacted whether or not their household was listed in a telephone directory. Approximately 54% of the total number of valid households responded to the survey. A random sample of 1,209 is considered accurate within +/- 2.8, 19 times out of 20.
Measures
Leisure-Time Physical Activity
The Godin Leisure-Time Exercise Questionnaire [32] was used to estimate LTPA levels. Self-reported weekly frequencies of strenuous, moderate, and light activities were multiplied by their estimated value in METs (nine, five, and three respectively). Total weekly LTPA was calculated by adding the products of the separate components. Albertans were considered sufficiently physically active if they expended 38 METs a week for men or 35 METs a week for women. These cutoff values were derived based upon the work of several researchers [33-35]. According to Jacobs et al. [34], these values equal 300–400 MET-minutes per day. This number of MET-minutes is equivalent to a weekly energy expenditure of 2,000 kcals [33]. An energy expenditure of 2,000 kcals or more per week is associated with a reduced risk of heart disease [35]. Based on this criterion, we created a dichotomous variable for LTPA: inactive or active.
Perceived environment
Nine questions from the International Physical Activity Prevalence Study Environmental Survey Module [36] were used to assess respondents' perceptions of neighbourhood characteristics that may influence LTPA. The module consists of a series of items that reflect current thinking in assessment of environmental factors for walking and bicycling in one's neighbourhood and for which reliability and validity have been assessed [36]. Since walking (82%) is the primary source of LTPA among Canadians and bicycling is reported by a significant proportion (45%) of the population [37], the environment scale was deemed to be an appropriate measure of neighbourhood influences on LTPA.
Specifically, respondents were asked to indicate how much they agree or disagree with the following items, on a 4-point scale where 1 means strongly disagree and 4 means strongly agree: "Many shops, stores, or other places to buy things I need are within easy walking distance of my home"; "There are sidewalks on most of the streets in my neighbourhood"; "In and around my neighbourhood, there are facilities for bicycling such as special bicycle lanes, bicycle paths, and shared use trails for cyclists and pedestrians"; "My neighbourhood has several free or low cost recreation facilities, such as parks, walking trails, bike paths, playgrounds, and recreation centres"; "The crime rate in my neighbourhood makes it unsafe to go for walks at night"; "There is so much traffic on the streets that it makes it difficult or unpleasant to walk in my neighbourhood"; "I see many people engaging in physical activity in my neighbourhood doing things like walking, jogging, cycling, or playing sports and active games"; "There are many interesting things to look at while walking in my neighbourhood." In addition, respondents were asked to rate on the same 5-point scale their agreement with the following statement: "I have easy access to places where I can get physical activity." For the purposes of this study, the responses to each of the environmental variables were collapsed into categories of disagree (1 or 2) or agree (3 or 4).
Sociodemographic
Sociodemographic variables included age (coded as continuous variable), education (less than secondary, secondary, post-secondary), annual household income (< $30,000;$30,000–60,000; > $60,000), and location (urban, rural).
Self-Efficacy
Based upon previous research [38], a self-efficacy score was derived by averaging the respondents' ratings on three items assessing their confidence in overcoming, respectively, fatigue, bad weather, and time constraints to participating in moderate LTPA. The Cronbach's alpha coefficient for these three items was 0.77.
Statistical Analyses
All data were cleaned and edited following standard quality control procedures. The analyses were conducted with the Statistical Package for the Social Sciences (SPSS), version 13.0. Data were weighted to account for the circumstance that the sample sizes of completed interviews for Edmonton, Calgary, and other Alberta, were not proportional to the Alberta population. Specifically, the area samples were multiplied by a weighting factor to account for the slight over representation of residents from Edmonton and Calgary and the slight under representation of residents from other parts of Alberta. However, according to the index of dissimilarity [39], little difference existed between the age groupings of our sample and that of the Alberta population [40]. In addition, the proportion of men and women in our sample was very similar to that of the Canadian and Alberta populations [40]. Thus, the sample adequately reflected the population from which it was drawn.
Descriptive statistics were calculated for sociodemographic variables, self-efficacy, and LTPA. Differences between women and men were tested with chi-square analysis, in the case of categorical variables, and t tests in the case of continuous variables. Gender differences in responses to each environmental item were tested with chi-square analysis. Because PA scores are typically not normally distributed [41], non-parametric procedures were adopted to analyse the LTPA data. Specifically, unconditional logistic regression analyses, with LTPA as the dependent variable, were used to calculate unadjusted and adjusted ORs and the 95% CIs for each environmental variable. To calculate the adjusted ORs, LTPA was regressed on the sociodemographic variables (age, education, income, and location) and, then, self-efficacy was entered on the second step. The decision about which variables to include in the regression analysis was guided by ecological models of PA [9,16] and other research showing relations between sociodemographic variables and LTPA [42-44]. Conducting separate analyses for males and females allowed us to examine unique patterns of association between the environmental variables and LTPA.
Results
Based on the cut-off values described earlier, 59.9% of men and 54.2% of women were considered physically active. As Table 1 also shows, men displayed significantly higher levels of self-efficacy (t = 6.02, p < 0.001) and had higher annual household income than women (χ2 = 43.46, p < 0.001). Men were also slightly younger than women (t = -2.33, p < 0.05). As seen in Table 2, women were more likely than men to perceive their neighbourhood as unsafe to go for walks at night (χ2 = 67.46, p < 0.001) and to report seeing people being active in their neighbourhood (χ2 = 6.73, p < 0.01). Conversely, women had lower perceptions of easy access to places for PA (χ2 = 11.50, p < 0.01) and of availability of places to buy things within easy walking distance from home (χ2 = 4.30, p < 0.05).
Table 1 Characteristics of Participants
Male (n = 600) Female (n = 609) χ2 or t
Age
Mean 41.4 43.6 -2.33*
SD 15.4 16.2
Education (% in each category) 0.96
Less than high school 12.9 11.8
High school 19.7 21.7
Postsecondary 67.4 66.5
Income (% in each category) 43.46***
<$30,000 9.3 23.3
$30,000–60,000 30.9 34.9
>$60,000 59.8 41.8
Location (% in each category) 0.64
Urban 81.7 83.4
Rural 18.3 16.6
Self-efficacy
Mean 7.3 6.5 6.02***
SD 2.3 2.4
LTPA (% active) 59.9 54.2 4.16*
* p < .05., ***p < 0.001
Table 2 Gender Differences in Perceptions of the Physical Environment
Male Female
Number % Number % χ2 (df, n)
Shops 4.30* 1, 1205
Disagree 202 33.8 240 39.5
Agree 396 66.2 367 60.5
Transit stop 2.09 1, 1178
Disagree 199 34.1 179 30.1
Agree 385 65.9 415 69.9
Sidewalks 0.57 1, 1176
Disagree 102 17.6 95 15.9
Agree 478 82.4 501 84.1
Free or low cost 0.01 1, 1198
Disagree 137 23.0 140 23.3
Agree 459 77.0 462 76.7
Crime 67.46*** 1, 1178
Disagree 506 85.3 378 64.6
Agree 87 14.7 207 35.4
Traffic 0.71 1, 1195
Disagree 482 81.1 476 79.2
Agree 112 18.9 125 20.8
Seeing people 6.73** 1, 1196
active
Disagree 170 28.6 133 22.1
Agree 424 71.4 469 77.9
Interesting things 0.26 1. 1203
Disagree 204 34.1 198 32.7
Agree 394 65.9 407 67.3
Easy access 11.50** 1, 1066
Disagree 76 14.2 118 22.2
Agree 118 85.8 413 77.8
* p < .05., **p < .01., ***p < 0.001.
The results revealed some interesting differences between men and women in terms of the perceived neighbourhood characteristics that are associated with an increased likelihood of being physically active. As table 3 shows, men who perceived many interesting things to look at while walking in their neighbourhood were 83% more likely to be physically active than those who did not. This association remained statistically significant even after adjusting for sociodemographic variables (OR = 1.94) and self-efficacy (OR = 1.65). When adjusting for sociodemographic variables and self-efficacy, perceptions of presence of shops and places to buy things within easy walking distance from home, were associated with increased LTPA among men as well (OR = 1.73). Among women, perceptions of availability of free or low cost recreational facilities were linked with higher levels of LTPA (OR = 1.53). However, this relationship did not remain statistically significant after adjusting for sociodemographic variables and self-efficacy. Seeing many people engaging in PA in their neighbourhood also increased the likelihood of being physically active in women (OR = 1.50). This association remained statistically significant after adjusting for sociodemographic variables (OR = 1.78) but not after self-efficacy was included in the model.
Table 3 Perceived Environmental Correlates of Being Active Versus Inactive
Male Female
Unadjusted OR (95% CI) Adjusted1 OR (95% CI) Adjusted2 OR (95% CI) Unadjusted OR (95% CI) Adjusted OR1 (95% CI) Adjusted OR2 (95% CI)
Shops
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 1.15 (0.81–1.62) 1.49 (0.95–2.34) 1.73 (1.06–2.84)* 1.08 (0.78–1.50) 1.06 (0.68–1.65) 0.84 (0.52–1.38)
Transit stop
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 0.94 (0.66–1.33) 1.30 (0.81–2.08) 1.16 (0.69–1.93) 0.96 (0.68–1.37) 0.69 (0.40–1.19) 0.59 (0.32–1.07)
Sidewalks
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 1.04 (0.67–1.61) 1.25 (0.61–2.59) 1.21 (0.54–2.71) 0.93 (0.59–1.44) 1.02 (0.46–2.25) 0.62 (0.26–1.49)
Bicycle facilities
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 1.12 (0.79–1.59) 1.12 (0.74–1.70) 0.90 (0.57–1.42) 1.31 (0.93–1.84) 1.24 (0.80–1.94) 1.12 (0.69–1.80)
Free or low cost
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 1.09 (0.74–1.61) 0.97 (0.60–1.57) 0.86 (0.51–1.45) 1.53 (1.05–2.24)* 1.43 (0.86–2.38) 1.18 (0.67–2.05)
Crime
Disagree 1.16 (0.73–1.85) 1.20 (0.70–2.04) 0.83 (0.46–1.50) 1.32 (0.94–1.86) 1.40 (0.90–2.15) 1.50 (0.94–2.40)
Agree 1.00 1.00 1.00 1.00 1.00 1.00
Traffic
Disagree 0.91 (0.60–1.39) 0.94 (0.57–1.54) 0.87 (0.51–1.50) 1.04 (0.70–1.54) 1.06 (0.66–1.72) 0.98 (0.58–1.67)
Agree 1.00 1.00 1.00 1.00 1.00 1.00
Seeing people active
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 1.17 (0.81–1.68) 1.20 (0.78–1.83) 1.17 (0.74–1.85) 1.50 (1.01–2.20)* 1.78 (1.08–2.91)* 1.59 (0.93–2.71)
Interesting things
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 1.83 (1.29–2.58)** 1.94 (1.302.88)** 1.65 (1.08–2.53)* 1.20 (0.85–1.69) 1.16 (0.76–1.77) 0.94 (0.59–1.50)
Easy access
Disagree 1.00 1.00 1.00 1.00 1.00 1.00
Agree 2.40 (1.46–3.93)*** 2.49 (1.43–4.32)** 1.82 (1.00–3.31)* 2.63 (1.72- 2.72 (1.56- 1.61 (0.87–2.96)
* p < .05., **p < .01., ***p < 0.001.
Note1: Adjusted by age, education, income, and location (urban vs. rural).
Note2: Adjusted by age, education, income, location, and self-efficacy.
Perceptions of easy access to places for PA were associated with higher levels of LTPA in both men and women (OR = 2.40 and OR = 2.63, respectively). For men, the relationship was still statistically significant after adjusting for sociodemographic variables (OR = 2.49) and self-efficacy (OR = 1.82). For women, the association was still significant after adjusting for sociodemographic variables (OR = 2.72), but not after self-efficacy was entered in the model.
Discussion
This study explored gender differences in perceptions of the physical environment and in environmental correlates of LTPA in a representative sample of adults from the province of Alberta. Our results are consistent with a growing body of literature that suggests the existence of a link between several dimensions of the perceived environment and self-reported LTPA [20,31]. Further adding to this literature, our analyses show that other studies may have overlooked an important moderating effect of gender in the relationship between the perceived environment and LTPA.
Unlike previous work concerned with gender differences in the association between PA and environmental variables [26], women and men in our study did not differ, at least to a statistically significant degree, in perceptions of traffic as a potential problem for walking. Consistent with this research [26], women were more concerned than men about the safety of walking at night. Nevertheless, perceptions of low safety were not significantly associated with lower levels of LTPA in women, which is in-line with results from previous studies assessing specific correlates of PA in women [45-47].
Several interesting differences emerged between women and men in terms of the dimensions of the perceived environment that were linked with increased LTPA participation. The perceptions that "there are many interesting things to look at while walking" and that "there are many places to buy things within easy walking distance from home", were significantly associated with higher levels of LTPA in men but not in women. This is somewhat inconsistent with a recent study [24] that found both men and women reporting positive changes over time in aesthetics and convenience in their neighbourhood were at least twice as likely to increase their walking. Likewise, another recent study [26] found that convenience (i.e., availability of shops within walking distance) was positively associated with high levels of walking (>150 mins/week) among women. One possible explanation for our findings is that perceived environments indeed have a differential impact on LTPA according to gender. Another possible explanation is that the role of perceived environment in relation to behaviour may be moderated by cultural and national factors that vary by country and region.
Conversely, since men reported higher levels of LTPA than women, interpretations that PA can affect perceptions of the environment are plausible as well [45]. Offering some support for this hypothesis, we found that men were higher than women in perceptions of easy access to places for PA and of availability of places to buy things within easy walking distance from home. Thus, it could be suggested that individuals who are physically active may be more likely to perceive that they have access to places where they can get PA, or to places where they can buy necessary things, and that they can reach by physically active means (e.g., walking) [48]. Alternatively, these findings may have been due to chance, thus replication of them is necessary. In any case, the unexpected results suggest that the interaction between environmental influences and individual difference characteristics may be more complex than previously thought.
Perceiving that one's neighbourhood has several free or low cost recreation facilities was associated with higher levels of LTPA in women but not in men. This finding makes some sense when taking into account that the average annual household income of the women in our sample was lower than that of men. Studies have found that low amounts of leisure-time PA are strongly associated with low income [49,50], low education [51], and low socio-economic status [52,53]. Moreover, the lowest participation rates have been found amongst the poor and women of child-rearing age, many of whom are the same people [54]. In fact, low-income women identify a lack of access to PA in their community as a major factor inhibiting the development of healthy lifestyles for themselves and their families [54]. Thus in the case of our results, having access to places where one can participate in PA for free or at a low cost may have been an incentive (i.e., facilitator) for women to get involved in PA.
Likewise, seeing many people being active in one's neighbourhood was associated with increased levels of LTPA only in women. The positive impact of physically active role models on women's participation patterns has been documented in other studies investigating the influence of characteristics of the neighbourhood environment on PA [45,46]. Bandura's [55] social cognitive theory explains how role modelling can influence behaviour by enhancing an individual's sense of self-efficacy. Women in our sample had lower self-efficacy to overcome barriers to PA than men. Consistent with previous research showing the importance of self-efficacy on women's LTPA [16,47], this may help explain why physically active role models seem to have been more influential for women in our study. However, the circumstance that neither having access to places where one can participate in PA for free or at neither a low cost, nor seeing many people being active in one's neighbourhood reached statistical significance when adjusted by self-efficacy points to an interesting phenomenon. It seems indeed as if self-efficacy to overcome barriers to PA participation "in spite of" an environment that may facilitate or hinder participation was more influential among women in our sample than self-efficacy "because of" a supportive environment (i.e., one in which role models and low cost facilities are available).
Having easy access to places for PA was the environmental dimension most strongly associated with LTPA in both men (even after adjusting for sociodemographic factors and self-efficacy) and women (even after adjusting for sociodemographics). Once again, the influential role of self-efficacy on women's LTPA in our sample was evidenced when adjusting for this variable had the effect of eliminating the statistical significance of the association. Access to places for PA has previously been linked with increased levels of PA in some studies [48,56] but not in others [46,47]. As Huston et al. [48] have discussed, this measure may appear more strongly related with PA simply because it provides a more global indicator of whether or not an individual has access to suitable places for PA, whether in their neighbourhood or not. Alternatively, it could be that individuals who are physically active may be more likely to perceive that they have access to places or facilities where they can get PA [48]. As our study shows, accounting for gender differences (e.g., in self-efficacy levels) may help to partially explain the discrepancy in results across studies assessing the impact of perceptions of access to places for PA. Thus, interventions should be designed to increase women's self-efficacy to participate in PA when environmental supports may not be readily available. Our results also suggest that in order to encourage women to become more physically active, interventions could be designed to increase awareness and use of environmental supports [57] such as places were people can engage at low or no cost in PA while seeing socially and economically similar others doing the same.
This study has several limitations. First, our data is cross-sectional and therefore we cannot make causal inferences. We also relied on a telephone survey format, which may have resulted in some segments of the population (e.g., low income individuals lacking access to telephones) being underrepresented. Additionally, we used self-report measures of both LTPA and perceptions of the physical environment. It is thus possible that the data may have been subject to biases. Since it is not clear yet whether the actual or perceived environment is more influential [9,45,58], it is important that future studies include assessments of both dimensions in their designs.
Conclusion
To date, few studies have explored gender differences in perceptions of the environment and how these differences may have an impact on PA patterns of individuals. We found several gender relevant differences in the ways individuals perceive their physical environments and on the dimensions of these environments that are associated with LTPA. Thus, the results from this study add to the knowledge base of gender differences in environmental correlates of PA. Our findings provide further support for the need of using models in which gender is treated as a potential moderator of the link between the perceived environment and PA. Further, these results suggest the possibility of designing interventions to increase PA that address differentially both structural (e.g., income disparities) and social cognitive (e.g., self-efficacy) factors typically associated with gender.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
EGB participated in the conception and design of the study, performed the statistical analyses and drafted the manuscript. JCS participated in the conception and design of the study, took a coordination role, and helped to draft the manuscript. KRM coordinated the design of the survey and helped to draft the manuscript. All authors read and approved the final manuscript.
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Ruhling RO Why not exercise? Quest 2004 56 208 225
US Department of Health and Human Services Physical activity and health: A report of the Surgeon General 1996 Atlanta Centers for Disease Control and Prevention
Bouchard C Shephard RJ Stephens T Physical activity fitness and health: International proceedings and consensus statement 1994 Champaign: Human Kinetics
Gauvin L Spence JC Anderson S Rippe JM Exercise and psychological well-being in the adult population: Reality or wishful thinking? Textbook of medicine exercise nutrition and health 1999 Malden: Blackwell 957 966
Oja P Descriptive epidemiology of health-related physical activity and fitness Res Q Exerc Sport 1995 214 303 312 8775586
Colditz GA Economic costs of obesity and inactivity Med Sci Sports Exerc 1999 663 667 10.1097/00005768-199911001-00026
Katzmarzyk PT Gledhill N Shephard RJ The economic burden of physical inactivity in Canada CMAJ 2000 163 1435 1440 11192648
King AC Stokols D Talen E Brassington GS Killingsworth Theoretical approaches to the promotion of physical activity: Forging a transdisciplinary paradigm Am J Prev Med 2002 15 25 12133734 10.1016/S0749-3797(02)00470-1
Spence JC Lee RE Toward a comprehensive model of physical activity Psychol Sport Exerc 2003 4 7 24 10.1016/S1469-0292(02)00014-6
Saelens BE Sallis JF Frank LD Environmental correlates of walking and cycling: Findings from the transportation, urban design, and planning literature Ann Behav Med 2003 25 80 91 12704009 10.1207/S15324796ABM2502_03
Brownson RC Boehmer TK Luke DA Declining rates of physical activity in the United States: What are the contributos? Ann Rev Public Health 2005 26 421 443 15760296 10.1146/annurev.publhealth.26.021304.144437
Frank LD Engelke PO Schmid TL Health and community design: The impact of the built environment on physical activity 2003 Washington: Island Press
Ewing R Schmid T Killingsworth R Zlot A Raudenbush S Relationship between urban sprawl and physical activity, obesity, and morbidity Am J Health Prom 2003 18 47 57
Frank LD Andresen MA Schmid TL Obesity relationships with community design, physical activity, and time spent in cars Am J Prev Med 2004 27 87 96 15261894 10.1016/j.amepre.2004.04.011
Frank LD Schmid TL Sallis JF Chapman J Saelens BE Linking objectively measured physical activity with objectively measured urban form Findings from SMARTRAQ Am J Prev Med 2005 28 117 125 15694519 10.1016/j.amepre.2004.11.001
Grzywacz JG Marks NF Social inequalities and exercise during adulthood: Toward an ecological perspective J Health Soc Behav 2001 42 202 220 11467253
Bauman A Smith B Stoker L Bellew B Booth M Geographical influences upon physical activity participation: Evidence of a 'coastal effect' Aust New Zeal J Public Health 1999 23 322 324
Corti B Donovan RJ Holman CDJ Factors influenceing the use of physical activity facilities: Results from qualitative research Health Prom J Aust 1997 7 16 21
Rutten A Abel T Kannas L Von Lengerke T Luschen G Diaz JA Vinck J Van der Zee J Self-reported physical activity, public health and perceived environment: Results from a comparative European study J Epidemiol Community Health 2001 55 139 146 11154254 10.1136/jech.55.2.139
Humpel N Owen N Leslie E Environmental factors associated with adults' participation in physical activity: A review Am J Prev Med 2002 22 188 199 11897464 10.1016/S0749-3797(01)00426-3
Sallis JF Kraft K Linton LS How the environment shapes physical activity: A transdisciplinary research agenda Am J Prev Med 2002 22 208 11897466 10.1016/S0749-3797(01)00435-4
Parks SE Housemann RA Brownson RC Differential correlates of physical activity in urban and rural adults of various socioeconomic backgrounds in the United States J Epidemiol Community Health 2003 57 29 35 12490645 10.1136/jech.57.1.29
van Lenthe FJ Brug J Mackenback JP Neighbourhood inequalities in physical activity: The role of neighbourhood attractiveness, proximity to local facilities and safety in the Netherlands Soc Sci Med 2005 60 763 775 15571894 10.1016/j.socscimed.2004.06.013
Humpel N Marshall AL Leslie E Bauman A Owen N Changes in neighborhood walking are related to changes in perceptions of environmental attributes Ann Behav Med 2004 27 60 67 14979864 10.1207/s15324796abm2701_8
Humpel N Owen N Leslie E Marshall AL Bauman AE Sallis JF Associations of location and perceived environmental attributes with walking in neighborhoods Am J Health Prom 2004 18 239 242
Foster C Hillsdon M Thorogood M Environmental perceptions and walking in English adults J Epidemiol Community Health 2004 58 924 928 15483308 10.1136/jech.2003.014068
Blanchard CM McGannon K Spence JC Rhodes RE Nehl E Baker F Bostwick J Social ecological correlates of physical activity in normal weight, overweight, and obese individuals Int J Obes 2005 29 720 726 10.1038/sj.ijo.0802927
Pinto BM Marcus BH Clark MM Promoting physical activity in women: The new challenges Am J Prev Med 1996 12 395 400 8909651
Eyler AA Personal, social, and environmental correlates of physical activity in rural Midwestern white women Am J Prev Med 2003 86 92 14499814 10.1016/S0749-3797(03)00169-7
Jaffee L Lutter JM Rex J Hawkes C Buccacio P Incentives and barriers to PA for working women Am J Health Prom 1999 13 215 218
Duncan MJ Spence JC Mummery WK Perceived environment and physical activity: A meta-analysis of selected environmental characteristics Int J Behav Nutr Phys Act 2005 2 11 16138933 10.1186/1479-5868-2-11
Godin G Shephard RJ A simple method to assess exercise behavior in the community Can J Appl Sport Sci 1985 10 141 146 4053261
Elosúa R García M Aguilar A Molina L Covas MI Marrugat J Validation of the Minnesota Leisure Time Physical Activity Questionnaire In Spanish Women Med Sci Sports Exerc 2000 32 1431 1437 10949009 10.1097/00005768-200008000-00011
Jacobs DR Ainsworth BE Hartman TJ Leon AS A simultaneous evaluation of 10 commonly used physical activity questionnaires Med Sci Sports Exerc 1993 25 81 91 8423759
Paffenbarger RS Wing AL Hyde RT Physical activity as an index of heart attack risk in college alumni Am J Epidemiol 1978 108 161 175 707484
International Physical Activity Prevalence Study Environmental Survey Module
Candian Fitness Lifestyle Research Institute Physical Activity Monitor 2000 Ottawa, ON
Rodgers WM Sullivan MJL Task, coping, and scheduling self-efficacy in relation to frequency of physical activity J Appl Soc Psych 2001 31 741 753
Duncan OD Duncan B Residential distribution and occupational stratification Am J Sociol 1955 60 493 10.1086/221609
Statistics Canada 2001 Census of Canada 2002 Ottawa ON
Thomas JR Thomas K Welk GJ Physical activity data: Odd distributions yield strange answers Physical activity assessments for health-related research 2002 Champaign, IL: Human Kinetics 73 80
Clark DO Racial and educational differences in physical activity among older adults Gerontologist 1995 35 472 480 7557517
Potvin L Gauvin L Nguyen NM Prevalence of stages of change for physical activity in rural, suburban and inner-city communities JComm Health 1997 22 1 13 10.1023/A:1025161522683
Powell LM Slater S Chaloupka FJ The relationship between community physical activity settings and race, ethnicity and socioeconomic status Evidence-Based Prev Med 2004 1 135 144
King AC Castro C Wilcox S Eyler AA Sallis JF Brownson RC Personal and environmental factors associated with physical inactivity among different racial-ethnic groups of U.S. middle-aged and older-aged women Health Psychol 2000 19 354 364 10907654 10.1037//0278-6133.19.4.354
Wilcox S Castro C King AC Housemann R Brownson RC Determinants of leisure time physical activity in rural activity compared with urban older and ethnically diverse women in the United States J Epidemiol Community Health 2000 54 667 672 10942445 10.1136/jech.54.9.667
Eyler AA Matson-Koffman D Young DR Wilcox S Wilbur J Thompson JL Sanderson B Evenson KR Quantitative study of correlates of physical activity in women from diverse racial/ethnic groups: The Women's Cardiovascular Health Network Project – summary and conclusions Am J Prev Med 2003 93 103 14499815 10.1016/S0749-3797(03)00170-3
Huston SL Evenson KR Bors P Gizlice Z Neighborhood environment, access to places for activity, and leisure-time physical activity in a diverse North Carolina population Am J Health Prom 2003 18 58 69
Stachenko SJ Reeder BA Lindsay E Donovan C Lessard R Smoking prevalence and associated risk factors in Canadian adults Canadian Medical Association Journal 1992 146 Canadian Heart Health Surveys Research 1989 1996 1596848
Steenland K Passive smoking and the risk of heart disease JAMA 1992 267 94 99 1727204 10.1001/jama.267.1.94
Sternfeld B Ainsworth BE Quesenberry JCP Physical activity patterns in a diverse population of women Prev Med 1999 28 313 323 10072751 10.1006/pmed.1998.0470
Blanksby B Anderson M Douglas GA Recreational patterns, body composition and socioeconomic status of Western secondary school students Ann Human Biol 1996 23 101 112 8702209
Mensink GBM Losse N Oomen CM Physical activity and its association with other lifestyle factors Eur J Epidemiol 1997 13 771 778 9384266 10.1023/A:1007474220830
Frisby W Crawford S Dorer T Reflections on participatory action research: The case of low-income women accessing local physical activity services J Sport Management 1997 11 8 28
Bandura A The explanatory and predictive scope of self-efficacy theory J Soc Clin Psychol 1986 4 359 373
Brownson RC Baker EA Housemann RA Brennan LK Bacak SJ Environmental and policy determinants of physical activity in the United States Am J Public Health 2001 91 1995 2003 11726382
Addy CL Wilson DK Kirtland KA Ainsworth BE Sharpe P Kimsey D Associations of perceived social and physical environmental supports with physical activity and walking behavior Am J Public Health 2004 94 440 443 14998810
Sallis JF Johnson MF Calfas KJ Caparosa S Nichols JF Assessing perceived physical environmental variables that may influence physical activity Res Q Exerc Sport 1997 68 345 351 9421846
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Lipids Health DisLipids in Health and Disease1476-511XBioMed Central London 1476-511X-4-191619120010.1186/1476-511X-4-19ResearchPlasma lipid profiles and risk of cardiovascular disease in occupational lead exposure in Abeokuta, Nigeria Ademuyiwa Oladipo [email protected] Regina Ngozi [email protected] Florence [email protected] Olugbenga [email protected] Department of Biochemistry, University of Agriculture, Abeokuta, Nigeria2 Department of Biochemistry, Olabisi Onabanjo University, Ikenne, Nigeria2005 28 9 2005 4 19 19 31 8 2005 28 9 2005 Copyright © 2005 Ademuyiwa et al; licensee BioMed Central Ltd.2005Ademuyiwa 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 order to investigate the effects of lead exposure on risk of cardiovascular disease during occupational exposure to this metal, plasma cholesterol and its fractions as high-density liporotein (HDL), low-density liporotein (LDL) and triglyceride were determined in various artisans in Abeokuta, Nigeria who have been shown to be occupationally exposed to lead and these were related to blood lead levels. Increased risk of cardiovascular disease was observed in the artisans. Total cholesterol in the artisans was between 1.5 and 2.0 times higher in the artisans than that present in controls while LDL cholesterol was between 1.6 and 2.4 times higher in the artisans when compared with control subjects [p < 0.001]. HDL cholesterol and triglyceride levels were not affected [p > 0.05]. A significant positive correlation was observed between blood lead and total cholesterol on one hand [r = 0.372; p = 3.0 × 10-5] and blood lead and LDL cholesterol on the other hand [r = 0.283; p = 0.001]. LDL/HDL cholesterol ratio was also higher in the artisans when compared with control. Blood pressure (systolic and diastolic) and other anthropometric parameters were not significantly different between the artisans and the control subjects [p > 0.05]. Results suggest that lead exposure increases cholesterol synthesis and transport to peripheral tissues whereas reverse cholesterol transport to the liver is not affected.
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Introduction
Studies in both humans and animals indicate that lipid metabolism is altered in chronic lead exposure [1-6]. The pathophysiological mechanisms involved in this lead-induced alterations are not completely understood. Lead has been shown to accelerate lipid oxidation in the presence of hemoglobin or Fe2+ [7,8]. Lead was also shown to enhance Fe2+-initiated lipid oxidation in liposomes, erythrocytes, microsomal fractions and rat brain homogenates [1,7,9]. Altered fatty acid composition of erythrocyte membranes has also been demonstrated in chronic lead exposure [5,10,11]. It has also been demonstrated that lead exposure affects levels of galactolipid metabolic enzymes in the developing rat brain resulting in myelin defects [3]. All these observations are largely more suggestive than conclusive about lead-induced alterations in lipid metabolism.
Lead poisoning is presently becoming the most common disease of environmental origin and is increasing very rapidly in developing countries [12-16]. While lead exposure studies is a well-trodden subject in economically advanced countries [17-22], few published reports exist about lead poisoning and its associated effects in developing countries like Nigeria [12-14,16].
During the past years, numerous reports have appeared in the literature indicating that abnormal blood lipid levels like total cholesterol and other lipids and lipoproteins predispose individuals to atherosclerosis and cardiovascular diseases [23-27]. We have previously reported on chronic lead poisoning and its associated biochemical effects in auto-mechanics, petrol station attendants and some other artisans in Abeokuta, Nigeria [12-14,16]. To the best of our knowledge, data regarding the distribution of blood lipids and the risk of cardiovascular disease associated with this distribution in those who are occupationally exposed to lead in Nigeria, are lacking. In order to gain an insight into lead exposure and its effects on lipid profiles, we investigated the distribution of blood lipids in various artisans in Abeokuta, Nigeria, who have been shown to be occupationally exposed to lead.
Subjects and methods
Study population
A total of 110 male subjects participated in the study. Control subjects were made up of staff and students of University of Agriculture, Abeokuta, Nigeria, while lead-exposed subjects comprised of different artisans located in two mechanic workshops in the southern and northern parts of the city of Abeokuta, Nigeria. It is typical of mechanic workshops in Nigeria to find other groups of artisans located in these workshops in addition to the auto-mechanics, thereby complementing each other's services. In these workshops, there is always a preponderance of auto-mechanics. After explaining the objectives and the requirements of the study to them, a total of 92 artisans, including 50 auto-mechanics, consented to participate in the study. Seven male petrol station attendants from a petrol station in the southern part of the city were also included among the occupationally exposed subjects. Table 1 summarises the study population.
Table 1 Study population
Subject Number
Control 11
Auto-mechanics 50
Auto-electricians 8
Battery chargers 2
Drivers 2
Painters (Vehicle) 7
Panel beaters 15
Petrol station attendants 7
Upholsterers 4
Spare parts/Oil sellers 2
Welders 2
Anthropometric measurements and body composition
The weight of each subject was measured to the nearest 0.1 kg with a battery operated scale while the subjects were dressed in their underwear and height was measured to the nearest centimeter with the aid of a portable stadiometer. From these data, Body Mass Index (BMI), Body Surface Area (BSA), Body Fat Mass Index (BFMI), Fat Free Mass Index (FFMI),), Lean Body Mass (LBM), Body Fat Mass (BFM), Total Body Water (TBW), Intracellular Fluid (IF) and Extracellular Fluid (EF), were calculated [28-30,35]. Blood pressure was measured two times on the left arm in each subject in a supine position with a traditional sphygmomanometer (Cacosson Products, England) [31]. Each measurement was spaced twenty minutes apart and was usually performed before collection of blood samples. The average of the two measurements was used for all analyses.
Blood analyses
Venous blood samples were obtained between 8.00 a.m and 10.00 a.m from the subjects after an overnight fast. Aliquots of blood samples were separated for lead analysis and the remaining blood samples were centrifuged to separate plasma and red blood cells. Plasma concentrations of total cholesterol, LDL-cholesterol and triglycerides were determined with commercial kits (Randox Laboratories, Crumlin, England). HDL-cholesterol was determined in plasma with the same commercial kits for total cholesterol after very low density lipoproteins (VLDL) and low density lipoproteins (LDL) were precipitated with heparin-MnCl2 solution [32]. Blood lead analysis was performed using atomic absorption spectrophotometry. Details of this have been given elsewhere [13,14].
Statistical protocol
Results are expressed as mean ± S.D. One-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test were used to analyse the results with p < 0.05 considered significant [33]. The relationships between blood lead levels and plasma lipids and the anthropometric parameters were also analysed using Pearson correlations [33].
Results
Demographic and anthropometric characteristics
The demographic and anthropometric characteristics of the subjects are depicted in Table 2. Their ages ranged between 18 and 66 years. The years of experience on the job for each group also shows a wide variation ranging from 0.7 to 53 in the auto-mechanics and 1.5 to 5 in the petrol station attendants. With the exception of the drivers and spare parts dealers, majority of the artisans spend an average of 11 hours per day in their workshops. The petrol station attendants on the other hand, spend an average of 14.3 hours with their nozzle.
Table 2 Some demographic and anthropometric characteristics of the subjects. Values are mean ± S.D.
Subjects
Parameters Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (Vehicle) Panel beaters Petrol station attendants Upholsterers Spare parts/Oil sellers Welders
Age (years) 26.00 ± 6.65 28.02 ± 10.72 32.00 ± 10.17 27.00 ± 7.07 39.50 ± 12.02 34.00 ± 19.56 29.53 ± 13.03 28.71 ± 3.99 29.25 ± 8.96 30.50 ± 0.70 37.50 ± 3.54
Job experience (years) - 11.64 ± 10.92 13.25 ± 11.18 7.50 ± 0.71 4.00 ± 2.83 15.71 ± 19.96 12.47 ± 10.45 3.57 ± 1.54 12.38 ± 11.46 6.50 ± 2.12 25.50 ± 0.71
Hours spent/day in workshop 8.64 ± 0.92 11.62 ± 0.78 11.25 ± 1.16 11.50 ± 0.71 10.00 ± 0.00 11.14 ± 0.69 11.20 ± 0.68 14.29 ± 3.25 11.25 ± 1.50 10.50 ± 2.12 11.50 ± 0.71
Height (m) 1.69 ± 0.05a 1.67 ± 0.13a 1.72 ± 0.05a 1.70 ± 0.02a 1.68 ± 0.04a 1.73 ± 0.08a 1.72 ± 0.03a 1.71 ± 0.08a 1.69 ± 0.07a 1.68 ± 0.02a 1.73 ± 0.04a
Weight (kg) 60.64 ± 8.10a 62.44 ± 11.54a 62.38 ± 7.89a 57.50 ± 0.71a 67.00 ± 11.31a 54.33 ± 2.52a 63.71 ± 10.16a 68.00 ± 7.26a 58.25 ± 11.62a 58.00 ± 4.24a 67.50 ± 14.85a
BMI (kg/m2) 21.34 ± 3.07a 21.78 ± 3.76a 20.88 ± 2.06a 20.02 ± 0.25a 23.66 ± 2.81b 18.15 ± 1.72a 21.71 ± 3.73a 23.49 ± 3.39b 20.62 ± 4.34a 20.66 ± 0.99a 24.84 ± 0.90b
BSA (m2) 1.69 ± 0.11a 1.71 ± 0.14a 1.72 ± 0.10a 1.66 ± 0.03a 1.76 ± 0.16a 1.65 ± 0.08a 1.78 ± 0.12a 1.79 ± 0.10a 1.64 ± 0.20a 1.41 ± 0.29b 1.88 ± 0.09c
BFMI (kg/m2) 3.81 ± 1.45a 4.08 ± 2.29a 3.24 ± 1.19a 3.29 ± 0.72a 5.17 ± 1.92a 1.81 ± 0.91a 8.57 ± 4.39c 6.04 ± 2.30b 10.28 ± 8.24d 3.24 ± 0.60a 6.35 ± 0.21b
FFMI (kg/m2) 17.53 ± 1.63a 17.44 ± 3.64a 17.48 ± 0.84a 16.73 ± 0.47a 18.50 ± 0.91a 16.35 ± 0.81a 15.88 ± 3.75a 17.44 ± 1.08a 10.34 ± 12.45b 17.43 ± 0.40a 18.46 ± 1.08a
LBM (kg) 49.88 ± 4.66a 50.67 ± 5.78a 51.52 ± 4.72a 47.64 ± 2.03a 53.21 ± 6.61a 47.13 ± 2.37a 52.08 ± 4.92a 50.43 ± 3.14a 47.72 ± 8.42a 48.44 ± 2.81a 55.74 ± 6.56a
BFM (kg) 10.75 ± 3.82a 11.63 ± 6.75a 9.55 ± 3.70a 9.47 ± 1.88a 14.74 ± 6.17a 5.29 ± 2.16a 25.32 ± 12.80c 15.21 ± 3.77a 28.12 ± 22.80c 9.09 ± 1.92a 18.94 ± 0.06b
TBW (litre) 36.86 ± 2.76a 37.19 ± 3.78a 37.69 ± 2.82a 35.90 ± 0.85a 35.80 ± 1.70a 35.90 ± 2.01a 39.34 ± 3.82a 39.47 ± 2.74a 35.63 ± 5.01a 35.80 ± 1.70a 41.55 ± 2.33a
IF (litre) 20.26 ± 1.51a 20.56 ± 2.04a 20.73 ± 1.45a 19.85 ± 0.35a 21.25 ± 2.33a 19.73 ± 1.11a 21.64 ± 2.07a 21.67 ± 1.43a 19.60 ± 2.74a 19.75 ± 0.92a 22.85 ± 1.34a
EF (litre) 16.58 ± 1.24a 16.58 ± 2.05a 16.96 ± 1.19a 16.20 ± 0.28a 17.35 ± 1.91a 16.17 ± 0.90a 17.73 ± 1.68a 17.71 ± 1.18a 16.03 ± 2.27a 16.15 ± 0.78a 18.70 ± 1.13a
BP (systolic) (mm Hg) 123.64 ± 15.88a 112.82 ± 23.71a 134.63 ± 10.85a 128.00 ± 11.31a 115.00 ± 12.73a 113.14 ± 33.86a 114.60 ± 25.18a 114.29 ± 6.78a 120.00 ± 14.24a 117.50 ± 6.36a 110.50 ± 14.85a
BP (diastolic) (mm Hg) 81.09 ± 19.94a 69.12 ± 16.67a 75.88 ± 10.34a 61.50 ± 16.26a 65.50 ± 2.12a 60.71 ± 15.00a 66.20 ± 15.19a 79.57 ± 7.04a 67.00 ± 11.34a 73.00 ± 1.41a 77.00 ± 24.04a
Values in a row having no letter (a – d) in common are significantly different from each other (p < 0.05).
Analyses of the anthropometric parameters determined in the subjects revealed unsystematic statistically significant differences between the control and the artisans. BMI was higher in drivers, petrol station attendants and welders, but the increase was not statistically significant (p = 0.512). BSA was decreased in spare parts/oil sellers, whereas it increased in the welders (p < 0.05). BFMI was significantly increased in the panel beaters, petrol station attendants, upholsterers and welders (p < 0.05), whereas FFMI was decreased in the upholsterers (p < 0.05). While BFM was increased in panel beaters, upholsterers and welders (p < 0.05), no statistical significant differences were observed between control and the artisans in LBM, TBW, IF and EF.
Blood pressure
The mean blood pressures of the subjects as depicted in Table 2 indicate that the systolic and diastolic blood pressures of the subjects were in the normotensive range. The auto-electricians however had a borderline high systolic blood pressure (134.63 ± 10.85 mmHg). However, statistical analyses revealed no significant difference in both the systolic and diastolic blood pressures between the control and the artisans (p > 0.05).
Plasma lipid profiles
The mean values of the investigated blood lipids in the subjects are depicted in Table 3. With a few exceptions, the mean values of these lipids were within the reference ranges prescribed by the American Heart Association [26]. Total cholesterol was slightly higher in the battery chargers while LDL cholesterol was above the reference range in the battery chargers, drivers and petrol station attendants. When the values of the investigated lipids in the artisans were however compared with the controls, certain patterns emerged. With the exception of the painters, the mean plasma concentrations of total cholesterol were significantly higher in the artisans compared with control subjects (p < 0.001). Of the artisans, the battery chargers had the highest plasma total cholesterol (203.50 ± 48.51 mg/dl), followed by the spare parts/oil sellers and drivers respectively. Quantitatively, cholesterol levels of the artisans were between 1.5 and 2.0 times higher than that of the controls. Although there were statistically significant differences in the LDL-cholesterol between control and the artisans, the increases in this parameter exhibited a different pattern compared with total cholesterol. While LDL cholesterol was not significantly different in the painters, upholsterers, spare parts/oil sellers and welders when compared with controls (p > 0.05), values obtained for the auto-mechanics, auto-electricians, drivers, panel beaters, battery chargers and petrol station attendants were significantly higher than controls (p < 0.001). In these groups of artisans, the LDL cholesterol levels were between 1.6 to 2.4 times higher than control subjects. In contrast however, both triglyceride and HDL cholesterol concentrations were not significantly different between control and the artisans (p > 0.05), although the upholsterers tended to have lower HDL cholesterol concentrations when compared with others.
Table 3 Blood lead and blood lipid profiles of the subjects. Values are mean ± S.D.
Subjects
Parameter Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (Vehicle) Panel beaters Petrol station attendants Upholsterers Spare parts/Oil sellers Welders
Blood lead (μg/dl) 15.78 ± 2.84a 43.98 ± 10.54e 48.90 ± 19.11f 41.13 ± 11.55d 40.99 ± 7.00d 36.10 ± 9.64c 35.99 ± 11.08c 42.53 ± 5.90d 31.61 ± 7.78c 35.79 ± 6.30c 27.00 ± 1.05b
Total chol. (mg/dl) 106.17 ± 16.74a 170.42 ± 43.80c 166.83 ± 43.86b 203.50 ± 48.51e 184.88 ± 64.59d 127.69 ± 19.34a 176.78 ± 52.81c 157.29 ± 27.94b 152.70 ± 61.10b 187.08 ± 57.88d 157.96 ± 87.17b
Triglyceride (mg/dl) 64.17 ± 30.36a 77.29 ± 34.65a 66.90 ± 22.57a 75.29 ± 18.50a 84.19 ± 38.72a 104.85 ± 35.21a 65.47 ± 29.15a 60.58 ± 21.02a 81.61 ± 71.83a 104.50 ± 19.81a 69.68 ± 34.16a
HDL chol. (mg/dl) 50.65 ± 6.96a 45.01 ± 16.11a 51.32 ± 14.04a 40.56 ± 2.55a 39.78 ± 19.80a 49.58 ± 11.54a 41.49 ± 17.21a 40.55 ± 15.42a 27.68 ± 10.43a 52.26 ± 5.37a 73.03 ± 14.17b
LDL chol. (mg/dl) 62.96 ± 24.68a 99.93 ± 33.67b 101.12 ± 42.91b 147.00 ± 49.50c 133.85 ± 81.69b 90.72 ± 35.70a 114.50 ± 30.52b 148.52 ± 29.62c 82.22 ± 59.75a 69.54 ± 3.48a 83.39 ± 44.29a
Total chol./HDL chol. 2.15 ± 0.55a 4.49 ± 3.08b 3.46 ± 1.29a 4.99 ± 0.88b 5.77 ± 4.49b 2.65 ± 0.54a 4.70 ± 2.10b 4.28 ± 1.33a 5.48 ± 0.63b 3.66 ± 1.48a 2.09 ± 0.79a
LDL chol./HDL chol. 1.25 ± 0.47a 2.61 ± 1.49b 2.15 ± 1.18a 3.60 ± 1.00c 4.42 ± 4.26e 1.91 ± 0.82a 3.27 ± 1.71b 4.00 ± 1.11d 3.20 ± 2.03b 1.33 ± 0.07a 1.11 ± 0.39a
Values in a row having no letter (a – f) in common are significantly different from each other (p < 0.05).
The ratios of the lipids, total cholesterol/HDL cholesterol on one hand, and LDL cholesterol/HDL cholesterol on the other hand, are also depicted in Table 3. There were no statistical significant differences between the control and the artisans in total cholesterol/HDL cholesterol ratios (p > 0.05), although there was a tendency towards higher ratios in the artisans. In contrast however, LDL cholesterol/HDL cholesterol ratios were significantly increased in the artisans when compared with the control subjects (p < 0.001). Of the artisans, the highest ratio was observed in the drivers (4.42 ± 4.23), followed closely by the petrol station attendants with 4.00 ± 1.10.
Blood lead concentrations
The mean blood lead concentrations in the subjects are also depicted in Table 3. Lead levels in the blood of the artisans were significantly higher than that of control (p < 0.001). Of the artisans, the auto-electricians had the highest blood lead level of 48.90 ± 19.11 μg/dl, a value which was 3 times higher than that of control subjects.
Correlations between blood lead and anthropometric parameters, and blood lead and investigated lipids
There was no significant correlation between blood lead levels and any of the anthropometric parameters (p > 0.05) (Data not shown). However, a positive significant correlation was observed between blood lead and total cholesterol (r = 0.372; p = 3.0 × 10-5) and blood lead and LDL cholesterol (r = 0.283; p = 0.001). We also analysed our data to see whether any correlation existed among the anthropometric parameters on one hand and anthropometric parameters and investigated lipids on the other hand. The following were observed:
1. A significant positive correlation between age and systolic blood pressure (r = 0.325; p = 3.0 × 10-4).
2. A significant positive correlation between age and diastolic blood pressure (r = 0.232; p = 0.007).
3. A significant positive correlation between age and BMI (r = 0.449; p = 1 × 10-5).
4. A significant positive correlation between weight and triglyceride (r = 0.275; p = 0.006).
5. A significant positive correlation between age and weight (r = 0.418; p = 5 × 10-5).
6. A significant positive correlation between BMI and triglyceride (r = 0.354; p = 6 × 10-4).
7. A significant positive correlation between BSA and LDL cholesterol (r = 0.202; p = 0.035).
8. A significant positive correlation between age and BSA (r = 0.390; p = 1 × 10-4).
9. A significant positive correlation between BMI and systolic blood pressure (r = 0.371; p = 7 × 10-4).
10. A significant positive correlation between BMI and diastolic blood pressure (r = 0.424; p = 1 × 10-4).
Discussion
During the past decade, a vast amount of evidence has confirmed that lipid and lipoprotein abnormalities play a major role in the pathogenesis and progression of atherosclerosis and cardiovascular diseases [23-27]. These chronic degenerative disorders have become a growing health problem worldwide.
In African populations, dyslipidemia as a risk factor for cardiovascular disease and increasing incidents of death due to cardiovascular disease in both urbanised and underdeveloped rural countries have been reported [34]. There is also increasing evidence that environmental factors contribute to this dyslipidemia [35]. In this present study, we evaluated the distribution of some blood lipids in a population of artisans who are occupationally exposed to lead, an occupational and environmental pollutant. We found that majority of the artisans had HDL cholesterol and triglyceride levels not significantly different from controls. On the contrary, total cholesterol levels in the artisans were between 1.5 and 2.0 times higher than controls. In addition, LDL cholesterol in some of the artisans was considerably higher when compared with controls. To our knowledge, the distribution of blood lipids in artisans in Nigeria has not been reported in the literature. We were compelled to compare our data with the guidelines of risk factors for cardiovascular disease given by the American Heart Association [26]. According to these guidelines, blood pressure < 130/85 mmHg; total cholesterol < 200 mg/dl; triglycerides < 200 mg/dl; HDL > 40 mg/dl and LDL < 130 mg/dl, are favourable risk factors. In addition, certain lipid ratios like total cholesterol/HDL cholesterol and the LDL cholesterol/HDL cholesterol ratio also correlate with cardiovascular disease. The recommended ratios for the two are ≤ 3.5 [26]. Indications from this comparison are that while the HDL cholesterol and triglyceride concentrations of both the controls and artisans were within the acceptable range prescribed by the American Heart Association, the battery chargers with total cholesterol of 203.50 ± 48.51 mg/dl and LDL cholesterol of 147.00 ± 49.50 mg/dl, drivers with LDL cholesterol of 133.85 ± 81.69 mg/dl, and petrol station attendants with LDL cholesterol of 148.52 ± 29.62 mg/dl, seem to have unfavourable risk profiles for cardiovascular disease when compared with other artisans and control. Furthermore, the LDL/HDL ratio of the artisans indicate that battery chargers, petrol station attendants and drivers seem to have a greater risk of cardiovascular disease when compared with other artisans and controls. As regards total cholesterol/HDL cholesterol ratio, the auto-mechanics, battery chargers, drivers, panel beaters, petrol station attendants, spare parts/oil sellers and upholsterers, seem to be at risk of cardiovascular disease. Although cholesterol levels are lower in African population when compared with their American counterparts [36,37], the findings of the present investigation indicate that exposure to lead alters the metabolism of cholesterol and thus increases the risk of cardiovascular disease and atherosclerosis in lead-exposed subjects. Although correlations do not imply causality, the observation of a positive relationship between lead and total cholesterol on one hand, and lead and LDL cholesterol on the other hand, seems to support these experimental findings.
HDL and LDL are two of the four main groups of plasma lipoproteins that are involved in lipid metabolism and the exchange of cholesterol, cholesterol ester and triglycerides between tissues [38-40]. Numerous population studies have shown an inverse correlation between plasma HDL levels and risk of cardiovascular disease, implying that factors associated with HDL protect against atherosclerosis. Some of these factors appear to have anti-oxidant and anti-inflammatory effects which may obviate processes that initiate atherogenesis [41,42]. Epidemiological studies have also shown that elevated concentrations of total or LDL cholesterol in the blood are powerful risk factors for coronary disease [43]. Most extra-hepatic tissues, although having a requirement for cholesterol, have low activity of the cholesterol biosynthetic pathway. Their cholesterol requirements are supplied by LDL, which is internalised by receptor-mediated endocytosis. A major function of HDL cholesterol is to enhance reverse cholesterol transport by scavenging excess cholesterol from peripheral tissues followed by esterification through lecithin:cholesterol acyltransferase and delivering it to the liver and steroidogenic organs for subsequent synthesis of bile acids and lipoproteins and eventual elimination from the body [44,45]. This role of HDL has been shown to be responsible for its atheroprotective properties. HDL cholesterol also regulates the exchange of proteins and lipids between various lipoproteins [46]. In addition, HDL provides the protein components required to activate lipoprotein lipase which releases fatty acids that can be oxidised by the β-oxidation pathway to release energy [38-40]. Most importantly, HDL can inhibit oxidation of LDL as well as the atherogenic effects of oxidised LDL by virtue of its antioxidant property [45]. The observation of increased total plasma cholesterol and LDL cholesterol levels, and normal HDL cholesterol levels in the artisans in this study suggests that reverse cholesterol transport in these artisans was not affected by lead exposure, rather cholesterol synthesis and transport to the peripheral tissues might be affected. It is possible that lead increases the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (the rate-limiting enzyme in cholesterol biosynthesis) and reduces the number/affinity of LDL receptors for cholesterol. Further studies that we are pursuing in this laboratory are addressing this hypothesis.
Exposure to lead has been shown to be a significant risk factor for the development of hypertension [47,48] and this is supported by experimental animal studies in several species as well as epidemiological studies of blood pressure in relation to blood lead levels [47-49]. Most, but not all, of the studies have demonstrated that blood lead increases from 10 to 25 μg/dl are associated with systolic and diastolic blood pressure increases of 1.4–8 mmHg and 1.2–4 mmHg, respectively [48]. Mean blood lead levels observed in the artisans in this study were between 2.5 and 3.0 times higher than control subjects. Even though job experience indicated that lead exposure has been going on in these artisans for 4 to 26 years, only the auto-electricians had a borderline high systolic blood pressure as prescribed by the American Heart Association. It remains to be established in these artisans the threshold value of blood lead that might be associated with increased blood pressure.
In addition to blood lipids, other factors such as age, physical activity, genetics, body composition, alcohol intake, tobacco use and body fat distribution, contribute significantly to risk of cardiovascular disease [26,50]. All the subjects who participated in this study were non-smokers and all consume cassava-based meal as their carbohydrate source. Alcohol intake was limited to only six of the auto-mechanics who consume 1 bottle of beer per day. By virtue of the demands of their occupations, most of the artisans are physically active and this might account for the normal levels of HDL cholesterol observed in them [35]. The anthropometric parameters measured in the artisans did not reveal any statistically significant difference between the control subjects and artisans, thus suggesting that these factors may not play any significant role in the dyslipidemia observed in these artisans.
The increased plasma cholesterol levels observed in these artisans raises a few questions. Since erythrocyte cholesterol reflects plasma cholesterol, it might be interesting to investigate cholesterol levels in the erythrocytes in lead exposure and how this affects cholesterol:phospholipid ratio in both plasma and erythrocytes. The consequences of these alterations might probably explain alterations observed in cation fluxes in the erythrocyte membrane in lead exposure. Studies going on at present in this laboratory are directed towards addressing these questions.
Acknowledgements
The authors are grateful to the heads of the two mechanic workshops and the other subjects in the workshops who participated in the study.
Our thanks are also due to the medical and nursing teams of University of Agriculture Health Centre, Abeokuta, Nigeria, for their support in this study. The technical assistance of Mrs. J. O. Adebawa is also highly appreciated.
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Adonaylo VN Oteiza PI Pb2+ promotes lipid oxidation and alterations in membrane physical properties Toxicol 1999 132 19 32 10.1016/S0300-483X(98)00134-6
Bhattacharjee CR Dey S Goswami P Protective role of ascorbic acid against lead toxicity in blood of albino mice as revealed by metal uptake, lipid profiles and ultrastructural features of erythrocytes Bull Environ Contam Toxicol 2003 70 1189 1196 12756459 10.1007/s00128-003-0108-z
Deng W Poretz RD Lead exposure affects levels of galactolipid metabolic enzymes in the developing rat brain Toxicol Appl Pharmacol 2001 172 98 107 11298496 10.1006/taap.2001.9142
Ito Y Niiya Y Kurita H Shima S Sarai S Serum lipid peroxide dismutase activity in workers with occupational exposure to lead Arch Occup Environ Health 1985 56 119 127 10.1007/BF00379383
Osterode W Ulberth F Increased concentration of arachidonic acid in erythrocyte membranes in chronically lead-exposed men J Toxicol Environ Health Part A 2000 59 87 95 10653437 10.1080/009841000156998
Skoczynska A Smolik R Jelen M Lipid abnormalities in rats given small doses of lead Arch Toxicol 1993 63 200 204 8494500 10.1007/BF01973308
Quinlan GJ Halliwell B Moorehouse CP Gutteridge MC Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions Biochem Biophys Acta 1988 962 196 200 3167077
Rivarov SR Benov LC Benchev IC The effect of lead on hemoglobin-catalyzed lipid peroxidation Biochim Biophys Acta 1981 664 453 459 7272316
Oteiza PI Verstraeten SV Adonaylo VN Oxidative damage induced by metals without redox capacity in biological systems Cienc Cult 1995 47 330 335
Donaldson WE Knowles SO Is lead toxicosis a reflection of altered fatty acid composition of membranes? Comp Biochem Physiol 1993 104C 377 379
Lawton LJ Donaldson WE Lead-induced tissue fatty acid alterations and lipid peroxidation Biol Trace Elem Res 1991 28 83 97 1709034
Ademuyiwa O Arowolo T Ojo DA Odukoya OO Yusuf AA Akinhanmi TF Lead levels in blood and urine of some residents of Abeokuta, Nigeria Trace Elem Electro 2002 19 63 69
Ademuyiwa O Ugbaja RN Ojo DA Owoigbe AO Adeokun SE Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by vitamin C in occupational lead exposure in Abeokuta, Nigeria Environ Toxicol Pharmacol 2005 20 404 411 10.1016/j.etap.2005.04.002
Dosumu O Onunkwor B Odukoya O Arowolo T Ademuyiwa O Biomarkers of lead exposure in auto-mechanics in Abeokuta, Nigeria Trace Elem Electro 2005 22 185 191
Nriagu JO Blankson ML Ocran K Childhood lead poisoning in Africa: A growing public health problem Sci Total Environ 1996 181 93 100 8820380 10.1016/0048-9697(95)04954-1
Onunkwor B Dosumu O Odukoya OO Arowolo T Ademuyiwa O Biomarkers of lead exposure in petrol station attendants and auto-mechanics in Abeokuta, Nigeria: effects of 2-week ascorbic acid supplementation Environ Toxicol Pharmacol 2004 17 169 176 10.1016/j.etap.2004.04.003
Canfield RL Henderson CR JrCory-Slechta DA Cox C Jusko TA Lanphear BP Intellectual impairment in children with blood lead concentrations below 10 μg per deciliter N Engl J Med 2003 348 1517 1526 12700371 10.1056/NEJMoa022848
EPA Air quality criteria for lead (vols. I-IV EPA-600/8-83/02aF) 1986 US Environmental Protection Agency, Washington DC
Goyer RA Lead toxicity: current concerns Environ Health Perspect 1993 100 177 187 8354166
Meyer I Heinrich J Trekpa M The effect of lead in tap water on blood lead in children in a smelter town Sci Total Environ 1998 209 255 271 9514044 10.1016/S0048-9697(97)00324-0
Pirkle J Brody D Gunter E The decline in blood lead levels in the United States. The National Health and Nutrition Examination Surveys (NHANES) J Am Med Assoc 1994 272 284 291 10.1001/jama.272.4.284
Ponka A Lead in the ambient air and blood of children in Helsinki Sci Total Environ 1998 219 1 5 9770320 10.1016/S0048-9697(98)00209-5
Chrysohoou C Panagiotakos DB Pitsavos C Kosma K Barbetseas J Karagiorga M Ladi I Stefanadis C Distribution of serum lipids and lipoproteins in patients with beta thalassaemia major; an epidemiological study in young adults from Greece Lipids Health Dis 2004 3 3 15023232 10.1186/1476-511X-3-3
Ginsberg HN Lipoprorein metabolism and its relationship to atherosclerosis Med Clin North Am 1994 78 1 20 8283926
Glew RH Williams M Conn CA Cadena SM Crossey M Okolo SN Cardiovascular disease risk factors and diet of fulani pastorialists of northern Nigeria Am J Clin Nutr 2001 74 730 736 11722953
Glew RH Kassam HA Bhanji RA Okorodudu A VanderJagt DJ Serum lipid profiles and risk of cardiovascular disease in three different male populations in northern Nigeria J Health Popul Nutr 2002 20 166 174 12186197
Gotto AM Jr Pearson TA, Criqui MH, Luepker RV, Oberman A, Wilson M Lipid and lipoprotein disorder Primer in Preventive Cardiology 1994 Dallas Tex; American Heart Association 107 129
VanItallie TB Yan M-U Heymsfeld SB Funk RC Boileau RA Height normalised indices of the body's fat free mass and fat mass:potentially useful indicators of nutritional status Am J Clin Nutri 1990 52 953 959
James WPT Research on obesity 1976 Her Majesty Stationery Office, London
Hume R Weyers Relationship between total body water and surface area in normal and obese subjects J Clin Pathol 1971 24 234 238 5573437
Famodu AA Osilesi O Makinde YO Osonuga OA Blood pressure and blood lipid levels among vegetarian, semi-vegetarian and non-vegetarian native Africans Clin Biochem 1998 317 545 549 9812174 10.1016/S0009-9120(98)00067-8
Gidez LI Miller GJ Burstein M Slagle S Eder HA Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure J Lipid Res 1982 23 1206 1223 7175378
Sachs L Angewandte Statistik 1983 Springer Verlag, Berlin
van der Sande MA Inskip HM Jaiteh KO Maine NP Walraven GE Hall AJ McAdam KP Changing causes of death in the West African town of Banjul, 1942–1997 Bull World Health Organ 2001 79 133 141 11242820
Aguilar-Salinas CA Olaiz G Valles V Torres JMR Pérez FJG Rull JA Rojas R Franco A Sepulveda J High prevalence of low HDL cholesterol concentrations and mixed hyperlipidemia in a Mexican nationwide survey J Lipid Res 2001 42 1298 1307 11483632
Brotons C Ribera A Perich RM Abrodos D Magana P Pablo S Teradas D Fernadez F Permanyer G Worldwide distribution of blood lipids and lipoproteins in childhood and adolescence: a review study Atherosclerosis 1997 139 1 9 9699886 10.1016/S0021-9150(98)00056-2
Bansal D Bhatti HS Sehgal R Role of cholesterol in parasitic infections Lipids Health Dis 2005 4 10 15882457 10.1186/1476-511X-4-10
Gordon DJ Rifkind BM High-density lipoprotein: the clinical implications of recent studies N Eng J Med 1989 321 1311 1316
Sviridiv D Intracellular cholesterol trafficking Histol Histopathol 1999 14 305 319 9987675
McNamara DJ Dietary fatty acids, lipoproteins, and cardiovascular disease Adv Food Nutr Res 1999 36 253 1497850
Navab M Berliner JA Watson AD Hama SY Territo MC Lusis AJ Shih DM Van Lenten BJ Frank JS Demer LL Edwards PA Fogelman AM The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture Arterioscler Thromb Vasc Biol 1996 16 831 842 8673557
Oram JF Lawn RM ABCA1: the gatekeeper for eliminating excess tissue cholesterol J Lipid Res 2001 42 1173 1179 11483617
Law MR Lowering heart disease risk with cholesterol reduction:evidence from observational studies and clinical trials Eur Heart J Suppl 1999 1 S3 S8
Stein O Stein Y Atheroprotective mechanisms of HDL Atherosclerosis 1999 144 285 303 10407490 10.1016/S0021-9150(99)00065-9
Das DK Cardioprotection with high density lipoproteins. Fact or friction? Circ Res 2003 92 258 260 12595335 10.1161/01.RES.0000058881.44913.7B
Jacques PF Bostom AG Williams RR Ellison RC Eckfeldt JH Rosenberg IH Selhub J Rozen R Relation between folate status, a common mutation in methylene tetrahydrofolate reductase and plasma homocysteine concentrations Circulation 1996 93 7 9 8616944
Tsao DA Yu HS Cheng JT Ho CK Chang HR The change of beta adrenergic system in lead-induced hypertension Toxicol Appl Pharmacol 2000 164 127 133 10764625 10.1006/taap.1999.8871
Hertz-Picciotto I Croft J Review of the relation between blood lead and blood pressure Epidemiol Rev 1993 15 352 373 8174662
Schwartz J Lead, blood pressure and cardiovascular disease in men Arch Environ Health 1995 50 31 37 7717767
Brochu M Poehlman ET Savage P Ross S Ades PA Coronary risk profiles in men with coronary artery disease: effects of body composition, fat distribution, age and fitness Coron Artery Dis 2000 11 137 144 10758815 10.1097/00019501-200003000-00008
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Mol PainMolecular Pain1744-8069BioMed Central London 1744-8069-1-291619120310.1186/1744-8069-1-29MethodologyAn efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons Luo Miaw-Chyi [email protected] Dong-Qin [email protected] Shou-Wu [email protected] Yuan-Yuan [email protected] Sam J [email protected] Frank [email protected] Josephine [email protected] Department of Pharmacology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA2 Neuromics, Minneapolis, MN 55438, USA2005 28 9 2005 1 29 29 24 6 2005 28 9 2005 Copyright © 2005 Luo et al; licensee BioMed Central Ltd.2005Luo 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.
We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect™ (vehicle), and delivered as repeated daily bolus doses (0.5 μg to 4 μg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect™ complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.
ratantinociceptionsensory neuronsspinal cordknockdownRNA interference
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Background
RNA interference (RNAi) is an endogenous mechanism of RNA dependent degradation of specific mRNA by a protein complex called the RNA induced silencing complex (RISC) [1]. This mechanism was first characterized in the nematode Caenorhabditis elegans as an intrinsic protective response against invaded viral RNA [2]. The sequence specific substrate selectivity of RISC is dictated by its complex formation with certain double stranded, small interfering RNA (siRNA) that, in the nematode, is generated from viral RNA by an enzyme complex called DICER [3]. Subsequently, it was demonstrated that RNAi could be achieved in cultured mammalian cells by transfecting them with chemically synthesized siRNA [4], or by plasmid generated siRNA [5], thus establishing a new technology for functional genomics as well as drug target validation. In vertebrate experimental models, RNAi has been successfully applied to target exogenous and endogenous gene expression in organ systems such as liver, spleen, kidney, lung, and pancreas via systemic delivery [6-10]. However, this novel technology is not readily amenable to targeting nervous system genes because siRNA does not easily cross the blood brain barrier, and its uptake by neurons in vitro is poor [11]. While chemical modification of siRNA may enhance the efficiency of uptake of these molecules in cultured neurons as demonstrated recently [12], only a few studies to date succeeded in knocking down target genes in the central nervous system (CNS) [13-16]. We proposed that the limited advance made in the latter could be improved by establishing a paradigm that efficiently delivers the siRNA to the CNS and can be optimized for a variety of targets.
We have previously shown that the delta opioid receptor (DOR) can be effectively knocked down in vitro [17] and in vivo [18,19] by antisense oligodeoxynucleotide (ODN) treatment. In vivo, an antisense ODN to the DOR, at a dose of 12.5 μg (1.6 nmol), given twice daily, produced a robust inhibition, by day 3, of antinociception of the DOR selective agonist, [D-Ala2, Glu4]deltorphin II (DELT) when given intracerebroventricularly [18] or intrathecally [19]. We thus postulate that the DOR is a suitable prototypic nervous system gene target for the purpose of optimizing in vivo RNAi by siRNA based on our prior experience with delivery and dosing of ODN, time course of the knockdown and turnover rate of the DOR. Another critical advantage is the well-established protocols and reagents for measuring the expression and function of DOR. We show here that a low dose of 2 μg of siRNA when mixed with a transfection agent, i-Fect™, and given once daily by the intrathecal route, effectively abolishes the function of DOR in the lumbar spinal cord. A selective reduction in the transcript and protein level of DOR in the lumbar dorsal root ganglia (DRG) and in the lumbar dorsal horn of the spinal cord suggests that the inhibitory effect of the siRNA on DOR is specific to its knockdown of DOR expression. The uptake of the siRNA by cells in the DRG and the spinal cord is consistent with its site of action. The effect of the siRNA is abolished by scrambling its sequence, or by discontinuing the siRNA treatment. The effective dose of the siRNA requires the use of i-Fect™, which facilitates the uptake of the siRNA into target tissues.
Results
Intrathecal administration of a siRNA to DOR blocked spinal DELT antinociception
The siRNA that was designed was first evaluated for its effect on DOR expression in vitro. This was accomplished by transfecting the siRNA/i-Fect™ complexes into NG108-15 cells that express endogenous DOR (the DOR in the NG108-15 cells has the mouse genotype; while the siRNA sequence was designed based on the rat DOR sequence, it cross-reacts with the mouse sequence). Quantitative RT-PCR analysis of total RNA extracts from these cells harvested 48 hr after transfection showed an 82.4 ± 9.2% knockdown of the DOR transcripts compared with that in control, non-transfected cells. For in vivo delivery, an initial experiment was conducted to determine the dose response of the siRNA, or mismatch RNA on intrathecal DELT antinociception (Fig. 1). Rats were given vehicle, the siRNA or mismatch RNA once daily for 3 consecutive days, and spinal DELT antinociception was measured 24 hr after the last injection. In the vehicle pretreated rats, intrathecal DELT produced 60 ± 8% MPE, which was consistent with that previously published [19]. The siRNA at 2 μg and 4 μg (as two 2 μg doses, one at 0900 and one at 1600 hr) daily dose both significantly attenuated the antinociception of DELT when compared with DELT antinociception in the vehicle control, while a 0.5 μg daily dose had no effect. The antinociception by DELT observed in the 2 μg and 4 μg siRNA pretreated groups were not significantly different from each other (10 ± 8% MPE, 25 ± 5% MPE, respectively; P > 0.05). The mismatch RNA treated groups did not differ significantly from the vehicle control irrespective of dosage of the mismatch RNA. These data established the 2 μg daily as the approximate minimum effective dose of this siRNA. However, if this dose of the siRNA was given without the vehicle i-Fect™, the subsequent antinociceptive effect of intrathecal DELT was robust, and was not significantly different from that observed in the vehicle pretreated group, or mismatch RNA treated groups (Fig. 1). Notably, none of the RNA treatments had any effect on the baseline thermal nociceptive threshold when compared with that of the vehicle control group. The baseline thermal latencies for paw withdrawal in the vehicle, siRNA (2 μg/day for 3 days), and mismatch RNA groups prior to treatment were: 20 ± 1.5 sec, 21 ± 1.1 sec, and 20 ± 1.1 sec, respectively. Twenty-four hr after the last injection, the baseline thermal latencies were 20 ± 1.0 sec, 19 ± 1.5 sec, and 20 ± 1.0 sec, respectively. Neither vehicle nor RNA treatment precipitated overt signs of behavioral toxicity or motor impairment in the animals. All subsequent siRNA or mismatch RNA treatment employed the 3 consecutive daily dose of 2 μg given with i-Fect™.
Figure 1 Dose effect of intrathecal siRNA to DOR on the antinociceptive effect of the delta opioid agonist [D-Ala2, Glu4]deltrophin II (DELT). DELT was given intrathecally (30 μg). The antinociceptive efficacy of DELT is defined as % antinociception measured 30 min after drug administration (n = 6 rats/group). Intrathecal DELT antinociception was significantly blocked after 3 consecutive once daily dose of siRNA at a daily dose of 4 μg (2 μg b.i.d.) or 2 μg (*p < 0.05), when compared with that observed in the vehicle control group. Mismatch RNA at the same doses had no effect on DELT antinociception. The 2 μg daily dose of siRNA, when administered without i-Fect™, did not block intrathecal DELT antinociception. The 0.5 μg dose of the siRNA in i-Fect™ did not have any effect on intrathecal DELT antinociception. Statistical analysis was carried out by one-way ANOVA following Dunnett's multiple comparisons.
The inhibitory effect of siRNA treatment on DELT antinociception is transient and reversible
Whereas spinal DELT antinociception was attenuated after siRNA treatment, determined at 24 hr after the last siRNA injection, when the animals were tested again 48 hr later (i.e., 72 hr after the last injection), DELT antinociception was not different when compared with that in mismatch RNA and vehicle control groups (Fig. 2).
Figure 2 Spinal DELT antinociception tested 72 hours after the last RNA or vehicle injection (n = 6/group). There is no significant difference in the % antinociception among the three pretreatment groups (p > 0.05).
Knockdown of DOR in the spinal dorsal horn by the siRNA
Figure 3 shows the saturation analysis of DOR in the spinal dorsal horn by the selective antagonist, [3H]naltrindole. SiRNA, but not mismatch RNA, produced a significant reduction in the density of the DOR measured as the maximum specific binding (Bmax) of the radioligand when compared with that in the vehicle treated controls. The reduction represents ~70% knockdown of the DOR density after siRNA treatment. The dissociation constant (Kd) for [3H]naltrindole was not different in the three treatment groups. Immunohistochemical analysis using an anti-DOR antibody showed a significant reduction of immunoreactivity for DOR in the superficial laminae of the dorsal horn of the lumbar spinal cord from siRNA treated rats, but not from the mismatch RNA treated rats (Fig. 4). Lumbar spinal dorsal horn harvested from siRNA treated rats 72 hr after the last siRNA injection showed strong DOR immunoreactivity, which was not different from the vehicle or from the mismatch RNA controls. The recovery of DOR immunoreactivity by 72 hr after the last siRNA injection is consistent with the observed recovery of DELT antinociception in the siRNA treated rats at this time point shown in Fig. 2.
Figure 3 Saturation [3H]naltrindole binding in membranes prepared from rat dorsal lumbar spinal cord (n = 3 rats/group). The Bmax value in the siRNA group, but not the mismatch RNA group, was significantly lower than that in the vehicle treated group. The Kd values are not significantly different among the 3 groups (p > 0.05). Data are representative of 3 independent experiments.
Figure 4 DOR-immunoreactivity in the dorsal horn of the spinal cord after vehicle, siRNA or mismatch (MM) RNA treatment. The immunoreactivity of DOR was predominantly found in laminae I/II of the dorsal horn. The immunolabeling was significantly lower in tissue from siRNA treated rats. Tissues taken 72 hours after the last siRNA injection (siRNA recovery) show similar level of immunoreactivity for DOR when compared with vehicle or MM RNA controls. Each image is representative of multiple sections from 3 rats per treatment group.
siRNA treatment reduced the level of DOR transcripts in the DRG and the spinal dorsal horn
The mRNA levels of DOR in the lumbar spinal cord and L4/L5 DRGs were determined by quantitative RT-PCR at 24 hr after the last siRNA injection. In the siRNA treated group, there was a 38% reduction in DOR transcripts in the DRG (61.9 ± 5.7% of vehicle control), and 62% reduction in the lumbar spinal cord (38 ± 17.7% of vehicle control). Mismatch RNA pretreatment did not significantly alter the DOR mRNA level in either the DRG (109 ± 2.0% of vehicle control) or the lumbar spinal cord (90 ± 0.01% of vehicle control).
siRNA was taken up by DRG and spinal cord cells
AlexaFluor546-tagged siRNA fluorescence was clearly detected in both the lumbar DRG and spinal cord 24 hr after intrathecal delivery of 2 μg of the tagged RNA (Fig. 5A, B). In the DRG, fluorescence was present in cell bodies of various sizes. The labeling was punctate, perinuclear, and varied in intensity among the cells (Fig. 5A). In the spinal cord cross sections, fluorescence could be seen distributed over the entire cross sections, labeling a large number of cells in both the dorsal and the ventral horn, as well as the area around the central canal. Figure 5B shows an example of a 40 × magnification of the labeled spinal cord cells in the superficial laminae of the dorsal horn. The fluorescence is seen in the cytoplasm of the labeled cells, while nuclei typically appear as dark, unlabeled areas in the center of these cells. The localization of the fluorescence strongly suggests that the labeling is intracellular and thus due to the uptake of the tagged siRNA by the DRG and the spinal cord cells. Autofluorescence of the tissue sections was negligible under these imaging conditions when tissue sections from vehicle-injected rats were analyzed (Fig. 5C, D). Notably, when fluorescence-tagged siRNA was delivered into rats without i-Fect™, its uptake in the lumbar DRG (Fig. 5E) as well as in the lumbar spinal cord (Fig. 5F) was extremely poor.
Figure 5 AlexaFluor546-tagged siRNA uptake in lumbar DRG (A, C, E) and spinal cord (B, D, F). Vehicle (10 μL) (C, D), siRNA with vehicle (2 μg/ 10 μL) (A, B), or siRNA without vehicle (2 μg/ 10 μL of aqueous annealing buffer) (E, F) was given intrathecally as a single injection. Tissues were harvested and processed 24 hr later. Images of the DRG were taken using a 20× objective lens and that of the spinal cord taken using a 40× objective lens. In all the fluorescence labeled cells, the labeling was punctate and peri-nuclear, and labeling intensities varied among cells. In the spinal cord (B), labeled cells were distributed widely in both the dorsal horn and the ventral horn. The images in B, D, F were taken from laminae I/II of the dorsal horn. Scale bar for the DRG images shown in panel E is 50 μm; scale bar for the spinal cord images shown in panel F is 25 μm.
siRNA to DOR did not alter the function or expression of MOR
In vehicle-pretreated rats, DAMGO (0.5 μg) produced 66 ± 16% MPE, which is consistent with that previously published [20]. Neither siRNA to DOR, nor mismatch RNA treatment, had any effect on intrathecal DAMGO antinociception when compared with the vehicle control (79 ± 11% MPE, 73 ± 10% MPE, respectively to 66 ± 16% MPE; P > 0.05). There was also no significant difference in the MOR immunoreactivity in the dorsal horn of the spinal cord between siRNA, mismatch RNA, and vehicle treated groups (Fig. 6).
Figure 6 MOR-immunoreactivity in the dorsal horn of the spinal cord after vehicle, siRNA (to DOR) or mismatch (MM) RNA treatment. The MOR immunoreactivity is predominantly localized to laminae I/II of the dorsal horn. No significant difference in the immunoreactivity for MOR was seen among the 3 treatment groups. Tissue taken 72 hours after the last siRNA injection (siRNA recovery) also showed similar MOR immunoreactivity as the control groups. Each image is representative of multiple sections processed from 3 rats used in each group. The sections used in this figure and those used for DOR labeling shown in Figure 4 were from the same animals.
Discussion
The advances made recently toward the in vivo applications of RNAi in vertebrate systems are critical towards developing siRNA as therapeutics [21-23]. These in vivo applications, however, do not yet apply to central nervous system function because siRNA do not readily cross the blood brain barrier (BBB) via systemic delivery. Because drugs may bypass the BBB by delivering them directly into the CSF, such delivery routes may be exploited in order to determine whether neurons and other cells in the nervous system may be amenable to RNAi by siRNA in vivo. Two recent reports used intrathecal delivery to prevent hypoxia-induced expression of brain derived neurotrophic factor in the spinal cord [14], or to knockdown the expression of the purinergic receptor subtype, P2X3, in sensory primary afferent [15]. While both studies demonstrated that siRNA attenuated the intended gene target, the former was a short intervention (3.5 hr) while the experimental subject was under general anesthesia, whereas the latter required very high concentration and continuous infusion of siRNA (400 μg/day for up to 7 days) to elicit a modest knockdown of the target. It is not clear whether the high dose of siRNA needed to effect reflects an inefficient RNAi mechanism in neurons or whether the modulation of gene function cannot be sustained over time.
Our data show that in the presence of a suitable transfection agent, the siRNA to the DOR is highly effective at a low concentration of 2 μg, or 0.14 nmol, given once a day. This dose is 23 times lower than the amount of antisense ODN required to elicit a knock down of the DOR as previously published [18], and is substantially lower than the effective dose reported for the knock down of the P2X3 receptors in the DRG [15]. The siRNA treatment delivered without the transfection reagent was without effect, suggesting that the transfection reagent significantly enhanced the uptake of the siRNA into target cells, as verified by the detection of fluorescence uptake of spinal cord and DRG tissues after injecting the tagged siRNA. This observation is consistent with recent evidence that the uptake of siRNA by neurons in culture is likely a key limiting step in the siRNA mediated gene silencing [12]. The amount of RNA delivered as a bolus dose in transfection reagent tends to be limited by the solubility of the RNA. The transfection reagent used in the present study was i-Fect™, which is a cationic lipid mixture that has been optimized for the delivery of short oligonucleotides in vitro [24]. This reagent was chosen for the present study because the RNA/lipid complexes remain in suspension at a fairly high concentration in a volume that is suitable for intrathecal delivery. A maximum of 2 μg of RNA can be given in a 10 μL injection volume. Should a daily dose of >2 μg is desired, the delivery can be adjusted by giving multiple doses.
A significant knockdown of DOR transcripts by siRNA treatment is consistent with the proposed mechanism of action of RNAi [25,26]. In this regard, the knockdown of the DOR transcripts was observed in both the dorsal horn of the spinal cord as well as the lumbar DRG, demonstrating that the siRNA interferes with the synthesis of both the presynaptic and the postsynaptic populations of DOR, which is also highly consistent with the uptake pattern of the tagged siRNA. The effects of siRNA culminate in a significant reduction of DOR immunoreactivity and ligand binding capacity in the superficial laminae of the dorsal horn of the spinal cord where the functional receptors are predominantly located. The loss of functional DOR is evident by the loss of antinociceptive activity of the DOR selective agonist, DELT, given intrathecally. Together, these results justify our conclusion that we have established an effective method for delivering siRNA that interferes efficiently with the expression and function of target genes in both the peripheral nervous system (i.e., sensory primary afferent) and the central nervous system (i.e., spinal cord).
The use of a mismatch RNA confirms the specificity of the siRNA sequence for the DOR. The siRNA treatment had no effect on the expression or the function of the highly homologous receptor type, MOR, further supporting the target specificity of the siRNA employed here. Finally, the effect of the siRNA is fully reversible; thus the observed effects of the siRNA are specific to the use of siRNA, and the treatment paradigm does not precipitate any long-term effects due to toxicity such as motor impairment. This paradigm can be easily adjusted for dosage and duration of treatment, and is based on a well-established, relatively non-invasive method of drug delivery that has general applications for spinal and peripheral targets. The reagents required are minimal and economical, and can be adapted for other gene targets. Our findings support the hypothesis that siRNA can be effectively applied to modulate nervous system function. The significant knockdown of the target transcripts in both the DRG and the spinal cord conforms with the established mechanism of siRNA mediated gene silencing, and is consistent with the uptake of siRNA seen in both the peripheral neurons (i.e. DRG) and the central nervous system (i.e., spinal cord). The low dose of siRNA further suggests that the efficacy of RNAi depends critically on the efficient delivery of the siRNA to the target tissues. Unlike antisense oligodeoxynucleotides, siRNA may be delivered systemically [23]. Thus, chemical modifications that enhance systemic stability and facilitate siRNA transport across the BBB or the uptake of siRNA by neurons would greatly advance the potential of siRNA as therapeutic.
Methods
siRNA preparation
The siRNA sequence for the DOR (accession no. U00475, the only gene sequence that is defined as delta opioid receptor to date) was from nucleotides 364 to 384 relative to the start codon. The sequences were as follows: sense 5'-GGCUGUGCUCUCCAUUGACUU-3'; antisense 5'-GUCAAUGGAGAGCACAGCCUU-3'. A scrambled sequence was designed as a mismatch control: sense 5'-GGCGUGUCUCUCUUACGACUU-3' and antisense 5'-GUCGUAAGAGAGACACGCCUU-3'. BLAST search of the nucleotide sequences in the GenBank database showed no substantial homology with other genes. These 21-nucleotide RNA oligonucleotides were synthesized individually, deprotected and purified by RNase-free HPLC (Midland Certified Reagent Company). siRNA and mismatch RNA duplexes were formed in a concentration of 200 μM in annealing buffer as described [4] for 3 min at 90°C followed by 1 h at 37°C. The siRNA stocks were aliquoted and stored at -80°C. For fluorescence labeling of the siRNA, AlexaFluor 546 was conjugated at the 5' end of the sense strand by solid phase synthesis and the tagged siRNA was purified by HPLC (Qiagen) and resuspended to a concentration of 200 μM in annealing buffer.
In vitro analysis of knock down of DOR in NG108-15 cells by the siRNA
NG108-15 Cells were cultured in 5% fetal calf serum/ 5% newborn calf serum/ 45% Ham's F-12/ 45% DMEM/ 100 U mL-1 penicillin/ 100 μg mL-1 streptomycin (Invitrogen). Cells were maintained in 75 cm2 flasks in a humidified atmosphere with 95% air and 5% CO2. For experiments, cells were seeded in 6-well plates at 3 × 105 cells/well 24 hr before the siRNA treatment. Cells were transfected with 2 μg of siRNA/i-Fect™ complexes in 1:4 ratio (w/v) per well. Forty-eight hours after transfection, cells were harvested and total RNA was isolated. DOR transcripts were detected by real-time RT-PCR (see below).
Quantitative RT-PCR
Total RNA was isolated from the lumbar dorsal spinal cord, the L4 and L5 dorsal root ganglia using Aurum™ total RNA mini kit (Bio-Rad). Quantitative RT-PCR was performed using the iCycler iQ Multicolor Real-Time PCR Detection System with iScript cDNA Synthesis Kit and iQ SYBR Green Supermix (Bio-Rad). All samples were run in triplicate using an annealing temperature of 60°C. Primers for the amplification of DOR were: forward primer: 5'-GTTCACCAGCATCTTCACG -3'(nuc. 396~414); reverse primer: 5'-TGCATACCACTGCTCCATC -3'(nuc. 577~595). That for GAPDH were: forward primer: 5'-ATCATCCCTGCATCCACTG-3'(nuc. 610~628); reverse primer: 5'-GCCTGCTTCACCACCTTC -3'(nuc. 771–788). All primers were synthesized by Midland Certified Reagent Company, Inc. PCR efficiencies for DOR and GAPDH were 97% and 98%, respectively, with correlation coefficient of 0.999. Expression of DOR was normalized to expression of GAPDH. The differences of DOR mRNA expression between treatments were analyzed using the Comparative CT Method (ABI Prism 7700 Sequence Detection System User Bulletin #2, p11–15, 2001). The threshold cycle (CT) is defined as the cycle at which the amount of amplified PCR product from the target cDNA reaches a fixed threshold. In each treatment, ΔCT = CT for the target, DOR – CT for the endogenous reference, GAPDH. ΔΔCT = ΔCT,treatment – ΔCT,control. The equation, 2-ΔΔCT, denotes the ratio of the level of DOR transcripts in the treated group and that of the control group. This number is converted to percent of control, where control is set at 100.
Animal surgery and RNA administration
Male Sprague-Dawley rats, weighing between 200–220 g, were used in these experiments. All the procedures used in these experiments have been approved by the Institutional Animal Care and Use Committee. Rats were implanted with intrathecal (i.th.) catheters and allowed 7 days to recover from surgery prior to treatment. The rats were divided into siRNA, mismatch RNA and vehicle groups, with at least 6 rats per group. siRNA or mismatch RNA complexes were prepared immediately prior to administration by mixing the RNA solution (200 μM in annealing buffer) with a transfection reagent, i-Fect™ (Neuromics), in a ratio of 1:4 (w:v) [24]. At this ratio, the final concentration of RNA as an RNA/lipid complex was 2 μg in 10 μL. siRNA or mismatch RNA, or i-Fect™ alone (defined as vehicle) in 10 μL was delivered to the lumbar region of the spinal cord via the i.th. catheters. Injections were given daily for 3 consecutive days. Nociceptive testing and tissue harvest were carried out at 24 hr and 72 hr after the last injection. The fluorescence tagged siRNA was mixed with i-Fect™ and injected in the same manner as for the untagged RNA, except that only one injection was given and tissue harvested 24 hr later.
Nociceptive testing
Radiant heat paw withdrawal test using a movable infrared light source was employed as the nociceptive stimulus. The experimenter who conducted the nociceptive testing was blinded to the pretreatment of the experimental groups. The baseline latency for withdrawal of the left hindpaw was recorded from the experimental animals 24 hr prior to RNA or vehicle administration. Twenty-four hr following the last injection, the baseline latency was recorded again. The rats then received either 30 μg of [D-Ala2]deltorphin II (DELT) or 0.5 μg of [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO) (Sigma) intrathecally and the thermal latency were measured at 15-min interval over 60 min. A maximal cutoff latency of 33 s was used to prevent potential tissue injury. The % antinociceptive effect of DELT or DAMGO was defined as: [(treatment-baseline)/(cutoff-baseline)] × 100. The testing was repeated at 72 hr after the last injection.
Saturation analysis of DOR by [3H]naltrindole
Crude membranes were prepared from the lumbar dorsal spinal cords pooled from the rats in each treatment group, resuspended in 50 mM Tris buffer, pH7.2, and the protein content measured as previously described [18]. Saturation analysis of [3H]naltrindole (20 Ci/mmol, PerkinElmer) was carried out using 10 concentrations of [3H]naltrindole (31.3 pM to 5 nM), done in triplicates, incubated with 50 μg of membranes per assay tube in 0.5 mL buffer, at a total volume of 1.0 mL, in a shaking water bath at 25°C for 3 hr. Non-specific binding of the radioligands was defined by that in the presence of 10 μM naloxone. The reactions were terminated by rapid filtration through Whatman GF/B filters, followed by five washes with 4 mL of ice-cold saline. The radioactivity was determined by liquid scintillation counting. Data were analyzed by non-linear least squares regression analysis using GraphPad Prism 4 (GraphPad software).
Immunohistochemical analysis of DOR and MOR and fluorescence imaging
Rats were deeply anesthetized with ketamine HCl/xylazine (Sigma). The heart was exposed and transcardially perfused with 10 mM sodium phosphate-buffered saline (PBS, pH 7.4) until exudate ran clear, then switched to 10% buffered formalin for a further 15 min. Lumbar spinal cords and the L4 and L5 DRG were isolated and post-fixed in 10% buffered formalin for 24 hr and cryoprotected with 30% sucrose in 10 mM PBS. After pre-blocking with 10% normal goat serum (Vector Laboratories), frontal sections (20 μm) of the spinal cord were incubated with either a rabbit anti-rat primary antibody for DOR (1:2000) or a rabbit anti-rat primary antibody for MOR (1:5000) at 4°C for 48 hr (both antibodies were from Neuromics). Slides were rinsed with 2.5% normal goat serum/PBS and then incubated with a secondary antibody (AlexaFluor 594-conjugated goat anti-rabbit IgG (1:1000, Molecular Probes) for 1 hour at room temperature. Slides were again rinsed 3 times with the same buffer, dried, and mounted for microscopy. Fluorescent imaging of the samples was carried out using Nikon E800 fluorescence microscope equipped with 10×, 20× and 40×objective lenses and standard filters for Y-2E/C TX RED, coupled to a Hamamatsu C5810 color CCD camera (Hamamatsu Photonic System) for digital image acquisition using Adobe Photoshop software on a PC workstation. The same system was used for the imaging of slide mounted lumbar spinal cord and L4/L5 DRG (20 μm longitudinal sections) from animals that have been injected with the tagged siRNA, except that the filters for TRITC was used.
Statistical analysis
Significant differences among paw withdrawal latencies at 30-min time point after DELT or DAMGO injection were determined by ANOVA followed by the post hoc least significant differences test. Unpaired t-test was used for all other between group comparisons.
Abbreviations
RNAi RNA interference
siRNA Small interfering RNA
MM RNA Mismatch RNA
i.th. Intrathecal
DOR Delta opioid receptor
DELT [D-Ala2]deltrophin II
MOR Mu opioid receptor
DAMGO [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin
DRG Dorsal root ganglia
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgements
The authors thank Dr. David Bumcrot for critical review and discussion of the manuscript, and Wen-Jun Zhang for technical assistance. This work is supported by the National Institute on Drug Abuse Grant P01 DA06284.
==== Refs
Hammond SM Bernstein E Beach D Hannon GJ A genetic link between co-suppression and RNA interference in C. elegans Nature 2000 404 293 296 10749213 10.1038/35005107
Fire A Xu S Montgomery MK Kostas SA Driver SE Mello CC Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 1998 391 906 811 10.1038/35888
Bernstein E Caudy AA Hammond SM Hannon GJ Role for a bidentate ribonuclease in the initiation step of RNA interference Nature 2001 409 363 366 11201747 10.1038/35053110
Elbashir SM Harborth J Lendeckel W Yalcin A Weber K Tuschl T Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 2001 411 494 498 11373684 10.1038/35078107
Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells Science 2002 296 550 553 11910072 10.1126/science.1068999
McCaffrey AP Meuse L Pham T-TT Conklin DS Hannon GJ Kay MA Gene expression: RNA interference in adult mice Nature 2002 418 38 39 12097900 10.1038/418038a
Song E Lee S-K Wang J Ince N Quyang N Min J Chen J Shankar P Lieberman J RNA interference targeting Fas protects mice from fulminant hepatitis Nat Med 2003 9 347 351 12579197 10.1038/nm828
Sorensen DR Leirdal M Sioud M Gene silencing by systemic delivery of synthetic siRNAs in adult mice J Mol Biol 2003 327 761 766 12654261 10.1016/S0022-2836(03)00181-5
Zender L Hutker S Liedtke C Tillmann HL Zender S Mundt B Waltemathe M Gosling T Flemming P Malek NP Trautwein C Manns MP Kühnel F Kubicka S Caspase 8 small interfering RNA prevents acute liver failure in mice Proc Natl Acad Sci USA 2003 100 7797 7802 12810955 10.1073/pnas.1330920100
Kobayashi N Matsui Y Kawase A Hirata K Miyagishi M Taira K Nishikawa M Takakura Y Vector-based in vivo RNA interference: dose- and time-dependent suppression of transgene expression J Pharmaco Exp Ther 2004 308 688 693 10.1124/jpet.103.059931
Trülzsch B Wood M Application of nucleic acid technology in the CNS J Neurochem 2004 88 257 265 14690514
Davidson TJ Harel S Arboleda VA Prunell GF Shelanski ML Greene LA Troy CM Highly efficient small interfering RNA delivery to primary mammalian neurons induces microRNA-like effects before mRNA degradation J Neurosci 2004 24 10040 10046 15537872 10.1523/JNEUROSCI.3643-04.2004
Makimura H Mizuno TM Mastaitis JW Agami R Mobbs CV Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake BMC Neurosci 2002 3 18 23 12423556 10.1186/1471-2202-3-18
Baker-Herman TL Fuller DD Bavis RW Zabka AG Golder FJ Doperalski NJ Johnson RA Watters JJ Mitchell GS BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia Nat Neurosci 2003 7 48 55 14699417 10.1038/nn1166
Dorn G Patel S Wotherspoon G Hemmings-Mieszczak M Barclay J Natt FJC Martin P Bevan S Fox A Ganju P Wishart W Hall Jonathan siRNA relieves chronic neuropathic pain Nucleic Acids Res 2004 32 e49 15026538 10.1093/nar/gnh044
Thakker DR Natt F Husken D Maier R Muller M van der Putten H Hoyer D Cryan JF Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference Proc Natl Acad Sci 2004 101 17270 17275 15569935 10.1073/pnas.0406214101
Lai J Crook TJ Payne A Lynch RM Porreca F Antisense targeting of delta opioid receptors in NG108-15 cells: direct correlation between oligodeoxynucleotide uptake and receptor density J Pharmacol Exp Ther 1997 281 589 596 9103548
Bilsky EJ Bernstein RN Hruby VJ Rothman RB Lai J Porreca F Characterization of antinociception to opioid receptor selective agonists after antisense oligodeoxynucleotide-mediated "knock-down" of opioid receptor in vivo J Pharmacol Exp Ther 1996 277 491 501 8613959
Bilsky EJ Wang T Lai J Porreca F Selective blockade of peripheral delta opioid agonist induced antinociception by intrathecal administration of delta opioid receptor antisense oligodeoxynucleotide Neurosci Lett 1996 220 155 158 8994216 10.1016/S0304-3940(96)13262-6
Vanderah TW Gardell LR Burgess SE Ibrahim M Dogrul A Zhong C-M Zhang E-T Malan TP JrOssipov MH Lai J Porreca F Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance J Neurosci 2000 20 7074 7079 10995854
Wood MJA Trülzsch B Abdelgany A Beeson D Therapeutic gene silencing in the nervous system Hum Mol Genet 2003 12 R279 R284 12928477 10.1093/hmg/ddg275
Rossi JJ A cholesterol connection in RNAi Nature 2004 432 155 156 15538347 10.1038/432155a
Soutschek J Akinc A Bramlage B Charisse K Constien R Donoghue M Elbashir S Geick A Hadwiger P Harborth J John M Kesavan V Lavine G Pandey RK Racie T Rajeev KG Röhl I Toudjarska I Wang G Wuschko S Bumcrot D Koteliansky V Limmer S Manoharan M Vornlocher H-P Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs Nature 2004 432 173 178 15538359 10.1038/nature03121
More information about this reagent may be obtained on line at
Hannon GJ RNA interference Nature 2002 418 244 251 12110901 10.1038/418244a
Novina CD Sharp PA The RNAi revolution Nature 2004 430 161 164 15241403 10.1038/430161a
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J NanobiotechnologyJournal of Nanobiotechnology1477-3155BioMed Central London 1477-3155-3-91618803810.1186/1477-3155-3-9ReviewAtomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa Kumar Sunil [email protected] Koel [email protected] Prasenjit [email protected] Sujoy K [email protected] School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721 302, India2 School of Physical Sciences, Jawaharlal Nehru University, New Delhi-110067, India2005 27 9 2005 3 9 9 5 4 2005 27 9 2005 Copyright © 2005 Kumar et al; licensee BioMed Central Ltd.2005Kumar 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.
Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development.
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Introduction
Sperm morphology is regarded as a significant prognostic factor for fertilization and pregnancy [1]. Abnormal sperm morphology is one of the most common factors of male infertility. Morphological changes are also considered to be a potential target in contraceptive development. There is, therefore, an urgent need to analyze the morphological alterations of spermatozoa in their nearly physiological environment in greater detail.
Atomic force microscopy (AFM) has opened up new avenues of study in reproductive biology. AFM, invented by Binnig, Quate and Gerber in 1986, has evolved as a powerful imaging technique to obtain nanometer-resolved topographic data images. In brief, the sample surface is raster scanned by a flexible cantilever with a sharp tip at one end. A laser beam focused on the back of the cantilever is bounced off and is detected by a photodiode detector. The ability of this technique to image non-conductive living cells in physiological environment (aqueous solution) in 3D array without elaborate sample preparation or fixing of samples unlike conventional electron microscopy (which requires the cells to be fixed with aldehyde and stained) has made AFM a valuable tool to study various biomolecules [2-4], including sperm cells [5]. AFM imaging in air require cells to be fixed to avoid structural changes caused by drying forces on the cell [6]. But this fixing need not require post fixation like in electron microscopy. Conventional microscopy not only distorts sperm morphology, but is also unable to provide high-resolution 3D images owing to the small size of the spermatozoa. Optical microscopy provides valuable information only if the alterations are gross and of the order of a micron or fraction thereof.
AFM provides the advantage of directly observing spermatozoa in their native environment thereby opening the exciting possibility of analyzing their structural and functional aspects at the sub-molecular level. This article provides a review on morphological and topological images of sperm cells using AFM. Such high-resolution images are expected to provide a better understanding of male factor infertility, improve the success rate of ART procedures and also give a new direction towards contraceptive development.
Morphological and pathological changes of spermatozoa
Figure 1 shows the 2D and 3D images of the normal human spermatozoa using non-contact mode AFM; the graph indicates the head and the length profiles of the head region. Defects in the acrosomal region may often lead to the loss of functional competence of the spermatozoa. The major advantage of AFM in pathological studies of spermatozoa is that it allows the evaluation of position and form of the acrosome. Electron microscopy investigation reveals the presence of nano-grooves or "channels" on top of the flagellum of healthy spermatozoa [7] whereas AFM provides precise topographical information. This technique has been successfully employed for studying human sperm in its natural environment and 3D images reconstructed which enhances the contrast to resolve details such as mitochondria that surround the axoneme at the sperm middle piece [4]. An organized structure in the flagellar axoneme region in addition to depressions of the membrane that could not be observed with the conventional microscope has been reported. The 3D image contrast mechanism has been utilized to study bovine sperm cells structures [8]. Results show that imaging spermatozoa in physiologic conditions provides more native views of the cells due to the retention of cytoplasmic structures, which are otherwise easily disrupted by drying forces.
Figure 1 AFM of normal human spermatozoa. a. 2D image (8.00 × 8.00 μm scan) of normal sperm head. b. Height profile of the head region of spermatozoa showing clear difference in the head and the acrosomal region. c. Power spectrum of the above profile showing the scaling of roughness. d. Histogram plot of the height of the head region. e. 3D image of the head region of the spermatozoa.
AFM has been used for morphologic and morphometric analyses of acrosome intact and acrosome-reacted human sperm heads [9]. Structural changes of the hamster sperm head surface associated with maturation, capacitation and acrosome reaction has also been studied using this technique [10]. Changes in the plasma membrane over the head region of mammalian spermatozoa during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle have been reported [11]. Morphological and topological alterations in human spermatozoa induced by a non-hormonal polyelectrolytic male contraceptive in vitro have been examined using AFM which suggested almost complete disintegration of the plasma membrane with subsequent rupture of the acrosomal membrane leading to dispersion of acrosomal contents [12]. A more recent study by Takano et al. (2004) provides the details of surface structure changes in spermatozoa from mouse epididymis associated with maturation [13]. Saeki et al. (2004) have provided similar details about sperm head including acrosome, equatorial segment, post acrosomal region and neck during acrosome reaction induced by lysophosphatidylcholine are given [14]. In addition, a numerical analysis carried out by the research group indicates that the area of medial sagittal plane of the anterior portions of acrosome-reacted sperm heads is approximately 40% less than those of intact heads.
Morphological alterations in spermatozoa leading to oligoasthenoteratozoospermia (OAT) and asthenozoospermia have been analyzed using AFM [15]. This study clearly indicates alteration in the infected sperms and provides extensive information on morphological changes in the head, neck and flagellum. Similar studies have shown dimensional changes in the head and defective neck and flagellum in spermatozoa from patients reporting with varicocele [16]. Recent work in this field includes the application of AFM to study the morphological and topographical changes caused by HIV and the effects of highly active antiretroviral therapy (HAART) on spermatozoon of HIV infected patients [17]. The study was so effective that even minute details, such as position of the viral particles located on the sperm membrane and their merging on the surface of spermatozoa were detected with high precision. Unlike electron microscopy, and other conventional microscopes, one of the biggest advantages of AFM is that it images virions in their nearly natural environment, which may be highly beneficial in determining interaction of virions with the host.
Detailed topology of bovine spermatozoa and force vs. distance curves has been obtained using contact mode AFM [18]. The acrosome, midpiece, postacrosomal segments and flagellum were clearly distinguishable due to the local height variations. A model of the overall mechanical response of the cell that allows separating out the mechanical response from the local surface interactions is presented. This model differs from traditional Hertzian contact models, commonly used in AFM, by explicitly taking into account the mechanics of the biomembrane and cytoskeleton [19]. With this mathematical model it is possible to determine the extent of membrane deformation due to net forces generated by the AFM tip on spermatozoa. Similar modeling is reported for analyzing deformation of living bovine spermatozoa [20]. A model to measure the mechanical response of the cells during recognition force microscopy (RFM), where specific molecules attached to the AFM tip scan the cell surface, which, in turn, provides vital information on intermolecular interaction has been proposed.
Axonemal imaging
Axoneme, a specific "9 + 2" arrangement of the microtubules in which nine outer doublet microtubules surround a central pair of singlet microtubules, plays an important role in the movement of spermatozoa. The bending of cilia and flagella is attributed to dynein-induced sliding of microtubules, which is a key step in force generation. A transverse function of the microtubule is predicted on the basis of the 3D movement of dynein motors i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. This has been confirmed using optical trapping [21] and electron microscopy [22] which provided valuable information on the biomechanics of their movements. Recently Sakakibara et al. (2004) have successfully shown using AFM that these transverse motions occur in an oscillatory manner when the axonemes of sea-urchin sperm flagella adhere onto glass substrates [23]. They further reported that Mg-ATP significantly increases the high frequency oscillations of the flagellum. Both, a horizontal as well as a vertical component of oscillation is observed when the AFM tip is in contact with the axonemes. A similar study on the structural details and the carbon density in the flagellum of sea urchins sperm has been carried out by Tomie et al. (1991) [24].
Sperm chromatin studies
DNA, present in a highly condensed state in mammalian sperm cells, was imaged successfully for the first time in both, air and liquids by Allen et al. (1993) [25]. The highly compact chromatin state in the sperm heads of octopus E. cirrhosa has been studied in great detail [26]. A simple, effective air-drying sample preparation technique for AFM of demembranated Xenopus sperm chromosomes has been suggested [27]. Artefact-free, high resolution nuclear reassembly images were obtained by this technique. Chromosomal banding pattern of height using AFM similar to that of G-banding by conventional optical microscopy is reported by De Grooth et al. (1992) [28]. The potential ability of AFM in localizing the DNA probes on in situ hybridized chromosomes using the height pattern has been studied. Synaptonemal complex from rat spermatocytes providing structural details of the protein has also been reported by this group.
Protamines, small arginine-rich protein, are the major DNA-binding proteins in the nucleus of spermatozoa of most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. The binding of protamine to sperm chromatin generates a large dense hydrophobic complex making the sperm chromatin structure difficult for microscopic examination. AFM imaging of the well-spread isolated sperm nuclei subjected to prior hypotonic treatment showed large nodular structures and a smaller nucleosome like particle near the periphery of the nucleus present in the chromatin [29]. Similarly, a toroidal shaped packaging unit for mammalian sperm chromatin has also been observed [30]. A novel method for reconstituting sperm chromatin to investigate condensation of DNA by protamine 1 is proposed [31]. Here the structures formed are found to be highly dependent on various conditions of sample preparation used for reconstitution. A previous study by Allen et al. (1992) showed that ribbon like images obtained from chromatin complexes with protamine are due to the convolutions of the imaging tip and the sample morphology [32]. A similar study on structural organisation of chromatin subunits from spermatozoa of two marsupial species, Smithopsis crassicaudata and Trichosurus vulpecula has been carried out using AFM [33]. The results indicate that the nucleohistone region consists of clusters of bigger nodules when compared to nucleoprotamine core region. A very interesting AFM study on sperm chromatin and synthetic DNA-protamine complexes is reported by Balhorn et al. (2000) [34]. The complex mimics increased resistance and structural similarity to the native sperm chromatin.
AFM has been used to perform volume measurements of the human sperm nuclei by Lee et al. (1997) [35]. Their results indicate that normal sperm and the seven of the nine classes of head-shape abnormalities studied have identical nuclear volumes, though the projected areas and shapes of the nuclei may vary widely. It is interesting to mention here that the results showed 25–40% of the sperm head morphologies found are not caused by factors that affect the volume of sperm chromatin, such as the DNA content of the sperm nucleus, differences in chromatin organization, or the extent of DNA compaction. Similar studies on volume changes in mouse and bull sperm nucleus have been carried out using scanning force microscopy by Allen et al. (1996) [36]. Their results provided details of the extent of hydration of sperm chromatin in its native state. Hence, volume of natively hydrated sperm nuclei can be easily determined.
Future Prospects
Constant force applied on the soft biological samples may damage the cell and thus change its morphology. Considering this, various imaging modes have been developed such as resonance based tapping mode, lift mode, force modulation imaging, nanoindenting, scratching and lateral force microscopy. The development of small micro-mechanized cantilever and optical fiber tips reduce thermal noise by providing a better ratio of cantilever stiffness and resonance frequency and improved imaging bandwidth. An important development would be the construction of antibody modified tips which could be useful in localizing antigens (by vertical/lateral force detection) on the plasma membrane. This may be helpful in studying molecular interactions in greater detail.
AFM, in itself, has proved to be a powerful instrument in nanoscopic analysis of biological samples. Nevertheless, more information with finer details may be achieved if combined with various other techniques such as optical microscope [37] and optical tweezers [38] as these techniques allow direct manipulation of individual cell. Advances in Cryo-AFM are very promising for imaging spermatozoa preserved in liquid nitrogen [39,40]. This technique, in combination with freeze-etching and freeze-fracture techniques, may be used to obtain high resolution images of preserved spermatozoa.
Time lapse AFM imaging has been used to observe the conformational changes in supercoiled DNA [41] and in chaperone complex (GroEl-GroEs) analysis [42]. Small cantilevers with high resonance frequencies have been developed by Walters et al. (1996) [43]. In addition, small spring constants and electronic devices of wide bandwidth have been included in AFM to obtain a powerful useful movie mode for scanning biomolecules successively in aqueous solution [44]. This sophisticated imaging mode may be applied for a better understanding of sperm oocyte interaction.
Scanning Near-field Optical Microscope (SNOM) is an emerging technique and is still in infancy with respect to the imaging of biological cells. This technique utilizes the near field, non-propagating component of light to scan the samples with optical tips, which can be applied in the tapping or contact mode. A resolution of few tens of nanometers is achievable by SNOM. High resolution topographic and optical images of sea urchin sperm flagellum have been obtained using fluorescent probe as a light [45]. Electrostatic Force Microscopy (EFM), Magnetic Force Measurements (MFM) and Scanning Thermal Microscopy (SThM) are relatively new techniques and may play a significant role in determining fluid dynamics and biomechanics of the sperm cells in their natural micro-environment. AFM, in combination with surface potential spectroscopy, has been applied to measure the surface charges of P. falciparum merozoites [46]. This methodology can also be applied to study the negative charge distribution on the sperm head, which is known to play a vital role in fertilization.
Recent developments in AFM have made it a powerful tool for analyzing biomolecules. Over the last decade, AFM has emerged as a valuable technique with extensive applications in the field of sperm biology. AFM is expected to provide a better understanding of various biological pathways and intermolecular interactions which will open up new avenues in reproductive medicine.
Authors' contributions
SK has contributed to the acquisition of a part of the data by carrying out AFM experimental studies on spermatozoa treated with the contraceptive, RISUG. PS has also assisted in acquiring data and analyzing it. In addition, he has done extensive literature survey and collected the data/research papers. KC has conceived the study and contributed to the design, analysis, co-ordination and interpretation of data. SKG, the inventor of RISUG, has also participated in the design, revised the manuscript critically for important intellectual content and has given final approval of the version to be published. All authors read and approved the final manuscript.
Acknowledgements
The authors are thankful to the Department of Biotechnology, Government of India for providing necessary financial support for the study.
==== Refs
Franken RD The clinical significance of sperm-zona pellucida binding Front Biosci 1998 3 247 253
Hansma HG Kim KJ Laney DE Garcia RA Argaman M Allen MJ Parsons SM Properties of biomolecules measured from atomic force microscope images: a review J Struct Biol 1997 119 99 108 9245749 10.1006/jsbi.1997.3855
Shao Z Mou J Czajkowsky DM Yang J Yuan JY Biological atomic force microscopy: What is achieved and what is needed Adv in Phy 1996 45 1 86
Hörber JKH Miles MJ Scanning Probe Evolution in Biology Science 2003 302 1002 1005 14605360 10.1126/science.1067410
Joshi N Medina H Colasante C Osuna A Ultrastructural investigation of human spermatozoon by using atomic force microscope Arch Androl 2000 44 51 57 10690765 10.1080/014850100262416
Lee WMJ Mah-Lee Ng A nano-view of West Nile virus-induced cellular changes during infection J Nanobiotechnology 2004 2 6 15225378 10.1186/1477-3155-2-6
Schaller M Panhans GA Benzold G Korting HS Wolff H Ultrastructural defects in acquired immotile sperm flagella Fertil Steril 2000 73 351 10685542
Allen MJ Bradbury EM Balhorn R The natural subcellular surface structure of the bovine sperm cell J Struct Biol 1995 114 197 208 7662487 10.1006/jsbi.1995.1019
Mai A Wattana W Mauro T Danial DMW George W Balhorn R Arthur L Jean LC Nongnuj T Use of atomic force microscopy for morphological and morphometric analyses of acrosome intact and acrosome-reacted human sperm Mol Repro Dev 2002 63 471 479 10.1002/mrd.10195
Takano H Kazuhiro A Changes in the surface structure of the hamster sperm head associated with maturation, in vitro capacitation and acrosome reaction: an atomic force microscopic study J Electron Microsc 2000 49 437 443
Ellis DJ Shadan S James PS Henderson RM Michael EJM Hutchings A Jones R Post-testicular development of a novel membrane substructure within the equatorial segment of ram, bull, boar, and goat spermatozoa as viewed by atomic force microscopy J Struct Biol 2002 138 187 98 12217657 10.1016/S1047-8477(02)00025-4
Kumar S Chaudhury K Sen P Guha SK AFM study of surface structure changes in human spermatozoa treated with RISUG: a new male contraceptive Proceedings of International Symposium on Advanced Materials and Processing ISAMAP2K4: 6–8 December 2004; IIT Kharagpur, India 2004
Takano H Abe K AFM study of surface structure changes in mouse spermatozoa associated with maturation Methods Mol Biol 2004 242 85 94 14578515
Saeki K Sumitomo N Nagata Y Kato N Hosoi Y Matsumoto K Iritani A Fine Surface Structure of Bovine Acrosome-Intact and Reacted Spermatozoa Observed by Atomic Force Microscopy J Reprod Dev 2004
Joshi N Honorio M Ibis C Jesus OMD Determination of the ultrastructural pathology of human sperm by atomic force microscopy Fertil Steril 2001 75 961 965 11334909 10.1016/S0015-0282(01)01755-1
Joshi NV Medina H Osuna JA Ultrastructural pathology of varicocele spermatozoa by using atomic force microscopy (AFM) Arch Androl 2001 47 143 52 11554686 10.1080/014850101316901352
Barboza JM Medina H Doria M Rivero L Hernandez L Joshi NV Use of atomic force microscopy to reveal sperm ultrastructure in HIV-patients on highly active antiretroviral therapy Arch Androl 2004 50 121 129 14761843
McElfresh M Eveline B Rod B James B Michael JA Robert ER Combining constitutive materials modeling with atomic force microscopy to understand the mechanical properties of living cells Proc Natl Acad Sci 2002 99 6493 6497 11983924 10.1073/pnas.082520599
Hassan AE Heinz WF Antonik MD D'Costa NP Nageswaran S Schoenenberger CA Hoh JH Relative microelastic mapping of living cells by atomic force microscopy Biophys J 1998 74 1564 1578 9512052
Rudd RE McElfresh M Baesu E Balhorn R Alleny M Belak J Modeling of the Deformation of Living Cells Induced by Atomic Force Microscopy NanoTech 2002 2 73 76
Shingyoji C Higuchi H Yoshimura M Katayama E Yanagida T Dynein arms are oscillating force generators Nature 1998 393 711 714 9641685 10.1038/31520
Sakakibara H Kojima H Sakai Y Katayama E Oiwa K Inner-arm dynein c of Chlamydomonas flagella is a single-headed processive motor Nature 1999 400 586 590 10448863 10.1038/23066
Sakakibara HM Kunioka Y Yamada T Kamimura S Diameter oscillation of axonemes in sea-urchin sperm flagella Biophys J 2004 86 346 352 14695276
Tomie T Shimizu H Majima T Yamada M Kanayama T Kondo H Yano M Ono M Three-dimensional readout of flash x-ray images of living sperm in water by atomic-force microscopy Science 1991 252 691 693 2024121
Allen MJ Lee C Lee JD IVPogany GC Balooch M Siekhaus WJ Balhorn R Atomic force microscopy of mammalian sperm chromatin Chromosoma 1993 102 623 630 8306824 10.1007/BF00352310
Diaspro A Beltrame F Fato M Palmeri A Ramoino P Studies on the structure of sperm heads of Eledone cirrhosa by means of CLSM linked to bioimage-oriented devices Microsc Res and Tech 1997 36 159 164 9080405 10.1002/(SICI)1097-0029(19970201)36:3<159::AID-JEMT3>3.0.CO;2-K
Yang N Chen Z Zhang Z Zhu X Zhai Z Tang X Atomic force microscopy observation on nuclear reassembly in a cell-free system Chinese Science Bulletin 2003 48 2284 2287 10.1360/03ww0086
De Grooth BG Putman CA High-resolution imaging of chromosome-related structures by atomic force microscopy J Microsc 1992 168 239 247 1484376
Allen MJ Lee JD IVLee C Balhorn R Extent of sperm chromatin hydration determined by atomic force microscopy Mol Reprod Develop 1996 45 87 92 10.1002/(SICI)1098-2795(199609)45:1<87::AID-MRD12>3.0.CO;2-U
Hud NV Allen MJ Downing KH Lee J Balhorn R Identification of the elemental packing unit of DNA in mammalian sperm cells by atomic-force microscopy Biochem Biophys Res Commun 1993 193 1347 1354 8323555 10.1006/bbrc.1993.1773
Allen MJ Bradbury EM Balhorn R AFM analysis of DNA-protamine complexes bound to mica Nucleic Acids Res 1997 25 2221 2226 9153324 10.1093/nar/25.11.2221
Allen MJ Hud NV Balooch M Tench RJ Siekhaus WJ Balhorn R Tip-radius-induced artifacts in AFM images of protamine-complexed DNA fibers Ultramicroscopy 1992 42 1095 1100 1413246 10.1016/0304-3991(92)90408-C
Soon LL Bottema C Breed WG Atomic force microscopy and cytochemistry of chromatin from marsupial spermatozoa with special reference to Sminthopsis crassicaudata Mol Reprod Dev 1997 48 367 374 9322249 10.1002/(SICI)1098-2795(199711)48:3<367::AID-MRD10>3.0.CO;2-T
Balhorn R Brewer L Corzett M DNA condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules Mol Reprod Dev 2000 56 230 234 10824973 10.1002/(SICI)1098-2795(200006)56:2+<230::AID-MRD3>3.0.CO;2-V
Lee JD IVAllen MJ Balhorn R Atomic force microscope analysis of chromatin volumes in human sperm with head-shape abnormalities Biol Reprod 1997 56 42 49 9002631
Allen MJ Bradbury EM Balhorn R The chromatin structure of well-spread demembranated human sperm nuclei revealed by atomic force microscopy Scanning Microsc 1996 10 989 994 9854851
Vesenka J Mosher C Schaus S Ambrosio L Henderson E Combining optical and atomic force microscopy for life sciences research Biotechnique 1995 19 240 253
Kricka JL Optical Tweezers and Immunoassay Clin Chem 1997 43 251 253 9023126
Shao Z Zhang Y Biological Cryo Atomic Force Microscopy: A Brief Review Ultramicroscopy 1996 66 141 152 9195750 10.1016/S0304-3991(96)00087-3
Zhang Y Sheng S Shao Z Imaging Biological Structures with the Cryo Atomic Force Microscope Biophysical Journal 1996 71 2168 2176 8889193
Nagami. F Zuccheri G Samori B Kuroda R Time-lapse imaging of conformational changes in supercoiled DNA by scanning force microscopy Anal Biochem 2002 300 170 176 11779108 10.1006/abio.2001.5435
Johannes HK Jackey AC Thomas G Paul KH New atomic force microscopies (AFM) for the study enzymatic properties and processes AAPPS Bulletin 2003 13 8 11
Walters DA Cleveland JP Thomson NH Hansma PK Wendman MA Gurley G Elings V Short cantilevers for atomic force microscopy Rev Sci Instrum 1996 67 3583 3590 10.1063/1.1147177
Ando T Kodera N Takai E Maruyama D Saito K Toda A A high-speed atomic force microscope for studying biological macromolecules Proc Natl Acad Sci 2001 98 12468 12472 11592975 10.1073/pnas.211400898
Rothery AM Gorelik J Bruckbauer A Yu W Korchev YE Klenerman D A novel light source for SICM-SNOM of living cells J Microsc 2003 209 94 101 12588526 10.1046/j.1365-2818.2003.01122.x
Akaki M Nagayasu E Nakano Y Aikawa M Surface charge of Plasmodium falciparum merozoites as revealed by atomic force microscopy with surface potential spectroscopy Parasitol Res 2002 88 16 20 11822732 10.1007/s00436-002-0690-8
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-501618803110.1186/1477-7827-3-50ResearchEffect of rabbit doe-litter separation on 24-hour changes of luteinizing hormone, follicle stimulating hormone and prolactin release in female and male suckling pups Cano Pilar [email protected]énez-Ortega Vanesa [email protected] Álvarez Maria [email protected]ño Mario [email protected] Daniel P [email protected] Ana I [email protected] Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense de Madrid, Spain2 Departamento de Biología Celular, Facultad de Medicina, Universidad Complutense de Madrid, Spain3 Departamento de Producción Animal, E.T.S.I. Agrónomos, Universidad Politécnica de Madrid, Spain4 Departamento de Fisiología, Facultad de Medicina, UBA, Buenos Aires, Argentina2005 27 9 2005 3 50 50 8 8 2005 27 9 2005 Copyright © 2005 Cano et al; licensee BioMed Central Ltd.2005Cano 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 daily pattern of nursing of the rabbit pup by the doe is the most important event in the day for the newborn and is neatly anticipated by them. Such anticipation presumably needs a close correlation with changes in hormones that will allow the pups to develop an appropriate behavior. Although a number of circadian functions have been examined in newborn rabbits, there is no information on 24-h pattern of gonadotropin release or on possible sex-related differences in gonadotropin or prolactin (PRL) release of pups. This study examined the 24-h changes of plasma luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in 11 days old suckling female and male rabbits left with the mother or after short-term (i.e., 48 h) doe-litter separation.
Methods
Animals were kept under controlled light-dark cycles (16 h – 8 h; lights on at 08:00 h). On day 9 post partum, groups of 6–7 female or male rabbit pups were separated from their mothers starting at 6 different time intervals in the 24 h cycle. Pups were killed 48 h after separation. At each time interval groups of male or female pups that stayed with the mother were killed as controls. Plasma, LH, FSH and PRL levels were measured by specific radioimmunoassays.
Results
In pups kept with their mother plasma FSH and LH maxima occurred at the first and second part of the light phase (at 13:00 and 17:00 – 21:00 h, respectively) (females) or as two peaks for each of the hormones (at 13:00 and 01:00 h) (males). PRL release was similar in female and male rabbit pups kept with their mother, showing a 24-h pattern with two peaks, at 13:00 and 01:00 h, respectively. Mean 24-h values of gonadotropins and PRL did not differ between sexes. Isolation of pups for 48 h augmented circulating gonadotropin and PRL levels and distorted hormone 24-h pattern to a similar extent in both sexes.
Conclusion
Significant sex differences in 24-h changes in LH and FSH, but not in PRL, release occurred in rabbit pups kept with the doe. Separation of newborn pups from their mother augmented circulating gonadotropin and PRL levels and disrupted 24-h rhythmicity of gonadotropin and PRL release similarly in both sexes. The effect of pups' isolation can be attributed either to a modification of the circadian pacemaker or to a masking effect on some of its output overt rhythms.
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Background
In contrast with an elaborate nest-building process, following parturition, maternal care in rabbits is restricted to a single, brief (around 3–5 min) nursing bout per day, an activity that is displayed with circadian periodicity during the dark phase of daily photoperiod (around 02:00 h) [1]. Despite the short duration of each nursing bout, the altricial rabbit pups (which are blind for the first 10 days of life) can locate the mother's nipples and suckle milk due to the perception of an olfactory signal that is emitted from the mother's ventrum [2].
Thus the daily pattern of nursing of the rabbit pup by the doe is the most important event in the day of newborn and is neatly anticipated by them [3,4]. The newborn rabbits spend most of the time between feeds lying quietly together, becoming increasingly active and gradually exposed 1–2 h in advance to nursing. These events presumably need a close correlation with changes in hormones that will allow the pups to develop an appropriate behavior. In fact, an earlier development of the circadian secretory pattern of prolactin (PRL) occurs in infantile rabbits [5,6] as compared to other laboratory species e.g., the albino rat [7,8]. Although a number of circadian functions have been examined in newborn rabbits [1,9-12] there is no information on 24-h pattern of gonadotropin release nor on possible sex-related differences in gonadotropin or PRL release of pups.
When litters are separated from their mother and deprived of one nursing they display the usual pattern of anticipatory behavior on the first day of separation, but when the doe fails to arrive, gradually become less active and covered over again with nest material [1]. The following day, approximately 45–48 h after last nursing, the pups again become aroused, uncovered and are able to suckle normally. Therefore, after 48-h doe-litter separation, rabbit pups exhibit a usual daily pattern of behavior, as compared to controls.
The present study was designed to answer the following questions: (1) are there sex differences in gonadotropin and PRL release in rabbit pups?; (2) does separation from the mother modify gonadotropin and PRL release in pups? To achieve this, the 24-h changes in plasma LH, FSH and PRL were examined in 11 days old suckling female and male rabbits left with the mother or after short-term (i.e., 48 h) doe-litter separation.
Results
Figure 1 shows the plasma levels of LH throughout the day in female and male pups kept with or separated from the doe. A multivariate ANOVA of the whole set of observations indicated significant effects of treatment (F = 13.8, p < 0.0001) and of time of day (F = 4.1, p < 0.02), when assessed as main factors. Mean values of LH were 6.13 ± 1.37 and 6.97 ± 1.57 ng/mL (SD) in control and isolated females (p < 0.02, Student's t test) and 5.64 ± 1.71 and 6.53 ± 1.22 ng/mL in control and isolated males (p < 0.03, Student's t test). Significant interactions of "gender × time of day" (F = 10.9, p < 0.0001), "treatment × time of day" (F = 28.1, p < 0.0001), "treatment × gender" (F = 7.5, p < 0.007) and "treatment × time of day × gender" (F = 6.6, p < 0.001) were found. In female pups kept with their mother, plasma LH peaked at the second part of the light phase of daily photoperiod whereas two peaks were observed in males, i.e., at 13:00 and 01:00 h, respectively. Pup isolation from the mother distorted the circulating LH rhythm in a similar way in both sexes, exhibiting a peak at the second part of the scotophase (at 05:00 h) (Fig. 1).
Figure 1 24-Hour changes in plasma LH levels in 11 days old rabbit pups kept with their mother or isolated for 48 h. Groups of 6–7 female or male pups were killed by decapitation at 6 different time intervals throughout a 24 h cycle. Bar indicates scotophase duration. Results are the means ± SEM. For statistical analysis, see text.
Figure 2 depicts the circulating FSH concentration. A multivariate ANOVA showed significant effects of treatment (F = 155.3, p < 0.0001) and time of day (F = 24.2, p < 0.0001). Mean values of FSH were 66.1 ± 13.6 and 89.9 ± 22.6 ng/mL in control and isolated females (p < 0.001, Student's t test) and 63.6 ± 15.2 and 95.5 ± 38.2 ng/mL in control and isolated males (p < 0.001, Student's t test). Significant interactions of "gender × time of day" (F = 4.9, p < 0.0001), "treatment × time of day" (F = 48.4, p < 0.0001), and "treatment × time of day × gender" (F = 4.3, p < 0.01) were detected. In female pups kept with their mother, plasma FSH peaked at 13:00 h while two peaks were seen in males, i.e., at 13:00 and 01:00 h, respectively. Doe-litter separation distorted pups' plasma FSH rhythm to a similar extent in both sexes, with peaks at 09:00, 21:00 and 05:00 h. At certain time points, i.e. at 17:00 and 21:00 h, significant sex differences were seen (Fig. 2).
Figure 2 24-Hour changes in plasma FSH levels in 11 days old rabbit pups kept with their mother or isolated for 48 h. Groups of 6–7 female or male pups were killed by decapitation at 6 different time intervals throughout a 24 h cycle. Bar indicates scotophase duration. Results are the means ± SEM. For statistical analysis, see text.
Plasma PRL levels in control pups, and their responses to the separation from the doe, are depicted in Fig. 3. In the multivariate ANOVA the effects of treatment (F = 51.1, p < 0.0001) and of time of day (F = 9.3, p < 0.0001) were significant. Mean values of PRL were 46.6 ± 14.1 and 55.6 ± 12.2 ng/mL in control and isolated females (p < 0.01, Student's t test) and 47.6 ± 11.1 and 56.8 ± 12.4 ng/mL in control and isolated males (p < 0.002, Student's t test). PRL release was essentially similar in control female and male rabbit pups, i.e., a 24-h pattern with two peaks, at 13:00 and 01:00 h, respectively. The only significant interaction detected in the multivariate ANOVA was that of "treatment × time of day" (F = 37.9, p < 0.0001), namely, the isolation of pups brought about a significant increase of plasma PRL as well as a phase-advance of 3 h in both peaks (Fig. 3).
Figure 3 24-Hour changes in plasma PRL levels in 11 days old rabbit pups kept with their mother or isolated for 48 h. Groups of 6–7 female or male pups were killed by decapitation at 6 different time intervals throughout a 24 h cycle. Bar indicates scotophase duration. Results are the means ± SEM. For statistical analysis, see text.
Discussion
The questions posed in the Introduction can now be answered: (1) significant sex differences in 24-h rhythmicity of LH and FSH, but not of PRL release, occurred in rabbit pups kept with the doe; (2) separation of newborn pups from their mother augmented circulating gonadotropin and PRL levels and disrupted 24-h rhythmicity of their release to a similar way in both sexes.
Foregoing results document the existence of sex differences in 24-h rhythmicity of gonadotropin but not of PRL release in newborn rabbits. Mean value of gonadotropin and PRL release did not differ between sexes in rabbit pups. This is in contrast with data obtained in newborn rats demonstrating that newborn female rats have significantly higher plasma FSH and LH levels than their male counterparts [13]. Indeed, the crucial difference between a reflex ovulator (like the rabbit) and a spontaneous ovulator (like the rat) is that the former lacks the capacity to generate the gonadotropin ovulatory surge in response to estrogen or progesterone injection [14,15]. It is supposed that reflex ovulators have the genetic information for the organization of a cyclic gonadotropin releasing mechanism but its development is hindered by the action of sex steroids produced by the fetal gonads [16]. If this tenet is true, the differences between male and female rabbit pups would be less defined than in rodents, as found in the present study. That the process of defeminization of the female brain by perinatal gonadal steroids occurs in rabbits is indicated by studies in pregnant female rabbits treated with testosterone propionate from day 17–29 of gestation [17]. While there was no apparent effect on gestation and maternal behaviors of injected mothers, the female offspring showed anatomical and behavioral masculinization.
Healthy rabbit pups can, and often do, miss a feed and survive. Thus, separation of litters from their mother provides an appropriate model for investigating the ontogeny of gonadotropin and PRL 24-h rhythms in view that maternal influence can be nullified for a short period without disrupting the normal neonatal environment [1,18,19]. The foregoing results indicate that separation of litters from their mother augmented circulating mean gonadotropin and PRL levels. It also distorted circulating gonadotropin and PRL 24-h rhythms to a similar extent in both sexes.
In previous studies we examined some of the neuroendocrine mechanisms associated with the 24-h plasma PRL rhythm in male and female rabbit pups [5,6]. The PRL rhythm reported in control male and female pups herein is similar to that reported previously.
Circadian rhythms of developing mammals can be entrained by the rhythmicity of their mother [20,21]. Previous data demonstrated that when litters remained separated from the doe and deprived of two nursings they show the usual pattern of anticipatory behavior and at the appropriate time [1,18,19]. This occurs regardless of the significant changes in gonadotropin and PRL release demonstrated herein. Indeed, after doe-litter separation for 48 h, the rabbit pups show a normal daily pattern of behavior but a disrupted 24-h variation of plasma LH, FSH and PRL release and a significant increase in their plasma concentration, indicating a certain degree of internal desynchronization produced by the isolation procedure. It must be noted that the experimental design employed does not allow discerning whether the circadian phase of separation initiation is critical or doe-litter separation affects circadian profile of hormone release regardless time of day of separation. Studies including a larger cohort of pups all separated at one circadian time point, with subgroups sacrificed at different time intervals later will be helpful to clarify this point.
Temporal organization is an important feature of the biological systems and its main function is to facilitate adaptation of the organism to the environment [22,23]. Stress is capable of perturbing this temporal organization by affecting the shape and amplitude of a rhythm or by modifying the intrinsic oscillatory mechanism itself. In particular, social stress in rodents has been found to cause disruptions of the circadian rhythms of body temperature, heart rate, locomotor activity and hormone release [24-28]. The present results confirm previous studies in rodents showing that maternal separation evokes marked alteration in neuroendocrine control mechanisms also in rabbits [29]. Further experiments are needed to assess whether the changes in mean value as well in timing of 24-h rhythms of gonadotropins and PRL seen in isolated rabbit pups can attributed the effect of stress on either the endogenous clock that modulates the circadian variation of hormone release or via an interfering ("masking") effect on some output(s) of the clock. Additionally, to what extent the increase of stress hormones like ACTH and glucorticoids vary in a circadian pattern after isolation should be examined to provide a basis for physiological explanation of the phenomenological data hereby presented.
Methods
Animals
This study was performed using 84 multiparous, lactating Californian × New Zealand White crossbreed female rabbits. Animals were housed in the research facilities of the Departamento de Producción Animal, Universidad Politécnica de Madrid. They were maintained under controlled light-dark cycles (16 h – 8 h; lights on at 08:00 h), housed in individual metal cages, fed at libitum using a commercial pellet diet [Lab Rabbit Chow, Purina Mills, Torrejón de Ardoz, Madrid, Spain] and had free access to tap water. On day 1 after parturition, litter size was standardized to 8–9 by adding or removing kits to assure similar lactation conditions during the experiment. Nursing visit of does under these conditions occurred during the dark phase of the photoperiod (around 02:00 am) for 3–5 min [1], this fact being observed under our experimental conditions. The does and the pups that did not observed this feeding schedule were not used for this study. On day 9 post partum, groups of 6–7 female or male rabbit pups were separated from their mothers starting at different time intervals in the 24 h cycle, i. e., at 09:00, 13:00, 17:00, 21.00, 01:00 or 05:00 h. Pups were killed 48 h after separation from their mothers. At each time interval groups of male or female pups that stayed with the mother were killed as controls. The study was performed according to the CEE Council Directive [86/609, 1986] for the care of experimental animals.
Hormone assay
Plasma, LH, FSH and PRL levels were measured by specific RIA methods [30] using AFP-3120489, AFP-472176 and AFP-991086 antibodies for, LH, FSH and PRL, respectively, as supplied by the National Institute of Health [NIH, Bethesda, MD, USA] and Dr. A.F. Parlow [Harbour-UCLA Medical Center, CA, USA]. The antibody titers used were, 1:250000 for LH, 1:45000 for FSH and 1:62500 for PRL assays, respectively. The volume of plasma used was 100 μL for LH, 75 μL for FSH and 10 μL for PRL. Staphylococcus aureus was used to precipitate the bound fraction. The assays were previously validated in our laboratory [30]
All samples were measured in the same assay run to avoid inter-assay variations. The limit of detection for LH, FSH and PRL was 0.05, 0.48 and 0.125 ng/mL respectively. The intra-assay coefficient of variation, calculated using a pool of plasma measured ten times in the same assay, was <5%.
Statistics
After determining that the homogeneity-of-variance assumption was tenable and that the distribution appeared unimodal and nonskewed, the statistical analysis of results was performed by a multifactorial factorial analysis of variance (ANOVA). Generally, the multifactorial ANOVA included assessment of the treatment effect (i.e. the occurrence of differences in mean values between control and separated groups), of sex effects (differences in mean values between female and male pups), of time of day effects (the occurrence of daily changes) and of the interactions among the three factors (from which inference about differences in timing and amplitude could be obtained). Student's t tests to compare 24 h mean serum hormone values were performed when appropriate. P values lower than 0.05 were considered evidence for statistical significance.
Acknowledgements
This work was supported by grants from DGES, BFI2000-0614, Ministerio de Educación y Cultura, Spain.
==== Refs
Jilge B Hudson R Diversity and development of circadian rhythms in the European rabbit Chronobiol Int 2001 18 1 26 11247109 10.1081/CBI-100001275
Hudson R Distel H Pheromonal release of suckling in rabbits does not depend on the vomeronasal organ Physiol Behav 1986 37 123 128 3737709 10.1016/0031-9384(86)90394-X
Jilge B The ontogeny of circadian rhythms in the rabbit J Biol Rhythms 1993 8 247 260 8280913
Hudson R Labra-Cardero D Mendoza-Soylovna A Sucking, not milk, is important for the rapid learning of nipple-search odors in newborn rabbits Dev Psychobiol 2002 41 226 235 12325137 10.1002/dev.10073
Alvarez MP Cardinali DP Jimenez V Alvariño M Esquifino AI 24-Hour rhythm of plasma prolactin in female rabbit pups. Correlation with hypothalamic and adenohypophysial dopamine, serotonin, gamma-aminobutyric acid and taurine content Animal Reprod Sci
Alvarez MP Cardinali DP Cano P Rebollar P Esquifino AI Prolactin circadian rhythm in male lactating rabbits J Circad Rhythms 2005 3 1 10.1186/1740-3391-3-1
Esquifino AI Arce A Villanua MA Cardinali DP Development of 24-hour rhythms in serum prolactin and luteinizing hormone levels in rats neonatally administered melatonin Chronobiol Int 1998 15 21 28 9493711
Garcia-Bonacho M Esquifino AI Castrillon PO Toso CR Cardinali DP Age-dependent effect of Freund's adjuvant on 24-hour rhythms in plasma prolactin, growth hormone, thyrotropin, insulin, follicle-stimulating hormone, luteinizing hormone and testosterone in rats Life Sci 2000 66 1969 1977 10821121 10.1016/S0024-3205(00)00522-1
Jilge B Stahle H The internal synchronization of five circadian functions of the rabbit Chronobiol Int 1984 1 195 204 6600026
Jilge B Hornicke H Stahle H Circadian rhythms of rabbits during restrictive feeding Am J Physiol 1987 253 R46 R54 3605390
Jilge B Restricted feeding: a nonphotic zeitgeber in the rabbit Physiol Behav 1992 51 157 166 1741443 10.1016/0031-9384(92)90218-Q
Jilge B A feeding-entrainable circadian oscillator system in the rabbit Ann Ist Super Sanita 1993 29 511 520 7985917
Ojeda SR Urbanski HF Ahmed CE The onset of female puberty: studies in the rat Recent Prog Horm Res 1986 42 385 442 3090657
Kanematsu S Scaramuzzi RJ Hilliard J Sawyer CH Patterns of ovulation-inducing LH release following coitus, electrical stimulation and exogenous LH-RH in the rabbit Endocrinology 1974 95 247 252 4600114
Dufy-Barbe L Dufy B Vincent JD Serum gonadotropin levels in the ovariectomized rabbit: effect of acute and chronic administration of estradiol Biol Reprod 1978 18 118 124 626762
Ramírez VD Soufi WL Knobil E, Neill J The neuroendocrine control of the rabbit ovarian cycle The physiology of reproduction 1994 New York: Raven Press 595 611
Anderson CO Zarrow MX Denenberg VH Maternal behavior in the rabbit: Effects of androgen treatment during gestation upon the nest-building behavior of the mother and her offspring Horm Behav 1970 1 337 345 10.1016/0018-506X(70)90026-7
Hudson R Distel H Sensitivity of female rabbits to changes in photoperiod as measured by pheromone emission J Comp Physiol [A] 1990 167 225 230 2213657
Jilge B Ontogeny of the rabbit's circadian rhythms without an external zeitgeber Physiol Behav 1995 58 131 140 7667410 10.1016/0031-9384(95)00006-5
Nishihara E Nagayama Y Inoue S Hiroi H Muramatsu M Yamashita S Koji T Ontogenetic changes in the expression of estrogen receptor alpha and beta in rat pituitary gland detected by immunohistochemistry Endocrinology 2000 141 615 620 10650942 10.1210/en.141.2.615
Seron-Ferre M Torres C Parraguez VH Vergara M Valladares L Forcelledo ML Constandil L Valenzuela GJ Perinatal neuroendocrine regulation. Development of the circadian time-keeping system Mol Cell Endocrinol 2002 186 169 173 11900892 10.1016/S0303-7207(01)00682-7
Moore-Ede MC Physiology of the circadian system: Predictive versus reactive homeostasis Am J Physiol 1986 250 R737 R752 3706563
Hastings MH Reddy AB Maywood ES A clockwork web: circadian timing in brain and periphery, in health and disease Nat Rev Neurosci 2003 4 649 661 12894240 10.1038/nrn1177
Greco AM Gambardella P Sticchi R DÆAponte D Di Renzo G de Franciscis P Effects of individual housing on circadian rhythms of adult rats Physiol Behav 1989 45 363 366 2474174 10.1016/0031-9384(89)90141-8
Sgoifo A Pozzato C Meerlo P Costoli T Manghi M Stilli D Olivetti G Musso E Intermittent exposure to social defeat and open-field test in rats: acute and long-term effects on ECG, body temperature and physical activity Stress 2002 5 23 35 12171764
Spani D Arras M Konig B Rulicke T Higher heart rate of laboratory mice housed individually vs in pairs Lab Anim 2003 37 54 62 12626072 10.1258/002367703762226692
Esquifino AI Alvarez MP Cano P Chacon F Reyes Toso CF Cardinali DP 24-Hour pattern of circulating prolactin and growth hormone levels and submaxillary lymph node immune responses in growing male rats subjected to social isolation Endocrine 2004 25 41 48 15545705 10.1385/ENDO:25:1:41
Esquifino AI Chacon F Jimenez V Reyes Toso C Cardinali DP 24-Hour changes in circulating prolactin, follicle-stimulating hormone, luteinizing hormone and testosterone in male rats subjected to social isolation J Circad Rhythms 2004 2 1 10.1186/1740-3391-2-1
Meaney MJ Diorio J Francis D Widdowson J LaPlante P Caldji C Sharma S Seckl JR Plotsky PM Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress Dev Neurosci 1996 18 49 72 8840086
Ubilla E Alvarino JMR Esquifino A Agrasal C Effects of induction of parturition by administration of a prostaglandin F2[alpha] analogue in rabbits: possible modification of prolactin, LH and FSH secretion patterns Animal Reprod Sci 1992 27 13 20 10.1016/0378-4320(92)90066-M
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-541616475210.1186/1742-4690-2-54ResearchThe HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix Fraedrich Kirsten [email protected]üller Birthe [email protected] Ralph [email protected] Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany2005 15 9 2005 2 54 54 12 7 2005 15 9 2005 Copyright © 2005 Fraedrich et al; licensee BioMed Central Ltd.2005Fraedrich 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 Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4.
Results
To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity.
Conclusion
Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.
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Background
The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is an essential regulator of viral replication and a critical determinant of the HTLV-induced diseases. These include the aggressive and fatal malignancy of CD4+ T-lymphocytes termed adult T-cell leukemia (ATL) [1-3]. Several lines of evidence indicate that p40tax is the oncogene responsible for viral lymphocyte-transforming and leukemogenic properties [4-7]. Mechanistically, several biochemical features of the protein can cooperate to transform, among them transcriptional stimulation of cellular signal transducers, cytokines [8-11] and anti-apoptotic effectors. Tax' capacity to stimulate aneuploidy and to interfere with DNA repair [12] could indirectly support malignant progression. A major mechanistic explanation for the mitogenic and immortalizing effects of the Tax oncoprotein is provided by its ability to stimulate the G1- to S-phase transition in T-cells [6,13-15].
In mammalian cells, G1-progression is controlled by the sequential activation of several cyclin-dependent kinases (CDKs), starting with CDK4, CDK6 and CDK2. Tax activates CDK4, CDK6 and CDK2 leading to phosphorylation of retinoblastoma (Rb) tumor suppressor proteins and liberation of the transcription factor E2F [6,16]. Moreover, Tax may also induce Rb degradation [17] and increases cellular E2F synthesis [18,19]. Several indirect effects of Tax and features of HTLV-infected cells may support the impact of Tax on CDK. For example, HTLV-1-infected T-cells contain increased levels of cyclin D2 [16,20,21], which upon binding to CDK4 forms functional holoenzyme complexes. Cyclin D2 expression is upregulated by interleukin-2 receptor (IL2-R) signals [22-24]. Tax may cooperate with interleukin-2 (IL-2) signaling either indirectly through stimulating the expression of IL-2Rα or directly by activating the cyclin D2 promoter [21,25]. Furthermore, expression of CDK inhibitory proteins, like p18INK4C [20], p19INK4D and p27Kip1[16,26] is reduced in the presence of Tax. By contrast, the inhibitory protein p21CIP1 is strongly upregulated in Tax-containing cells [20,27]. Tax also represses the function of distinct tumor suppressor proteins which interfere with G1- to S-phase transition. These include p16INK4A, p15INK4B [26,28,29] and p53 [30-35].
The protein-protein contact with the components of the cyclin D/CDK complexes provides a major explanation for the G1-phase stimulating effects of Tax. The Tax interaction with the CDK and cyclin component is direct and specific. This interaction is detectable in vitro, in transfected fibroblasts, HTLV-1-infected T-cells, and ATL-derived cultures [36,37]. The Tax-CDK complex represents an active holoenzyme. Direct association with Tax enhances CDK4 activity. This increased kinase activity in the presence of Tax may be explained by intensified association of CDK4 and its positive cyclin regulatory subunit and by resistance of the complex to inhibition by p21CIP1 [36,37].
To understand the molecular mechanism of the Tax-mediated CDK4 activation, the interacting domains of Tax and CDK4 were characterized. Here we show that a segment of 40 amino acids derived from the N-terminus of Tax is sufficient to bind CDK4 and cyclin D2. To define a Tax-binding domain, a series of CDK4 deletion mutants was tested in different assays. These point at two regions derived from the N- and C-terminus of CDK4 which upon deletion consistently result in reduced binding capacity. The potential of these isolated regions to interact with Tax was demonstrated by mammalian two-hybrid analysis. These experiments concurrently revealed Tax-binding at the N- and C-terminus of CDK4.
Results and discussion
Capacity of the isolated N-terminus of Tax to bind cyclin D2- and CDK4
N-terminal Tax mutants bind neither CDK4 nor cyclin D2 and are incapable to stimulate CDK holoenzyme activity. This indicates that the region is required for binding and activation. To investigate whether this segment is also sufficient for binding to cyclin D2 and CDK4, the coding sequence of the N-terminal fragment (codons 1–40) was cloned into the prokaryotic expression vector pET29b+ (Figure 1A). The corresponding protein (TaxM1-R40) and Taxwt were produced in E. coli and coupled to S-protein agarose (Figure 1B). To demonstrate direct interaction, in vitro binding assays were performed. For this purpose, 35S-labeled cyclin D2, CDK4 and, as a control, cyclin E were synthesized in vitro. All in vitro translation reactions resulted in major bands of the expected size in equal amounts (Figure 1C Input). Cyclin E was produced in two previously observed isoforms [38]. Bands of minor intensity are most probably due to incorrect in vitro translation products and were ignored for quantitation. For binding analysis aliquots of the agarose-coupled TaxM1-R40 and Taxwt (Figure 1B) were incubated with the in vitro-translated proteins. As Figure 1C (Precipitation) shows, incubation with TaxM1-R40 and Taxwt resulted in significant amounts of cyclin D2 and CDK4. By contrast, both of the cyclin E isoforms were significantly less precipitated. Three independent experiments were quantitated. They revealed a 3.5 – 5 fold increased protein binding of TaxM1-R40 to CDK4 and cyclin D2 compared to the cyclin E control (Figure 1D). The binding to CDK4 of the N-terminal peptide compared with full length Tax was slightly reduced. This may indicate structure differences rather than the contribution of other Tax regions in CDK4 binding. The interaction of the N-terminal Tax fragment with cyclin D2 could be reproduced with natural folded proteins in pull down experiments (Figure 1E). Cyclin D2- and cyclin E-containing lysates derived from transfected 293T cells were incubated with bacterially expressed TaxM1-R40 and Taxwt, immobilized on S-agarose (Figure 1B). Subsequent analysis of bound proteins by immunoblots revealed that the N-terminal Tax peptide interacted with cyclin D2 but not with cyclin E. In summary, these results demonstrate that a N-terminal peptide of Tax, spanning amino acids 1 – 40, is sufficient for direct and specific interaction with both, cyclin D2 and CDK4. These results are in agreement with the capacity of the 40 N-terminal amino acids of Tax to bind CDK4 in a yeast two-hybrid system and in pull down analyses [39]. In extension, we demonstrated the interaction with naturally folded CDK4 protein produced in human cells. The binding of both, CDK4 and cyclin D2, by this Tax domain could cause a spacially close positioning of these proteins and thus stimulate CDK4 – cyclin D2 holoenzyme formation. This could be part of the mechanistic explanation for the enhancement of CDK4 kinase activity induced by a synthetic N-terminal Tax peptide [39]. Furthermore, this may explain the increased affinity of cyclin to CDK in the presence of Tax [36]. In addition, Tax could influence kinase activity through mediating cyclin phosphorylation by its direct contact [14]. This phosphorylation appears in cyclins which are actively complexed to cognate CDKs [40,41] and may impair cyclin degradation via the ubiquitin proteasome pathway [42].
Figure 1 Binding of the isolated Tax N-terminus to CDK4 and cyclin D2. A) Physical map of Tax's functional domains and the position of the N-terminal peptide B) Taxwt and TaxM1-R40 were produced in E. coli and coupled to S-protein agarose. The figure depicts a coomassie brilliant blue-stained SDS-PAA gel loaded with the purified protein coupled to S-protein agarose and samples before and after induction with IPTG. C) CDK4, cyclin D2 and cyclin E were translated in vitro and incubated with S-agarose coupled, E.coli-produced Taxwt and TaxM1-R40. Bound proteins were detected in gels by phosphoimaging (precipitation). To control for equal inset, aliquots of the radioactive proteins were subjected to gel electrophoresis (input). D) The radioactive signals of bound proteins of two independent experiments were quantitatively evaluated. The figure depicts the mean relative binding. E) For in vivo pull-down analysis, cyclin D2 and cyclin E plasmids were transfected into 293T cells. Lysates were incubated with S-agarose coupled to Taxwt or the N-terminal peptide (TaxM1-R40). Bound proteins and aliquots of the lysates were subjected to gel electrophoresis and immunoblotting, using polyclonal cyclin D2 and cyclin E antibodies.
Relevance of N- and C-terminal CDK4 regions for Tax-binding in vitro
In order to understand whether domains, which are relevant for regulating CDK4 activity, are affected by Tax, Tax-binding CDK4 sequences were defined. For this purpose, a series of deletion mutants was generated which cover the complete coding region of CDK4 (Figure 2A). To identify CDK4 sequences, which are relevant for Tax-binding in the absence of other cellular components, in vitro binding assays were performed. Aliquots of the S-protein agarose matrix coupled Taxwt (Figure 1B) were incubated with the in vitro-translated, 35S-labeled CDK4 mutants. Subsequently, Tax-bound CDK4 mutants were collected (Figure 2B Pull down). Equal inset of the in vitro-translated proteins was verified (Figure 2B Input). As a background control, uncoupled S-protein agarose was incubated with the in vitro-translated proteins. The immobilized proteins were subjected to gel electrophoresis and quantitated by measuring the radioactivity of the specific bands. To determine relative Tax-binding, the ratio between the specific signal and the background was calculated. The results of three independent experiments (Figure 2C) show reduced relative binding compared to wild-type of three CDK4 deletion mutants in two regions. Two of them, CDK4dM1-F31 and CDK4>dH30-V72, affected a N-terminal region. In addition, a C-terminal mutant CDK4dL272-E303 did interact at reduced levels with Tax. Thus, the N-terminal region from amino acids 1–72 and the C-terminal region from amino acids 272–303 of the CDK4 protein directly interact with Tax. Alternatively, the deletion of these regions may reduce the protein's affinity to Tax by affecting its conformation.
Figure 2 Identification of a CDK4 region important for direct Tax interaction. A) For binding assays, CDK4 mutants were constructed via PCR and cloned into the mammalian expression vector pcDNA3.1MycHis. B) CDK4 and its mutants were translated in vitro and reacted with S-agarose-coupled Taxwt. As a control, translated proteins were also incubated with uncoupled S-Agarose. Examples of resulting phosphorimager scans are shown. C) The diagram shows the mean Tax binding and standard deviation of three independent experiments that were quantitatively evaluated.
Relevance of the N-terminal CDK4 domain for binding in vivo
In order to characterize CDK4 sequences relevant for in vivo interaction, Tax and the CDK4 deletion mutants were coexpressed in transfected 293T cells in equal amounts (Figure 3A, lysates). Subsequently, coimmunoprecipitation experiments were performed (Figure 3A, α-Tax-IP) using a Tax-specific antibody. The resulting immunoblots were stained with CDK4 and Tax-specific immune reactions. These revealed a reduced affinity of Tax to some mutants, in particular to CDK4dH30-V72 and CDK4dA182-K211. To quantitate binding, the amounts of coimmunoprecipitated CDK4- and Tax-proteins were determined. The ration of both was taken as relative binding. The mean from two independent experiments shows that three CDK4 deletion mutants (CDK4dH30-V72, CDK4dS150-R181, CDK4dA182-K211) in two regions have significantly reduced binding affinity to Tax (Figure 3B). The mutants CDK4dH30-V72 and CDK4dM1-F31, which also appears to be reduced in binding, represent the same N-terminal region, which was identified in the in vitro binding assays. In addition, two mutants in the central part of CDK4 (CDK4dS150-R181, CDK4dA182-K211) resulted in reduced Tax binding. Since this central region was not required in vitro, its deletion may affect the CDK4 structure in vivo, thus rendering it inaccessible for Tax-binding. The deletion of the C-terminal amino acids (CDK4dL272-E303) did not affect Tax-binding, indicating that this part is not essential for in vivo-binding and may be replaced by cellular factors. Moreover, this result may indicate that in vivo the N-terminus is sufficient for Tax-binding. Thus, the in vivo binding experiments confirmed the relevance of the N-terminal CDK4 region for Tax-binding.
Figure 3 Deletion of two regions in CDK4 interferes with Tax-binding in vivo. A) Tax and CDK4 mutants were coexpressed in transfected 293T cells. The complexes were immunoprecipitated by monoclonal Tax antibodies and protein A sepharose. To detect Tax-bound CDK4 mutants, complexes and lysate controls were subjected to gel-electrophoresis and Western blotting. One representative experiment is shown. B) Luminescence emitted by specific bands of two independent experiments was quantitative evaluated and the mean relative Tax binding was calculated.
Tax-binding activity of isolated CDK4 regions in vivo
To investigate the affinity to Tax of those CDK4 regions, which upon deletion affected Tax-binding, mammalian two-hybrid assays were performed. All corresponding CDK-sequences were cloned into the DNA-binding domain containing vector(Figure 4A). The N-terminal region, which was found to be important for Tax-binding in vitro and in vivo, is included in plasmid pCDK4M1-V71. The other regions, which affected Tax-binding in only one assay, are represented by the constructs CDK4V242-E303 (C-terminal region) and CDK4S150-K211 (central region). As a control, CDK4L100-T149 was constructed, which contains a region whose deletion did not affect Tax-binding in all assays. In addition, the deletion mutant CDK4dH30-V72 was inserted into the two-hybrid vector. The coding sequence of the CDK4-binding Tax domain (amino acids M1 – R40) was assembled into the DNA activation domain containing other two-hybrid vector. To test for interaction, human fibroblasts (293 cells) were co-transfected with these constructs and luciferase assays were performed. Whereas Firefly luciferase indicated the binding activity, Renilla luciferase, which is constitutively expressed from one plasmid, was analyzed as internal transfection control. Relative luciferase activity was calculated as the ratio of Firefly to Renilla luciferase activity. The mean relative luciferase activity of three independent experiments is shown in Figure 4B. Only two of the CDK4 constructs, CDK4M1-V71 and CDK4V242-E303, yielded significant amounts of relative luciferase activity, indicating direct interaction with TaxM1-R40. This demonstrates that the N-terminal region of CDK4 (peptide CDK4M1-V71), which upon deletion reduced binding affinity in vivo and in vitro, bound TaxM1-R40 in the two-hybrid assay. In agreement with the notion that the binding domain is absent, the mutant CDK4dH30-V72, lacking 42 of these amino acids, consistently showed no binding capacity in all assays. The peptide CDK4S150-K211, which represents the CDK4 region affecting Tax-binding exclusively in vivo, revealed no binding in the two-hybrid assay. In contrast, the C-terminal peptide CDK4V242-E303, representing the region of CDK4 affecting Tax-binding in vitro, bound TaxM1-R40. In agreement with the other assays, the peptide CDK4L100-T149 did not bind. Taken together, the results of all binding assays consistently identified the CDK4 N-terminus as main interaction domain for Tax (Figure 5A). The CDK4 C-terminus, which could directly interact with Tax, may cooperate with the N-terminus, although it was not essential for Tax-binding in vivo.
Figure 4 Interaction of CDK4 and Tax peptides in an eukaryotic two-hybrid assay. A) The coding sequence of CDK4 peptides and a CDK4 deletion mutant were constructed via PCR and assembled into the GAL4 DNA-binding domain-expressing vector. The sequence of the CDK reactive N-terminus of Tax was inserted into the VP16 activation domain-expressing vector. B) To test for interaction, CDK4-containing plasmids were co-transfected with the Tax plasmid into 293 cells and luciferase assays were performed. The mean of three independent experiments is shown.
Figure 5 Model of Tax-CDK4 interaction. A) Map of CDK4 regions relevant for Tax-binding. The N-terminal region of CDK4 is relevant in all binding assays, suggesting that it is the major binding region. In addition, the C-terminus is considered as a second possible binding region. Red: regions, which upon deletion result in reduced binding; green: regions, which bind to Tax. B) Tertiary structure prediction of CDK4. The structure was calculated from the amino acid sequence at Swiss Model . The resulting pdb file was visualized with rasmol. The prediction shows the proximity of N- and C-terminal regions in the folded CDK4 protein. Thus, it is conceivable that both represent a non-contiguous binding domain for Tax. Red: C-terminal segment; blue:N-terminal segment
To get an impression about the molecular interaction with the folded protein, a three-dimensional structure of CDK4 was calculated (Figure 5B). It resembles the structure of cdk2, which was determined from crystallized protein by X-ray diffraction [43]. As cdk2, the predicted structure is bi-lobated, containing a β-sheet-rich N-terminal and a alpha-helix-rich C-terminal region. This structure reveals that the N- and C-terminus of CDK4 are neighbouring. Thus, it is possible that both together provide a non-continuous binding domain for Tax. The N-terminus contains the PSTAIRE helix of CDK4, which is part of the CDK's cyclin D2 binding domain. Its rotation during the activation of CDK4 is required to unblock the catalytic cleft of the kinase [44]. Binding of Tax to this region may influence its spacial arrangement. Thus, Tax in cooperation with cyclin D2 could support formation of the active conformation and stimulate CDK4 activity by influencing the PSTAIRE helix.
Conclusion
The 40 N-terminal amino acids of Tax are sufficient to bind cyclin D2 and CDK4. Within CDK4 a N- and a C-terminal domain are relevant for Tax binding. These domains are neighbouring in the predicted three dimensional protein structure. Taken together, these findings suggest that Tax stimulates G1- to S-phase transition by supporting the association of CDK4 and cyclin D2. Furthermore, they support the conclusion that CDK4 activity is stimulated through conformational changes of the enzyme directly mediated by Tax.
Methods
Generation of CDK4 deletion mutants
All CDK4 deletion mutants were generated via PCR [45]. In order to introduce the internal deletions, 16 different primers were used, two outside 28-mer oligonucleotides spanning the 5' and 3' ends of the CDK4 open reading frame (CDK4S and CDK4AS) and 14 chimeric oligonucleotides designed to carry the 5' and 3' sequences flanking the deleted regions. After three rounds of PCR with Pwo polymerase (Roche, Mannheim, Germany), the deleted clones CDK4dH30-V72, CDK4dV70-L100, CDK4dR101-L120, CDK4dM121-S150, CDK4dS150-R181, CDK4dA182-K211, CDK4dK211-D241, CDK4dV242-M275 were created. To engineer the N- terminal CDK4dM1-F31 and C-terminal CDK4dL272-E303 deletion clones, one round of PCR was performed by using an internal 5' primer or 3' primer in combination with the corresponding outside primer. To engineer the CDK4 full length construct one round of PCR was performed with the outside primers. The resulting PCR products were digested with BamHI and HindIII and ligated via these sites into the pcDNA3.1(-)/Myc-His A expression vector (Invitrogen, Karlsruhe, Germany). The resulting clones were verified by nucleotide sequencing.
Coimmunoprecipitation
Human 293T cells were kept and transfected for coimmunoprecipitations as described [36]. Briefly, cells were lysed in buffer containing 50 mM Tris, 150 mM NaCl, 0.2% Tween 20, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml aprotinin. To immunoprecipitate Tax and associated proteins cleared protein supernatant (0.7 to 1 mg whole protein) were incubated for 1 h at 4°C with 1 μg of monoclonal Tax antibody and the immune complexes were collected by protein A-Sepharose CL4B (Pharmacia) beads (1 h at 4°C). Beads with the precipitated proteins were washed three times with lysis buffer. An aliquot of protein supernatant was taken as lysate control (40 μg whole protein). Immunoprecipitates and lysate controls were separated on gels and electro-blotted. Subsequently, membranes were incubated with 5% nonfat dry milk to block unspecific binding before reacting them with a 1: 200 dilution of monoclonal Tax antibody for 1 h at room temperature. Membranes were washed and incubated with a 1:2.500 dilution of an anti-mouse immunoglobulin G-horse-radish peroxidase conjugate (Amersham, Freiburg, Germany). Bound antibodies were visualized with an enhanced chemiluminescence detection system (Amersham) and CCD-camera. The luminescence of specific bands was quantitated from the digitalized image by using the program AIDA (raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany).
In vitro binding and pull down assays
35S-methionine labeled CDK4 and mutants were produced in vitro with a rabbit reticolocyte-based in vitro transcription/translation system (Promega, Mannheim, Germany). To prevent the expression of the myc/his-tag, the inset plasmids were digested with HindIII prior to translation. Tax was produced in E.coli and coupled to S-protein-agarose as previousely described [36]. For a binding assay 5–10 μl of the in vitro-translated protein was diluted in 500 μl of RIPA buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1 % Nonidet P-40, 0.5 % desoxycholat, 0.1 % sodium dodecyl sulfate). An aliquot of 10 μl was taken as an inset control. The S-protein-agarose-bound Tax protein (15 μl) was incubated with the radioactive proteins for 1 h at 4°C, washed with RIPA-buffer and recovered by boiling the beads in loading buffer. Proteins were sized on an SDS-12% polyacrylamide gel, quantitated and visualized by a phosphorimager.
TaxM1-R40 was generated via PCR, using the primers TaxM1-R40 -pet-S and TaxM1-R40 -pet-AS and plasmid pcTax [46] as template. Resulting PCR products and the pet 29b + vector (Novagen, Bad Soden, Germany) were digested with BamHI and HindIII and ligated. Resulting clones were verified via sequencing. Cyclin D2 and cyclin E were transfected in 293T cells and lysates were prepared as previously described [36]. A lysate control was performed with 40 μg whole protein. Lysates containing 0.5 – 1 mg whole protein were incubated with E.coli-produced Taxwt or TaxM1-R40, coupled to Ni-NTA agarose for 1 h, washed with lysis buffer and recovered by boiling the beads in loading buffer. Proteins were sized on a 12%-SDS-PAA gel, transferred onto a nitrocellulose transfer membrane and stained with specific antibodies.
Mammalian two-hybrid assay
All constructs for mammalian two-hybrid assay were generated via PCR. The TaxM1-R40 construct PCR was performed with the primer TaxM1-R40 -M2H-S and TaxM1-R40 -M2H-AS using the plasmid pcTax as a template. For the CDK4 constructs CDK4dV70-L100 the pcDNA3.1(-)/Myc-His A construct was used as a template. The resulting PCR products were digested with KpnI and XbaI. For the other CDK4 constructs CDK4M1-V71, CDK4L100-T149, CDK4S150-K211 and CDK4V242-E303 the CDK4 full length pcDNA3.1(-)/Myc-His A construct was used as template. The resulting PCR products were digested with BamHI and XbaI. The digested products were ligated into the vectors pBind and pAct (CheckMate Mammalian two-hybrid system, Promega). The vector pG5luc contains the reporter gene (Firefly luciferase). Human 293 cells were transfected with the plasmids using Lipofectamine reagents (Invitrogen). The luciferase-assay was performed with the Dual-Luciferase reporter assay (Promega) using a microplate luminometer.
Oligonucleotides
Designation for primers correspond to the plasmid names. The oligonucleotides sequences were as follows:
CDK4S, 5'-ATTTACGGATCCACCATGGCTACCTCTC-3' (outer primer);
CDK4AS, 5'-ATCCCCAAGCTTCTCCGGATTACCTTCA-3' (outer primer);
CDK4dM1-F31S, 5'-ATTTACGGATCCATGGTGGCCCTCAAGA-3';
CDK4dH30-V72S, 5'-CACAGTGGCCACTTTGTCCGGCTGSTGGAC-3';
CDK4dH30-V72AS, 5'-GTCCATCAGCCGGACAAAGTGGCCACTGTG-3';
CDK4dV70-L100S, 5'-GCTTTTGAGCATCCCAATAGGACATATCTGGACAAG-3';
CDK4dV70-L100AS, 5'-CTTGTCCAGATATGTCCTATT GGATGCTCAAAAGC-3';
CDK4dM121-S150S, 5'-GAAACGATCAAGGATCTGGGTGGAACAGTCAAGCTG-3';
CDK4dM121-S150AS, 5'-CAGCTTGACTGTTCCACCCAGATCCTTGATGGTTTC-3';
CDK4dS150-R181S, 5'-AACATTCTGGTGACAAGTGTTACACTCTGGTACCGA-3';
CDK4dS150-R181AS, 5'-TCGGTACCAGAGTGTAACACTTGTCACCAGAATGTT-3';
CDK4dA182-K211S, 5'-GCTCCCGAAGTTCTTCTGCCTCTCTTCTGTGGAAAC-3';
CDK4dA182-K211AS, 5'-GTTTCCACAGAAGAGAGGCAGAAGAACTTCGGGAGC-3';
CDK4dK211-D241S, 5'-GCAGAGATGTTTCGTCGAGATGTATCCCTGCCCCGT-3';
CDK4dK211-D241AS, 5'-ACGGGGCAGGGATACATCTCGACGAAACAGCTCTGC-3';
CDK4dV242-M275S, 5'-GATGACTGGCCTCGAGATCTGACTTTTAACCCACAC-3';
CDK4dV242-M275AS, 5'-GTGGGTGTTAAAAGTCAGATCTCGAGGCCAGTCATC-3';
CDK4dL272-E303AS, 5'-ATTTAGAAGCTTCAGCAGCTGTGCTCCC-3';
TaxM1-R40 -M2H-S, 5'-TCATCTAGAATGGCCCATTTCCCAGGGTT-3'(outer primer);
TaxM1-R40 -M2H-AS, 5'-ATTGGTACCTAGGCGGGCCGAACATAGTC-3'(outer primer);
CDK4-M2H-S, 5'-CCTTGGATCCTAATGGCTACCTCTC-3'(outer primer);
CDK4-M2H-AS, 5'-GCATTCTAGACGCCTCCGGATTACCTT-3'(outer primer);
CDK4M1-V71-AS, 5'-GCATTCTAGACGCAACATTGGGATGCTCAAA-3';
CDK4L100-T149-S: 5'-CCTTGGATCCTACTAAGGACATATCTGGAC-3';
CDK4L100-T149-AS: 5'-GCATTCTAGACGCTGTCACCAGAATGTTCTC-3';
CDK4S150-K211 -S: 5'-CCTTGGATCCTAAGTGGTGGAACAGTCAAG-3';
CDK4S150-K211 -AS: 5'-GCATTCTAGACGCCTTTCGACGAAACATCTC-3';
CDK4V242-E303-S: 5'-CCTTGGATCCTAGATGTATCCCTGCCCCGT-3';
TaxM1-R40 -pet-S: 5'-GATCGGATCCGATGGCCCATTTCCCAGGGTT-';
TaxM1-R40 -pet-AS: 5'-CTAATTAAGCTTTAGGCGGGCCGAACATAGTCCCCCAGAGATG-3',
Competing interests
The author(s) declare, that they have no competing interests.
Authors' contributions
KF performed most of the experiments. BM did experiments shown in Figure 1. Both KF and RG participated in experimental design, data interpretation and writing of manuscript. All authors have critically read the manuscript and approved the final version to be published.
Acknowledgements
We thank Kerstin Haller and Ewa Blazejewska for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (SFB466-C3) and the Wilhelm Sander-Stiftung (2004.019.1).,
==== Refs
Osame M Pathological mechanisms of human T-cell lymphotropic virus type I-associated myelopathy (HAM/TSP) J Neurovirol 2002 8 359 364 12402162 10.1080/13550280260422668
Matsuoka M Human T-cell leukemia virus type I and adult T-cell leukemia Oncogene 2003 22 5131 5140 12910250 10.1038/sj.onc.1206551
Matsuoka M Human T-cell leukemia virus type I (HTLV-I) infection and the onset of adult T-cell leukemia (ATL) Retrovirology 2005 2 27 15854229 10.1186/1742-4690-2-27
Grassmann R Berchtold S Radant I Alt M Fleckenstein B Sodroski JG Haseltine WA Ramstedt U Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture J Virol 1992 66 4570 4575 1351105
Akagi T Shimotohno K Proliferative response of Tax1-transduced primary human T cells to anti-CD3 antibody stimulation by an interleukin-2-independent pathway J Virol 1993 67 1211 1217 8437212
Schmitt I Rosin O Rohwer P Gossen M Grassmann R Stimulation of cyclin-dependent kinase activity and G1- to S-phase transition in human lymphocytes by the human T-cell leukemia/lymphotropic virus type 1 Tax protein J Virol 1998 72 633 640 9420268
Azran I Schavinsky-Khrapunsky Y Aboud M Role of Tax protein in human T-cell leukemia virus type-I leukemogenicity Retrovirology 2004 1 20 15310405 10.1186/1742-4690-1-20
Wäldele K Schneider G Ruckes T Grassmann R Interleukin-13 overexpression by tax transactivation: a potential autocrine stimulus in human T-cell leukemia virus-infected lymphocytes J Virol 2004 78 6081 6090 15163701 10.1128/JVI.78.12.6081-6090.2004
Chung HK Young HA Goon PK Heidecker G Princler GL Shimozato O Taylor GP Bangham CR Derse D Activation of interleukin-13 expression in T cells from HTLV-1-infected individuals and in chronically infected cell lines Blood 2003 102 4130 4136 12920029 10.1182/blood-2003-04-1043
Azimi N Brown K Bamford RN Tagaya Y Siebenlist U Waldmann TA Human T cell lymphotropic virus type I Tax protein trans-activates interleukin 15 gene transcription through an NF-kappaB site Proc Natl Acad Sci USA 1998 95 2452 2457 9482906 10.1073/pnas.95.5.2452
Ruckes T Saul D Van Snick J Hermine O Grassmann R Autocrine antiapoptotic stimulation of cultured adult T-cell leukemia cells by overexpression of the chemokine I-309 Blood 2001 98 1150 1159 11493464 10.1182/blood.V98.4.1150
Marriott SJ Lemoine FJ Jeang KT Damaged DNA and miscounted chromosomes: human T cell leukemia virus type I tax oncoprotein and genetic lesions in transformed cells J Biomed Sci 2002 9 292 298 12145525 10.1159/000064998
Liang MH Geisbert T Yao Y Hinrichs SH Giam CZ Human T-lymphotropic virus type 1 oncoprotein tax promotes S-phase entry but blocks mitosis J Virol 2002 76 4022 4033 11907241 10.1128/JVI.76.8.4022-4033.2002
Neuveut C Low KG Maldarelli F Schmitt I Majone F Grassmann R Jeang KT Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb Mol Cell Biol 1998 18 3620 3632 9584203
Neuveut C Jeang KT Cell cycle dysregulation by HTLV-I: role of the tax oncoprotein Front Biosci 2002 7 d157 d163 11779707
Iwanaga R Ohtani K Hayashi T Nakamura M Molecular mechanism of cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I Oncogene 2001 20 2055 2067 11360190 10.1038/sj.onc.1204304
Kehn K Fuente CL Strouss K Berro R Jiang H Brady J Mahieux R Pumfery A Bottazzi ME Kashanchi F The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation Oncogene 2005 24 525 540 15580311 10.1038/sj.onc.1208105
Ohtani K Iwanaga R Arai M Huang Y Matsumura Y Nakamura M Cell type-specific E2F activation and cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I J Biol Chem 2000 275 11154 11163 10753922 10.1074/jbc.275.15.11154
Lemasson I Thebault S Sardet C Devaux C Mesnard JM Activation of E2F-mediated transcription by human T-cell leukemia virus type I Tax protein in a p16(INK4A)-negative T-cell line J Biol Chem 1998 273 23598 23604 9722600 10.1074/jbc.273.36.23598
Akagi T Ono H Shimotohno K Expression of cell-cycle regulatory genes in HTLV-I infected T-cell lines: possible involvement of Tax1 in the altered expression of cyclin D2, p18Ink4 and p21Waf1/Cip1/Sdi1 Oncogene 1996 12 1645 1652 8622884
Santiago F Clark E Chong S Molina C Mozafari F Mahieux R Fujii M Azimi N Kashanchi F Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells J Virol 1999 73 9917 9927 10559304
Martino A Holmes JH Lord JD Moon JJ Nelson BH Stat5 and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2 J Immunol 2001 166 1723 1729 11160217
Moon JJ Rubio ED Martino A Krumm A Nelson BH A permissive role for phosphatidylinositol 3-kinase in the Stat5-mediated expression of cyclin D2 by the interleukin-2 receptor J Biol Chem 2004 279 5520 5527 14660677 10.1074/jbc.M308998200
Fung MM Chu YL Fink JL Wallace A McGuire KL IL-2- and STAT5-regulated cytokine gene expression in cells expressing the Tax protein of HTLV-1 Oncogene 2005 24 4624 4633 15735688 10.1038/sj.onc.1208507
Huang H Hu-Li J Chen H Ben Sasson SZ Paul WE IL-4 and IL-13 production in differentiated T helper type 2 cells is not IL-4 dependent J Immunol 1997 159 3731 3738 9378959
Suzuki T Narita T Uchida-Toita M Yoshida M Down-regulation of the INK4 family of cyclin-dependent kinase inhibitors by tax protein of HTLV-1 through two distinct mechanisms Virology 1999 259 384 391 10388662 10.1006/viro.1999.9760
Cereseto A Diella F Mulloy JC Cara A Michieli P Grassmann R Franchini G Klotman ME p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells Blood 1996 88 1551 1560 8781409
Suzuki T Kitao S Matsushime H Yoshida M HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards EMBO J 1996 15 1607 1614 8612584
Low KG Dorner LF Fernando DB Grossman J Jeang KT Comb MJ Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a J Virol 1997 71 1956 1962 9032327
Akagi T Ono H Tsuchida N Shimotohno K Aberrant expression and function of p53 in T-cells immortalized by HTLV-I Tax1 FEBS Lett 1997 406 263 266 9136898 10.1016/S0014-5793(97)00280-9
Mulloy JC Kislyakova T Cereseto A Casareto L LoMonico A Fullen J Lorenzi MV Cara A Nicot C Giam C Franchini G Human T-cell lymphotropic/leukemia virus type 1 Tax abrogates p53-induced cell cycle arrest and apoptosis through its CREB/ATF functional domain J Virol 1998 72 8852 8860 9765430
Pise-Masison CA Choi KS Radonovich M Dittmer J Kim SJ Brady JN Inhibition of p53 transactivation function by the human T-cell lymphotropic virus type 1 Tax protein J Virol 1998 72 1165 1170 9445014
Reid RL Lindholm PF Mireskandari A Dittmer J Brady JN Stabilization of wild-type p53 in human T-lymphocytes transformed by HTLV-I Oncogene 1993 8 3029 3036 8414503
Ariumi Y Kaida A Lin JY Hirota M Masui O Yamaoka S Taya Y Shimotohno K HTLV-1 tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBP sequestration Oncogene 2000 19 1491 1499 10734308 10.1038/sj.onc.1203450
Van PL Yim KW Jin DY Dapolito G Kurimasa A Jeang KT Genetic evidence of a role for ATM in functional interaction between human T-cell leukemia virus type 1 Tax and p53 J Virol 2001 75 396 407 11119608 10.1128/JVI.75.1.396-407.2001
Haller K Wu Y Derow E Schmitt I Jeang KT Grassmann R Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein Mol Cell Biol 2002 22 3327 3338 11971966 10.1128/MCB.22.10.3327-3338.2002
Kehn K Deng L De La FC Strouss K Wu K Maddukuri A Baylor S Rufner R Pumfery A Bottazzi ME Kashanchi F The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells Retrovirology 2004 1 6 15169570 10.1186/1742-4690-1-6
Resnitzky D Gossen M Bujard H Reed SI Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system Mol Cell Biol 1994 14 1669 1679 8114703
Li J Li H Tsai MD Direct binding of the N-terminus of HTLV-1 tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity Biochemistry 2003 42 6921 6928 12779347 10.1021/bi034369n
Matsushime H Roussel MF Ashmun RA Sherr CJ Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle Cell 1991 65 701 713 1827757 10.1016/0092-8674(91)90101-4
Steiner P Philipp A Lukas J Godden-Kent D Pagano M Mittnacht S Bartek J Eilers M Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes EMBO J 1995 14 4814 4826 7588611
Diehl JA Zindy F Sherr CJ Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquintin-proteasome pathway Genes & Development 1997 11 957 972 9136925
De Bondt HL Rosenblatt J Jancarik J Jones HD Morgan DO Kim SH Crystal structure of cyclin-dependent kinase 2 Nature 1993 363 595 602 8510751 10.1038/363595a0
Pavletich NP Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors J Mol Biol 1999 287 821 828 10222191 10.1006/jmbi.1999.2640
Pont-Kingdon G Creation of chimeric junctions, deletions, and insertions by PCR Methods Mol Biol 2003 226 511 516 12958538
Rimsky L Hauber J Dukovich M Malim MH Langlois A Cullen BR Greene WC Functional replacement of the HIV-1 rev protein by the HTLV-1 rex protein Nature 1988 335 738 740 3262832 10.1038/335738a0
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-551616805110.1186/1742-4690-2-55ResearchIdentification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage Strappe Padraig M [email protected] David W [email protected] Douglas [email protected] Begona [email protected] Maeve [email protected] James W [email protected] Andrew ML [email protected] Department of Medicine, University of Cambridge Addenbrooke's Hospital Cambridge CB2 2QQ, UK2 Centre for Brain Repair, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QQ, UK2005 16 9 2005 2 55 55 26 5 2005 16 9 2005 Copyright © 2005 Strappe et al; licensee BioMed Central Ltd.2005Strappe 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
Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells.
Results
HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors.
Conclusion
This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.
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Background
Viral vectors based on primate and non-primate lentiviruses have been shown to be efficient for gene delivery to a variety of cell types both in vitro and in vivo and may offer considerable advantages in gene therapy strategies [1,2]. Lentiviral vectors can provide stable gene expression following integration into the host chromosome and pseudotyping of these vectors with heterologous envelopes such as the G protein of Vesicular stomatitis virus (VSV) has provided a broad cell tropism [3]. Lentiviral vectors are particularly suited for transduction of non-dividing cells [4] such as those of the central nervous system [5] exemplified by successful therapeutic gene transfer to the brain of primates for treatment of experimentally induced Parkinson's disease [6]. Packaging of unspliced vector mRNA in the producer cell line is a key part in process of lentiviral vector production and measures to increase the packaging efficiency and to reduce self packaging of the Gag-Pol or other helper construct have contributed to increased vector titre and biosafety [7]. Lentiviral RNA packaging is achieved by an interaction between an RNA structure known as the packaging signal or psi and the nucleocapsid (NC) domain of the Gag structural polyprotein. This highly specific process results in the selection of unspliced viral mRNA from a high background of cellular mRNA. The packaging signals of several lentiviruses have been mapped by deletion and mutational analysis. For HIV-1, sequences between the major splice donor and the start codon of Gag have been shown to be important for efficient packaging [8]. HIV-1 may be the exception amongst lentiviruses since for HIV-2 and SIV, sequences upstream of the splice donor predominantly contribute to mRNA packaging [9,10] and in FIV regions in U5 and in the Gag coding sequence appear to be the major signals [11,12]. RNA packaging in HIV-2 has been shown to involve two novel mechanisms to increase specificity, cotranslational packaging and competition for limiting Gag polyprotein [13]. These differences in the location of the major packaging determinants may contribute to the ability of viral mRNA to be cross packaged by a heterologous Gag protein. The localisation of RNA capture in the cell is unclear although recent evidence suggests that the centrosome may be the primary site [14] and that the psi signal may act as a subcellular localisatio signal as well as a high affinity binding site for Gag. The resulting RNA-protein complex is then targeted to the plasma membrane where virion budding takes place.
The ability of one lentiviral Gag to cross-package the unspliced mRNA of another lentivirus species has been well demonstrated for HIV-1, which can cross-package HIV-2 [15], SIV [16,17] and FIV [18]. Both SIV and FIV Gag-Pol have been shown to cross-package HIV-1 mRNA [16,18], however HIV-2 Gag-Pol is unable to package HIV-1 mRNA [15]. How closely this reduced efficiency correlates with the effectiveness of gene transfer of cross-packaged vectors has not been assessed, in particular in appropriate primary cells. Cross-packaged lentiviral vectors have been shown to infect predominantly dividing cells in culture but transduction of neurons and CD34+ lymphocytes has only been shown qualitatively [16]. However chimeric vectors based on an SIV genome and an HIV-1 core were unable to transduce dendritic cells and had a reduced ability to transduce primary macrophages [19].
The production of lentiviral vectors for clinical trials requires that preparations do not contain replication competent lentiviruses (RCL). Development of PCR and sensitive culture based methods for detection of RCLs have confirmed the absence of RCLs in large production lots [20,21]. Production of RCLs can occur through homologous recombination, thus limiting the sequence similarity between the Gag-Pol construct and gene transfer vector will reduce the possibility of a recombination event. Gag-Pol and gene transfer vectors based on different lentiviruses will significantly reduce the risk of RCL production.
Transduction of the cells of the central nervous system (CNS), both brain and spinal cord, with lentiviral vectors has been well documented and long term therapeutic transgene expression has been reported with only a low level or transient immune/inflammatory response [22,23]. Furthermore, transduction of neural stem cells with lentiviral and adeno associated viral vectors expressing therapeutic genes that will affect differentiation and serve as markers of cell fate is a promising approach for procuring cells for transplantation into degenerated or damaged areas of the brain. Such cells have the potential to be useful for the treatment of Parkinson's disease, spinal cord injury and other inflammatory or destructive conditions of the CNS[24,25].
We investigated the cross packaging ability of the Gag-Pol components of HIV-1, HIV-2 and SIV and found a unique non-reciprocal packaging relationship between SIV Gag-pol and vectors based on HIV-2.
In this paper the tropism of these viruses is quantitated by examining the ability of a series of cross-packaged lentiviral vectors based on HIV-1, HIV-2 and SIV to transduce primary mixed glial cells which, are the predominant cell type in the injured brain or spinal cord. Qualitative data is also presented on the transduction of primary neuronal embryonic stem cells with cross-packaged vectors.
Results
Cross-Packaging of lentiviral RNA
Following concentration of viral vectors by ultracentrifugation, viral vector particle number was assessed by the reverse transcriptase assay, which gives a quantitative measure of RT in ng. The concentration of each viral vector was normalised to 4 ng/ μl following previous optimisation. The level of vector RNA in the producer cells was comparable as judged by fluorescence of the cells caused by expression of the transfected GFP containing vector. The levels of RNA packaged in virions were assessed by RT-PCR of the packaged transgene GFP, using specific primers. Figure 3A and 3B shows a limiting dilution PCR analysis of virion extracted RNA, reverse transcribed to cDNA and diluted serially from 1/10 and 1/20 to 1/40. Electrophoresis of PCR products reveals a limit of positivity and signal strength. In Figure 3(A) HIV-1 Gag-Pol is seen to efficiently package HIV-1 RNA and can also cross package HIV-2 vector RNA at similar levels, both to a limiting dilution of 1/20. In comparison cross packaging of SIV vector RNA by HIV-1 Gag-Pol is reduced and is similar to levels of SIV vector RNA packaged by SIV Gag-Pol to only a limiting dilution of 1/10. In Figure 3(B), SIV Gag-Pol efficiently cross packages HIV-2 vector RNA to a limiting dilution of 1/40, which is greater than the SIV homologous vector system (1/10) and the SIV Gag-pol + HIV-GFP vector system (1/10, data not shown). The ability of HIV-2 Gag-Pol to cross package HIV-1 and SIV vector RNA is significantly reduced compared to the homologous HIV-2 system which showed similar levels of packaged RNA to the HIV-1 homologous vector system.
Figure 3 Limiting dilution RT PCR of Virion associated GFP RNA. For each viral vector, four PCR s were performed containing a target cDNA at neat, 1/10, 1/20 and 1/40 dilution. A: Lanes 1–4, HIV-1 Gag-pol + HIV-1 Vector, Lanes 5–8, HIV-1 Gag-pol + SIV vector, Lanes 9–12, HIV-1 Gag-pol + HIV-2 vector, Lanes 13–16, SIV Gag-pol + SIV vector. B: Lanes 1–4, SIV Gag-pol + HIV-2 vector, Lanes 5–8, HIV-2 Gag-pol + HIV-2 vector, Lanes 9–12, HIV-2 Gag-pol + HIV-1 vector, Lanes 13–16, HIV-2 Gag-pol + SIV vector.
Gene transfer efficiency of cross packaged vectors
The semi quantitative PCR approach demonstrates levels of vector RNA packaged in comparable concentrations of virions, however the assay does not reflect the gene transfer efficiency of cross-packaged vectors. To address this, SVC2 cells were transduced with a range of vector-virion preparations at differing concentrations as measured by RT-assay. Figure 4 shows a series of FACS plots of GFP positive cells (lower right quadrant) following transduction with viral vector and this data is also described in tables 4A to 4C. HIV-1 Gag-Pol was used to package two separate HIV-1 vectors (+/-cPPT sequence), the gene transfer vector containing the cPPT demonstrated an increased transduction rate of SVC2 cells up to almost two fold with an input viral vector of 10 ng. Transfer of 20 ng of an HIV-2 vector packaged by HIV-1 Gag-Pol showed a similar transduction efficiency to that of the HIV-1 cPPT vector packaged by HIV-1 Gag-Pol, suggesting that the HIV-2 cPPT region also contributed to increased transduction. Transfer of an SIV vector expressing GFP, cross-packaged by HIV-1 Gag-Pol was significantly lower, almost six fold, compared to the homologous HIV-1 viral vector (-cPPT). It is not certain why this is nor why the homologous SIV system gave low/poorly reproducible results. Vector expression appeared comparable in producer cells. SIV Gag-Pol cross packaged and transferred an HIV-2 GFP vector at levels slightly higher than the homologous HIV-1 vector system. This is in contrast to the lack of gene transfer of a HIV-1 vector packaged by SIV Gag-Pol. The levels of HIV-2 vector RNA packaged by SIV Gag-Pol (Figure 3B) are also reflected in the high gene transfer efficiency. This packaging relationship between SIV and HIV-2 would appear to be non-reciprocal, with lower amounts of SIV vector RNA packaged by the HIV-2 Gag-Pol (Figure 3B) and no evidence of any significant gene transfer. Comparing the HIV-1 and HIV-2 homologous vector systems showed that levels of gene transfer to SVC2 cells were slightly higher for HIV-2 compared to a cPPT negative HIV-1 vector but lower when compared to the HIV-1 vector containing the cPPT region. HIV-2 Gag-Pol would appear to have no ability to cross-package and transfer HIV-1 vector, which is similar to a previous study [15] with no significant transduction of SVC2 cells.
Figure 4 FACS analysis of GFP expression in SV2 cells transduced with homologous and cross-packaged lentiviral vectors (10 ng of vector). Lower Right hand quadrant represents GFP positive cells. HIV-1 Gag-Pol + HIV-1GFP vector (A), HIV-1 Gag-Pol + HIV-1 cPPT-GFP vector (B), HIV-1 Gag-Pol + SIV GFP vector (C), HIV-1 Gag-Pol + HIV-2 GFP vector (D). HIV-2 Gag-Pol + HIV-2 GFP vector (E), HIV-2 Gag-Pol + SIV GFP vector (F), HIV-2 Gag-Pol + HIV-1 GFP vector (G). SIV Gag-Pol + SIV GFP vector (H), SIV Gag-Pol + HIV-2 GFP vector (I), SIV Gag-Pol + HIV-1 GFP vector (J).
One obvious difference between the vectors packaged is the presence of considerably more potential cis acting sequence in the HIV-2 based vector compared to the HIV-1 and SIV vectors. It is conceivable that the presence of extended cis acting sequence in the gag and pol genes alters the efficiency of packaging. From data using HIV-1 based vectors this would seem to be unlikely since the minimal HIV based vector packages at least as well as a less fully deleted version. Nevertheless to establish closer comparability we generated a series of further deletions in the HIV-2 based vector and compared gene transfer efficiency to that achieved with the minimally deleted vector. The vector series included one with near complete deletion of the Gag/Pol coding regions (pSVRΔGP-CMVGFP) and also the generation of a self inactivating (SIN) vector (pSVRΔSIN-CMVGFP) created by additional deletion in the 3' UTR. This will be copied into the 5'LTR during reverse transcription and thus inactivate the 5'LTR promoter such that expression of the transgene depends on the internal promoter. The deletion removing the Gag-Pol region extends into the first coding exons of Tat and Rev thus both of these vectors will be defective for these regulatory proteins and they are closely comparable to the HIV-1 and SIV vectors used. Using these HIV-2 based constructs we were able to demonstrate no difference in gene transfer ability with either the more extensively deleted or the SIN mutated vector. Examples of gene transfer efficiency are shown in Figure 6 in which the level of GFP expression on transfection and transduction of all of the HIV-2 vectors is comparable.
Figure 6 GFP expression from HIV-2 vectors following transfection inot produced cells and in cells transduced with the packaged vectors.
Transduction of CNS cell types
We decided to verify this unreported cross-packaging and gene transfer relationship between SIV Gag-Pol and a HIV-2 vector by first transducing rat primary mixed glial cultures. The cultures were transduced with either 40 ng or 20 ng of viral vector and the efficiency of transduction compared to that achieved with HIV-1 and HIV-2 homologous vector systems. Cells were immunostained for GFP expression and the astrocyte marker GFAP (Figure 7), and counted (Figure 8). Transducing the glial cultures with 20 ng of a SIV gag-pol+HIV-2 GFP viral vector resulted in GFP positivity in over 30% cells and approximately 80% of these positive cells were astrocytes. A similar transduction rate was seen with the HIV-1 homologous vector system, which lacks the cPPT sequence using 20 ng of viral vector. At the same viral vector concentration, the HIV-2 homologous vector system transduced approximately 25% of glial cells with 62% of these cells staining for GFAP. The effect of the cPPT sequence on HIV-1 viral vector transduction is evident with over 60% of glial cell expressing GFP with 20 ng of input vector and approximately 58% with 10 ng of vector. In summary, the gene transfer efficiency of the HIV-2 GFP vector to be cross packaged by SIV Gag-pol to glial cells was similar to both the HIV-1 and HIV-2 homologous vector systems.
Figure 7 Transduction of rat mixed glial cells with a HIV-2 based lentiviral vector packaged by SIV gag-pol. (A) GFP expression in lentivector transduced cells. (B) GFAP co-staining of astrocytes.
Figure 8 Transduction of Rat primary mixed glial cultures with Lentiviral vectors based on HIV-1 packaged by HIV-1 Gag-pol(A), HIV-1 +cPPT vector packaged by HIV-1 Gag-pol (B), HIV-2 vector packaged by SIV Gag-pol (C) and HIV-2 vector packaged by HIV-2 Gag-pol (D). Error bars indicate within experimental SEM.
Transduction of human embryonic neuronal stem cells was also performed using the HIV-1 and HIV-2 homologous vector system (not shown) and with the SIV Gag-Pol /HIV-2 GFP. The transduction efficiency was assessed qualitatively by fluorescence microscopy using 20 ng of viral vector, and Figure 9 shows that the SIV Gag-pol/HIV-2 GFP cross packaged vector system transduced both astrocytes and neurons post differentiation as demonstrated by immunostaining with GFAP (astrocytes) and beta-tubulin (neurons). The cross packaged vector system performed as well as the HIV-1 and HIV-2 homologous vector systems with astrocytes being transduced at a slightly higher efficiency.
Figure 9 Transduction of neural stem cells by a HIV-2 based GFP lentiviral vector packaged by SIV-2 Gag-Pol. (A) Phase contrast image through growing neurosphere (upper left). (B) Fluorescent image of neurosphere in A expressing GFP 72 hours post transduction (upper right). (C) Confocal image through neurosphere expressing GFP (lower left) (D) Neurons derived from human neurosphere 7 days post differentiation (lower right). Red represents β tubulin III, green – GFP, Hoechst stain (blue) nuclei. Arrow denotes double labelled cell. Magnification in A and B = 10×, in C = 100×, D = 40×
Discussion
Both lentiviruses and other retroviruses have shown an ability to cross package other viral genomes with HIV-1 Gag-Pol demonstrating the greatest cross packaging ability. Non-reciprocal packaging relationships such as have been demonstrated in HIV-1 and HIV-2 [15] or spleen necrosis virus and murine leukaemia virus [26] suggest that individual viruses have different packaging mechanisms relating possibly to the availability of the Gag protein or the position of the RNA packaging signal relative to the major splice donor or other as yet unknown factors. In this study we demonstrate for the first time a non-reciprocal packaging relationship between SIV and HIV-2. Interestingly, the major packaging determinant of both HIV-2 and SIV has been shown to be upstream of the major splice donor [9,10] and by inference one would expect SIV to demonstrate the same co-translational packaging process as HIV-2 [13]. SIV Gag-Pol has been previously reported to cross package HIV-1 and FIV unspliced vector mRNA [16,7,18] however the gene transfer ability of these chimeric vectors has been limited. We could not demonstrate any appreciable gene transfer of an HIV-1 based vector cross-packaged by SIV Gag-Pol, which is in contrast to a previous study [16], where transduction of both dividing and non-dividing cells was demonstrated. Nor was gene transfer of the HIV-1 GFP seen when packaged by HIV-2 Gag-Pol, in contrast to a previous report [15].
SIV Gag-Pol packaged similar levels of HIV-1 RNA compared to the homologous SIV vector system (Figure 3A and 3B), however a significant decrease in gene transfer was demonstrated with the SIV Gag-Pol/HIV-1 GFP vector when 4 ng of vector was used to transduce SVC2 cells (Figure 5B). A similar observation was demonstrated with HIV-2 Gag-Pol, which packaged equal levels of HIV-1 GFP and SIV GFP vector RNA and showed no appreciable gene transfer with 4 ng of vector. The RT-PCR data on virion extracted RNA suggests that low levels of RNA are being packaged. Why this does not translate into detectable gene transfer is not clear although the RT-PCR does not reveal whether complete or damaged genomes are being packaged. Gene transfer may be a threshold phenomenon in which many virions contain defective genomes and only a few have a full genomic RNA. Alternatively there may be an additional block in functional gene transfer either at reverse transcription or integration. Indeed, there is no reported data on the function of SIV reverse transcriptase or integrase in an HIV-1 backbone. The cross packaging ability of HIV-1 Gag-Pol was demonstrated by its ability to package both HIV-2 and SIV RNA and effect GFP gene transfer. HIV-1 Gag-Pol packaged a greater level of HIV-2 RNA than SIV RNA and a significantly greater number of cells were transduced with the HIV-1 Gag-Pol/HIV-2 GFP vector.
Figure 5 a Quantitative assessment of GFP transfer to SVC2 cells by FACS analysis using HIV-1 Gag-Pol to package gene transfer vectors based on HIV-1 (+/- cPPT sequence), HIV-2 and SIV. A range of Viral vector concentrations from 40 ng to 4 ng of Reverse Transcriptase was used. (Blank = No data). b Quantitative assessment of GFP transfer to SVC2 cells by FACS analysis using SIV Gag-Pol to package gene transfer vectors based on SIV, HIV-1 and HIV-2. A range of Viral vector concentrations from 20 ng to 4 ng of Reverse Transcriptase was used. c Quantitative assessment of GFP transfer to SVC2 cells by FACS analysis using HIV-2 Gag-Pol to package gene transfer vectors based on, HIV-2, HIV-2 and SIV. A range of Viral vector concentrations from 20 ng to 4 ng of Reverse Transcriptase was used.
One advantage of a chimeric lentiviral vector is a reduction in the risk of development of a replication competent retrovirus which may occur through a recombination event due to sequence homology between the Gag-Pol and gene transfer constructs. However it is important to assess the gene transfer capabilities of these chimeric vectors in suitable primary cells. This has been highlighted in a study where a gene transfer vector based on SIV packaged by HIV-1 Gag-Pol showed a reduced transduction efficiency of human dendritic cells associated with a post-entry defect. [19]. A second major advantage of this chimeric system is the ability to deliver a cross-packaged vector to a simian animal model with a vector based on SIV Gag-Pol packaging an HIV-2 genome. The same combination could subsequently be used in humans allowing biosafety and bio-distribution studies to be performed directly without the necessity for surrogate systems. This is not possible with an HIV-1 based system and would give the SIV/HIV-2 system considerable advantages over other primate lentiviral combinations.
Rat astrocytes are the major cell type associated with the glial scar resulting from injury to the CNS [27] and human fetal embryonic neural stem cells offer the potential for regenerating damaged areas of the CNS [28]. Engraftment of neural stem cells transduced with a lentiviral vector based on HIV-1 has been demonstrated with a high level and duration of transgene expression[29]. Our results demonstrate that both the HIV-1 and HIV-2 homologous GFP lentivectors efficiently transduced rat primary astrocytes. Similar to previous studies on the effect of the cPPT sequence on gene transfer [30,31] our data shows that the presence of the cPPT sequence in the HIV-1 vector results in a two fold increase in transduction efficiency, similar to the HIV-2 homologous vector system which also contains the HIV-2 cPPT in the pol sequence. The SIV Gag-Pol/ HIV-2 GFP vector also transduced primary astrocytes with efficiency similar to the HIV-1 cPPT homologous vector system, indicating no associated post-entry defects. Efficient transduction of human fetal embryonic neural stem cells was also shown with the cross packaged SIV Gag-Pol/HIV-2 GFP vector highlighting the ability of this vector to transduce human cells.
Conclusion
We have identified a non reciprocal cross packaging relationship between SIV Gag-Pol and a HIV-2 based GFP vector, which demonstrated equivalent transduction efficiencies in 293T cells, rat primary astrocytes and embryonic stem cells as that of homologous HIV-1 and HIV-2 vector systems. The efficiency of the combination correlates with the level of vector RNA packaged indicating that this is a major determinant of vector efficiency. It suggests that there are as yet unidentified differences in the RNA capture mechanisms of HIV-1, HIV-2 and SIV.
The implications for testing of lentiviral vector biosafety are potentially very important. Testing in appropriate animal models is a major concern associated with the use of lentiviral vectors in clinical trials. As HIV-1 only causes AIDS in humans, there is presently no animal model to test the safety of HIV-1 based vectors. However animal models based on Asian macaques and baboons exist for SIV and HIV-2, respectively which may be applicable to testing the biosafety of SIV cross packaged HIV-2 lentiviral vectors.
Methods
Lentiviral vectors
The lentiviral gene transfer vectors and Gag-Pol expression constructs are outlined in Figures 1 and 2. The constructs based on HIV-1 and SIV have been previously described [4,32] and were kind gifts of D. Trono and K. Uberla. The HIV-1 gene transfer vector HR'GFP was modified to include the HIV-1 central polypurine (cPPT) tract or DNA flap sequence. The sequence was PCR amplified and cloned into the unique Cla1 site upstream of the Rev Responsive Element (RRE) sequence using cPPT primer sequences described [30]. The HIV-2 gene transfer vector was also modified from the construct pSVRΔNBPuroΔH [13] by replacing the SV40-Puromycin construct with a CMV-GFP reporter gene construct to create pSVRΔ-CMVGFP. The HIV-2 Gag-Pol construct, (pSVRΔNBDM) contains a deletion in the 5'untranslated region, which has been shown to abrogate packaging [13].
Figure 1 Gag-Pol packaging constructs.
Figure 2 GFP gene transfer vectors. The dotted line indicates a deletion
Construction of minimal HIV-2 based vectors
pSVRΔNBΔH [13] was digested with BsmBI, and a ClaI linker was inserted into the site. ClaI and EcoRV digestion of this produced two DNA fragments, the smaller of which (nucleotides 1101–6128 encompassing gag and pol sequences) was discarded. The remaining fragment was religated and formed pSVRΔGP. CMVGFP was obtained from SalI digestion of pSVRΔ-CMVGFP and ligated into the SalI linker of dephosphorylated pSVRΔGP to give pSVRΔGP-CMVGFP.
The HIV-2 U3 region contains a TATA box, core enhancer regions, and Sp1, κB and peri-κB binding sites that are responsible for transcription from the 5'LTR. This 141 bp region (nucleotides 9329–9470) was deleted in the 3'LTR to produce a SIN vector as follows. The 3'LTR was removed from pSVRΔGP, by BamHI and XbaI digestion, and subcloned into pBluescript II KS. Site directed mutagenesis introduced BglII restriction sites at the 5' and 3' ends of the 141 bp region that was to be deleted. Mutagenesis was carried out as in two stages using the following primers: stage 1, upstream mutagenesis:- 5'-GGAATACCATTTAGTTAAAGATCTGAACAGCTATACTTGGTCAGGG-3' and :- 5'-CCCTGA CCAAGTATAGCTGTTCAGATCTTTAACTAAATGGTATTCC-3'; for stage 2, downstream mutagenesis, 5'-CGCCCTCATATTCTCTGTATAGATCTACCCGCTAGCTTGCATTG-3' and 5'-CAATGCAAGCTAGCGGGTAGATCTATACAGAGAATATGAGGGCG-3'.
The 141 bp U3 region was removed from the plasmid by BglII digestion and the plasmid religated. BamHI and XbaI digestion of the plasmid and religation of the Δ3'LTR into pSVRΔGP created the pSVRΔSIN vector. CMVGFP was inserted as described to produce the vector pSVRΔSIN-GFP
Lentiviral vector production
Lentiviral vectors were produced by calcium phosphate transfection of 293T cells grown in DMEM media and 10% FCS with 7 μg of the gene transfer vector, 7 μg of the Gag-Pol construct, 3 μg of a Rev expressor and 3 μg of the VSV-G heterologous envelope. For HIV-2 and SIV vector production the Rev expressor was omitted. 24 hours following transfection the media was replaced and supernatant containing recombinant virions was recovered 48 hours post transfection. Virions were concentrated by ultracentrifugation for 2.5 hours at 25,000 RPM in an SW28 Beckmann rotor. The viral pellet was resuspended in 300 μl of tissue culture media, aliquoted and stored at -70°C.
Lentiviral vectors were quantitated using a commercially available RT-assay (Cavid Tech, Uppsala) Vector preparations were measured in duplicate and normalised to a concentration of 8 ng of RT per μl. Although the sensitivity of the assay for different RTs may be slightly different the fact that each Gag-Pol construct is being used to package each vector provides an internal control.
Levels of RNA packaging were assessed by RT-PCR of Virion associated RNA. Virion RNA was extracted using the Qiagen Viramp kit from 10 ng of virus (RT levels). Following extraction the RNA was also treated with RNase Free DNase for 10 mins at 37°C and the DNase was in activated by incubation at 70°C for a further 10 mins. An aliquot of RNA was reverse transcribed to cDNA using the Promega Improm RT system with an antisense GFP primer (AAGTCGTGCTGCTTCATGTG). The cDNA was then serially diluted and amplified using a sense primer (GACGTAAACGGCCACAAGTT) and the antisense primer. Amplified products were resolved by agarose gel electrophoresis and EtBR staining.
The transduction efficiency of cross-packaged vectors was assessed by FACS analysis of GFP positive cells. A range of viral vector concentration from 40 ng to 4 ng was used to transduce 1 × 106 of fibroblast SV2C cells in a six well plate. Viral vector was diluted in DMEM containing 6 μg/ml polybrene and cells were exposed to virus for 5 hours. The media was then replaced and GFP expression was assessed at time periods after 72 hours post transduction.
Glial cell Culture and Stem cell culture
Primary mixed glial cultures were prepared from the brains of newborn rats > 3 days old by dissociation of whole cortex in trypsin, then cultured in poly-D-lysine coated flasks in DMEM/10% FCS. Mixed glial cultures were derived from these cells, once they were confluent, by trypsinisation. The cells were then resuspended in DMEM containing 10% FCS and 1% PSF and centrifuged at 10,000 RPM for 5 minutes. The supernatant was removed and cells were resuspended in DMEM/10% FCS and plated onto Poly-D-Lysine coated coverslips in 24 well plates. Transduction of glial cultures with lentiviral vectors was carried out as described for SV2C cultures. 72 hours post transduction; glial cultures were fixed in 4% paraformaldehyde and stored in PBS at 4°C prior to immunostaining.
Human fetal neuronal stem cell culture was performed as described previously [33]. Transduction of Stem cell cultures of cortical origin was performed with 20 ng of viral vector in DMEM/ HAMS F12 (2:1), 1% N2, EGF (20 ng/ml) FGF-2 (20 ng/ml) and heparin (5 mg/ml) for four hours followed by replacement of the media. Cells were allowed to differentiate on poly-L-lysine/laminin coated coverslips followed by replacement of the media 72 hours post transduction. Cells were fixed in 4% paraformaldehyde after a further 96 hours, followed by immunostaining for GFP, GFAP and β-Tubulin III.
Immunostaining
Lentiviral vector transduced mixed glial cultures were first blocked using 3% goat serum in TXTBS (0.2% triton X-100, in Tris Buffered Saline) for one hour. Monoclonal anti GFAP (Sigma, 1:500) and polyclonal goat anti rabbit GFP (Molecular Probes), 1: 1000) were diluted in TXTBS with 1% normal goat serum (NGS) for 2 hours. Cells were then washed in TBS for 3 × 10 minutes. Cells were then incubated with secondary antibodies, goat anti mouse Alexa (Molecular Probes, 1:500) and biotinylated goat anti rabbit (Amersham Biosciences, 1:500) for 90 minutes. Following a second 3 × 10 minute wash in TBS, Streptavidin-FITC (Serotec, 1:100) was added in TBS with 1% NGS and Bis-benzamide (Sigma, 1:5000). Coverslips were then mounted in Fluorosave reagent (Calbiochem). Cell counts of immunostained mixed glial cultures were performed from one edge of the coverslip all the way across to the other, horizontally and vertically. A 0.5 mm2 area was counted every 1.5 mm.
Competing interests
PS, DB and AML are inventors on various patents filed by the University of Cambridge containing usage claims for chimeric lentiviral vectors. There are no licences currently associated with these patents.
Authors' contributions
PS, AML and JWF jointly conceived of these studies. PS produced and titered the lentiviral vectors, performed FACS analysis and RT-PCR analysis and transduced cell lines, primary glial cells and neural stem cells. DWH produced the primary mixed glial cultures. DB cloned the CMV-GFP cassette into the HIV-2 vector and performed the comparative analysis of the HIV-2 vectors. BCG cloned the HIV-1 cPPT region into the HIV-1 vector. MC produced the neural stem cell cultures. PS drafted this manuscript, which was critically reviewed by AML and JWF.
Acknowledgements
This work was supported by a programme grant from the Medical Research Council (UK)
==== Refs
Trono D Lentiviral vectors: turning a deadly foe into a therapeutic agent Gene Therapy 2000 7 20 23 10680011 10.1038/sj.gt.3301105
Connolly JB Lentiviruses in gene therapy clinical research Gene Therapy 2002 9 1730 1734 12457288 10.1038/sj.gt.3301893
Zufferey R Nagy D Mandel RJ Naldini L Trono D Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo Nat Biotechnol 1997 871 875 9306402 10.1038/nbt0997-871
Naldini L Blomer U Gallay P Ory D Mulligan R Gage FH Verma IM Trono D In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector Science 1996 272 263 267 8602510
Blomer U Naldini L Kafri T Trono D Verma IM Gage FH Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector J Virol 1997 71 6641 6649 9261386
Kordower JH Emborg ME Bloch J Ma SY Chu Y Leventhal L McBride J Chen EY Palfi S Roitberg BZ Brown WD Holden JE Pyzalski R Taylor MD Carvey P Ling Z Trono D Hantraye P Deglon N Aebischer P Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease Science 2000 290 767 773 11052933 10.1126/science.290.5492.767
Dull T Zufferey R Kelly M Mandel RJ Nguyen M Trono D Naldini L A third generation lentivirus vetor with a conditional packaging system J Virol 1998 72 8463 8471 9765382
Lever A Gottlinger H Haseltine W Sodroski J Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions J Virol 1989 63 4085 4087 2760989
McCann EM Lever AM Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2 J Virol 1997 71 4133 4137 9094696
Strappe PM Greatorex J Thomas J Biswas P McCann E Lever AM The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2 J Gen Virol 2003 84 2423 2430 12917463 10.1099/vir.0.19185-0
Kemler I Barraza R Poeschla EM Mapping the encapsidation determinants of feline immunodeficiency virus J Virol 2002 76 11889 11903 12414931 10.1128/JVI.76.23.11889-11903.2002
Browning MT Mustafa F Schmidt RD Lew KA Rizvi TA Delineation of sequences important for efficient packaging of feline immunodeficiency virus RNA J Gen Virol 2003 84 621 627 12604814 10.1099/vir.0.18886-0
Griffin SD Allen JF Lever AM The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: Cotranslational RNA encapsidation and limitation of Gag protein confer specificity J Virol 2001 75 12058 12069 11711596 10.1128/JVI.75.24.12058-12069.2001
Poole E Strappe P Mok HP Hicks R Lever AM HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET Traffic 2005 in press 16101678
Kaye JF Lever AML Nonreciprocal packaging of human immunodeficiency virus type 1 and type 2 RNA: a possible role for the p2 domain of Gag in RNA encapsidation J Virol 1998 72 5877 5885 9621049
White SM Renda M Nam NY Klimatcheva E Zhu Y Fisk J Halterman M Rimel BJ Federoff H Pandya S Rosenblatt JD Planelles V Lentivirus vectors using human and simian immunodeficiency virus elements J Virol 1999 73 2832 2840 10074131
Rizvi TA Panganiban AT Simian immunodeficiency virus RNA is efficiency encapsidated by human immunodeficiency virus type 1 particles J Virol 1993 67 2681 2688 8474168
Browning MT Schmidt RD Lew KA Rizvi TA Primate and Feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions J Virol 2001 75 5129 5140 11333894 10.1128/JVI.75.11.5129-5140.2001
Goujon C Jarrosson-Wuilleme L Bernaud J Rigal D Darlix JL Cimarelli A Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virus derived genome display a specific postentry transduction defect in dendritic cells J Virol 2003 77 9295 9340 12915545 10.1128/JVI.77.17.9295-9304.2003
Sastry L Xu Y Johnson T Desai K Rissing D Marsh J Cornetta K Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses Mol Ther 2003 8 830 839 14599817 10.1016/j.ymthe.2003.08.003
Escarpe P Zayek N Chin P Borellini F Zufferey R Veres G Kiermer V Development of a sensitive assay for detection of replication-competent recombinant lentivirus in large-scale HIV-based vector preparations Mol Ther 2003 8 332 341 12907156 10.1016/S1525-0016(03)00167-9
Zhao C Strappe PM Lever AM Franklin RJ Lentiviral vectors for gene delivery to normal and demyelinated white matter Glia 2003 42 59 67 12594737 10.1002/glia.10195
Baekelandt V Eggermont K Michiels M Nuttin B Debyser Z Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain Gene Ther 2003 10 1933 1940 14528317 10.1038/sj.gt.3302094
Ruitenberg MJ Blits B Dijkhuizen PA Beek ET Bakker A van Heerikhuize JJ Pool CW Hermens WT Boer GJ Verhaagen J Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury Neurobiol Dis 2004 15 394 406 15006710 10.1016/j.nbd.2003.11.018
Ostenfeld T Tai YT Martin P Deglon N Aebischer P Svendsen Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons J Neurosci Res 2002 69 955 965 12205689 10.1002/jnr.10396
Certo JL Shook BF Yin PD Snider JT Hu WS Nonreciprocal pseudotyping: murine leukaemia virus proteins cannot efficiently package spleen necrosis virus based vector RNA J Virol 1998 72 5408 5413 9620995
Properzi F Fawcett JW Proteoglycans and brain repair News physiol Sci 2004 19 33 38 14739401 10.1152/nips.01449.2003
Tai YT Svendsen CN Stem cells as a potential treatment of neurological disorders Curr Opin Pharmacol 2004 4 98 104 15018846 10.1016/j.coph.2003.09.006
Englund U Ericson C Rosenblad C Mandel RJ Trono D Wictorin K Lundberg C The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS Neuroreport 2000 11 3973 3977 11192612
Manganini M Serafini M Banbacioni F Casati C Erba E Follenzi A Naldini L Bernasconi S Gaipa G Rambaldi A Biondi A Golay J Introna M A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors Hum Gene Ther 2002 13 1793 1807 12396613 10.1089/104303402760372909
Zennou V Serguera C Sarkis C Colin P Perret E Mallet J Charneau P The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain Nat Biotechnol 2001 5 446 450 11329014 10.1038/88115
Schnell T Foley P Wirth M Munch J Uberla K Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus Hum Gene Ther 2000 11 439 447 10697118 10.1089/10430340050015905
Svendsen CN ter Borg MG Armstrong RJ Rosser AE Chandran S Ostenfeld T Caldwell MA A new method for the rapid and long term growth of human neural precursor cells J Neurosci Meth 1998 85 141 152 10.1016/S0165-0270(98)00126-5
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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-3-361619119710.1186/1479-5876-3-36ResearchThe subcellular distribution of myeloid-related protein 8 (MRP8) and MRP14 in human neutrophils Stroncek David F [email protected] Raji A [email protected] Keith M [email protected] Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA2 3M Corporation, St. Paul, Minnesota, USA3 The Department of Medicine, The University of Minnesota Medical School and Masonic Cancer Center, Minneapolis, Minnesota, USA2005 28 9 2005 3 36 36 6 7 2005 28 9 2005 Copyright © 2005 Stroncek et al; licensee BioMed Central Ltd.2005Stroncek 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
Myeloid-related protein 8 (MRP8) and MRP14 are S100 family calcium binding proteins that form a heterodimer known as calprotectin or MRP8/14 that is present in the cytosol of neutrophils and monocytes. MRP8/14 becomes associated with endothelium at sites of monocyte and neutrophil adhesion and transmigration and induces a thrombogenic and inflammatory response by increasing the endothelial transcription of proinflamatory chemokines and adhesion molecules. The distribution of MRP8/MRP14 among neutrophil granules and plasma membranes is unclear and was investigated to better understand the role of this molecule in acute inflammation.
Study design
Three monoclonal antibodies specific for MRP8 and MRP14 were characterized and used in immunoblotting assays of neutrophil whole cell extracts, and isolated plasma membranes, primary granules, secondary granules and cytosol.
Results
MRP8 and MRP14 were detected in neutrophil cytosol, plasma membrane, primary granule and secondary granule fractions. MRP8/14 demonstrated a calcium-dependent adherence to plasma membranes and primary granules and could be removed by washing with EGTA in a high ionic strength buffer. In contrast, MRP8/14 was found within the contents of the secondary granules. Activated neutrophils released secondary granules and MRP8/14.
Conclusion
MRP8/14 is located in neutrophil cytosol and secondary granule fractions and is loosely associated with plasma membranes. MRP8/14 released with secondary granules by activated neutrophils likely binds to endothelium and plays an important role in acute inflammation.
Myeloid-related protein 8MRP8myeloid-related protein 14MRP14neutrophilsmacrophagescalprotectin
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Introduction
Calprotectin is a heterodimer of two calcium-binding proteins that belong to the S100 family: myeloid-related protein 8 (MRP8) and MRP14. MRP8 (10.8 kDa) is also known as p8, L1 light chain, calgranulin A, and cystic fibrosis antigen (CFA). MPR14 (13.2 kDa) is also referred to as p14, L1 heavy chain, and calgranulin B [1-7]. MRP8 and MRP14 bind both calcium and zinc. Calprotectin is also known as MRP8/14.
Neutrophils are the major producers of calprotectin, but monocytes and some macrophages express MRP8/14. Macrophages found at the sites of acute infection express MRP8/14, but resident tissue macrophages and those found at the sites of chronic inflammation do not [7]. MRP8/14 is found in the cytosol of neutrophils and macrophages[8,9]. It is the most abundant protein in neutrophil cytosol making up 30% to 60% of all cytosolic proteins[8], but it is much less abundant in monocytes and comprises about 1% of all monocyte cytosol protein [10].
MRP8/14 plays an important role in leukocyte interactions with endothelium. It is not expressed by endothelium, however, examination of tissue sections revealed that MRP8/14 becomes associated with endothelium at sites where monocytes and neutrophils pass through the endothelium [7]. In vitro studies have found that when activated monocytes come in contact with extracellular matrix proteins or inflamed endothelium intracellular calcium levels increase, protein kinase C is activated, and MRP8/14 is released [11,12]. Once MRP8/14 is released it binds to the endothelium. Two mechanisms of binding have been proposed. One group has shown that MRP14 binds to endothelial cell heparan sulfate proteoglycans[13] and another that MRP8/MRP14 binds to carboxylated N-glycans expressed by activated endothelial cells [14].
Once associated with the endothelium, MRP8/14 has important proimflamatory effects. The binding of MRP8/14 to endothelium induces a thrombogenic and inflammatory response by increasing the transcription of proimflamatory chemokines and adhesion molecules and by decreasing the expression of cell junction proteins and molecules involved in cell monolayer integrity [15].
Elevated levels of MRP8/14 have been found in many sites of inflammation and in the extracellular fluid of patients with many types of inflammatory conditions. The concentration of MPR8/14 in the blood is increased in patients with rheumatoid arthritis, Chron's disease, colorectal cancer, cystic fibrosis, multiple scerlosis, and HIV infections [1,2,4,6,16]. Extracellular MRP8/14 has antimicrobial, antigrowth and apoptotic effects. It suppresses the growth of some fungi and bacteria [1,2]. It also suppresses the proliferation of several different types of cells including: macrophages, lymphocytes, hematopoietic progentitors, and tumor cell lines. MRP8/14 can also induce apoptosis of some tumor cell lines [1,2].
The primary source of MRP8/14 in tissues and body fluids has been suggested to be the cytosol of dead or lysed neutrophils [2]. However, the source of neutrophil MRP8/MRP14 that becomes associated with endothelium is not certain. While MRP8/14 is the most abundant neutrophil cytosol protein, investigations of the distribution of MRP8/14 among neutrophil granules and plasma membranes have yielded conflicting results. Some studies have reported that MRP8/14 is present in neutrophil cytosol and plasma membranes, but the primary or secondary granules were not analyzed [3,17]. However, another study did not detect MRP8/14 in either the plasma membranes or granules [10].
This study analyzed the neutrophil subcellular distribution of MRP8 and MRP14 to better understand changes in expression of MRP8/14 by activated neutrophils and the role of neutrophil MRP8/14 in acute inflammation. The expression of MRP8/14 on neutrophil plasma membranes, primary granules, secondary granules and cytosol was analyzed.
Materials and methods
Isolation of Neutrophils
Neutrophils were isolated by dextran sedimentation and centrifugation over Ficoll-Hypaque using a modification of the method of Boyum [18,19] and suspended in phosphate-buffered saline (PBS) or Hank's balanced salt solution (HBSS; GIBCO, Grand Island, NY).
Production of Monoclonal Antibodies
The monoclonal antibodies (MoAbs) were produced using BALB/c mice as previously described [20]. MoAbs AHN-17, AHN-17.1, and 15H9 were produced using human neutrophils as the immunogen, and AHN-17.1 using human eosinophils obtained from a patient with hypereosinophilic syndrome.
Characterization of Monoclonal Antibodies
All 3 MoAbs were IgG1. The MoAbs were tested by immunostaining [21] against neutrophil slide preparations, prepared by cytocentrifugation and all three reacted with neutrophils. AHN-17 and AHN-17.1 were used as ascites and 15H9 as isolated immunoglobulin.
Immunoblotting of Neutrophil Proteins
Neutrophils (1 × 108 per mL of PBS) were treated with 5 mmol/L diisopropylfluorophosphate (DIFP) (Sigma Chemical Company, St. Louis, MO) for 10 minutes at 4°C and were washed twice with PBS. The cells were resuspended at a concentration of 5 × 107 cells/mL in a buffer containing 5 mM Tris (Sigma), 400 mM KCl, 1 mM EDTA (Sigma), 1% Triton-X-100, and 2 mM (PMSF) (Sigma), pH 8.2 (Extraction Buffer) [20]. The solution was then frozen, thawed, and centrifuged at 10,000 g, for 10 minutes at 4°C. The cell extract was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunoblotted as previously described [22]. SDS-PAGE was performed in a 13% gel and the cell extracts were analyzed under both reducing and non-reducing conditions. MoAb 15H9 was used at a final concentration of 10 μg/mL and the ascites containing AHN-17 and AHN-17.1 at a dilution of 1:300.
Subcellular Fractionation of Neutrophils
Subcellular fractionation of neutrophils was performed using a modification of the method described by Borregaard et al [22,23]. Briefly, neutrophils were resuspended in 10 mL ice cold Relaxation Buffer [100 mM KCI, 3 mM NaCl, 1 mM adenosine triphosphate (Na)2 (Sigma), 3.5 mM MgCl2, and 10 mM piperazine-N, N-bis (2-ethanesulfonic acid) (PIPES) (Sigma), pH 7.3]. This solution was then equilibriated for 20 minutes at 4°C with nitrogen at 350 PSI with constant slow stirring in a cell disruption bomb (Parr Instrument, Moline, IL Model no 4639). The suspension was then collected drop wise into 2.2 mL of 12.5 mM EDTA (Sigma), pH 7.4 in Relaxation Buffer. Nuclei and unbroken cells were removed by centrifugation at 500 g for 20 minutes at 4°C. Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) was adjusted to a density of 1.120 and 1.050 g/mL with relaxation buffer 10 times concentrated. The 1.050 density Percol was laid over the 1.120 density Percol, and the supernate was loaded onto the Percol gradients, which had been precooled at 4°C. The pelleted membranes and granules were resuspend in Relaxation Buffer and centrifuged at 100,000 g for 90 minutes at 4°C. The membranes and granules were washed again with relaxation buffer and analyzed by immunoblotting. The plasma membrane, primary granule, secondary granule, and cytosol fractions were analyzed by immunoblotting with AHN-1.1 (CD15) [20], AHN-10 (anti-elastase) [24], AHN-9 (anti-lactoferrin) [21], and AHN-11 (anti-cathepsin G) [21] to confirm that adequate separation had occurred.
In other studies the plasma membranes, primary, and secondary granules were centrifuged at 100,000 g for 90 minutes at 4°C and resuspended in 1 M NaCl and 10 mM EGTA (Sigma), pH 7.4 in Relaxation Buffer. The wash was repeated and then the plasma membranes, primary granules and secondary granules were disrupted by freeze thawing twice and sonicating (Microultrasonic Cell Disrupter, model KT40 Kontz) for 1 minute on ice. The membranes and soluble granule contents were separated by centrifugation at 120,000 g for 90 minutes at 4°C. Protein concentrations of the fractions were determined by bicinchoninic acid (BCA) protein assay C (Pierce Chemical Company, Rockford, IL).
Stimulation of Neutrophils
Neutrophils at a concentration of 107/mL in HBSS were incubated at 37°C for 15 minutes alone or with phorbol myristate acetate (PMA) (100 ng/mL) (Sigma) or N-formal-Met-Leu-Phe (FMLP) (10-6M) (Sigma). The solution was then centrifuged at 500 g for 10 minutes and the supernatant was analyzed by immunoblotting.
Isolation of Proteins
MoAbs AHN-17 and 15H9 were immobilized on a protein A column and crosslinked with dimethyl pimelimidate according to the manufacturer instructions (lmmuno Pure IgG Orientation kit (Pierce). Neutrophils or subcellular fractions were suspended at a concentration of 5 × 107/mL in Extraction Buffer. The solution was then frozen, thawed, and centrifuged at 10,000 g for 10 minutes at 4°C. The neutrophil extact was mixed 1:1 with 10 mM Tris, ph 7.5, and applied to the immunoaffinity column. After the column was washed with 150 mM NaCl, 0.2% Triton X-100, 10 mM Tris, pH 7.5 to remove unbound protein. Antigens were eluted with 0.1 M glycine, pH 2.8, containing 0.2% Triton X-100 and immediately neutralized with 2 M Tris-HCI, pH 7.6.
The 10 kDa and 14 kDa proteins isolated with MoAb AHN-17 were analyzed by SDS-PAGE and coomassie blue staining or immunoblotting. The 10 and 14 kDa proteins isolated with MoAb 15H9 were separated by reverse phase high pressure liquid chromatography (HPLC) using C-4 column (Vydac, Hesperia, CA) which had been pre-equilibrated with three column volumes of 0.1% trifluoroacetic acid. The bound proteins were eluted from the column by application of 0–60% gradient of acetonitrite over 50 minutes. Aliquots (1 mL) were collected and analyzed by SDS-PAGE and silver staining or immunoblotting with MoAb 15H9. The N- terminal amino acid sequence of the 10 and 14 kDa proteins were analyzed using an automated amino acid sequencer (ABl 470/477, Applied Biosystem Inc, Foster City, CA).
Tryptic Digestion of the 14 kDa Protein
The isolated 14 kDa protein was dried using vacuum centrifugation. The dried proteins were resuspended in 50 μL of a solution containing 8 M urea and 0.4 M ammonium bicarbonate and following the addition of 10 μL 45 mM dithiothreitol (Sigma) was incubated for 30 minutes at 50°C. After the solution cooled to 20°C, 10 μL of 100 mM iodacetamide (Sigma) was added and the solution was incubated for 30 minutes in the dark at 20°C. Next, trypsin (Worthington Biochemical Corp, Freehold, NJ) in 120 μL of water at 1:25 (w/w) ratio of enzyme to protein (determined by BCA) was added and the solution was incubated at 37°C for 24 hours. The resulting tryptic fragments were separated by high pressure liquid chromatography (HPLC) (C-18 Column, Vydac, Hesperia, CA). The N-terminal amino acid sequence of the tryptic fragments was analyzed using an automated amino acid sequence (Applied Biosystems).
Results
Characterization of MoAbs AHN-17, AHN-17.1, and15H9 as Specific for MRP8 and MRP14
All three MoAbs were tested in an immunoblotting assay against neutrophils analyzed by SDS-PAGE under non-reducing conditions. Multiple proteins of various apparent molecular weights were detected by AHN-17, AHN-17.1, and 15H9 and the pattern of proteins detected was similar for all three MoAbs (data not shown). AHN-17 and AHN-17.1 did not react by immunoblotting with proteins from extracts analyzed by SDS-PAGE under reducing conditions, but 15H9 reacted with two proteins of 10 kDa and 14 kDa (data not shown). These results suggest that the three antibodies react with same proteins, but the eptiopes recognized by AHN-17 and AHN-17.1 are confirmation dependent.
To determine if all three MoAbs reacted with the same proteins, the antigens recognized by AHN-17 were isolated by affinity chromatography with AHN-17 and were analyzed by immunoblotting with AHN-17, AHN-17.1, and 15H9. When the purified proteins were analyzed by SDS-PAGE under non-reducing conditions and coomassie blue staining, several bands were detected but when analyzed under reducing conditions only a 10 kDa and 14 kDa protein were detected (data not shown). When the isolated proteins were analyzed by SDS-PAGE under nonreducing conditions and immunoblotting, AHN-17 (Figure 1, lane A), AHN-17.1 (lane B) and 15H9 (lane C) reacted with several bands and the proteins identified by all three antibodies were similar. Immunoblotting with normal mouse serum (NMS) did not detect these proteins (lane D). These results indicate that all three antibodies recognized the same proteins.
Figure 1 Immunoblotting of proteins purified by affinity chromatography using monoclonal antibody AHN-17. Neutrophil proteins recognized by AHN-17 were isolated by immunoaffinity chromatography, analyzed by SDS-PAGE in a 13% gel under non-reducing conditions, transferred to nitrocellulose, and immunoblotted with AHN-17 (lane A), AHN-17.1 (lane B), 15H9 (lane C), and normal mouse serum (NMS) (lane D). Proteins used as molecular weight standards were: rabbit muscle phosphorylase B, 97,000; Bovine serum albumin 66,000; ovalbumin 45,000; glyceraldehyde 3 phosphate dehydrogenase, 36,000; bovine erythrocyte carbonic anhydrase 30,000; soybean trypsin inhibitor 20,000; and bovine lactalbumin 14,000.
The 10 and 14 kDa proteins purified by affinity chromatography with MoAb AHN-17 were separated by HPLC, and the N-terminal amino acid sequence of the 10 kDa protein was analyzed. A sequence of 29 amino acids was identified which was identical to that of MRP8 (Figure 2). The N-terminus of the 14 kDa protein was blocked, however, the amino acid sequences of two tryptic peptides were determined and found to be identical to MRP14 at 25 of 27 amino acids (Figure 2).
Figure 2 Comparison of the N-terminal amino acid sequences of the 10 kDa protein and MRP8 (panel A) and the amino acid sequences of the two tryptic peptides from the 14 kDa protein with MRP14 (panel B).
Subcellular Distribution of MRP8 and MRP14 within Neutrophil Granules
Nitrogen cavitation and density gradient separation was used to isolate neutrophil cytosol, plasma membranes, primary granules, and secondary granules. The secondary granule fraction also contains a distinct granule population termed tertiary or gelatinase granules [25]. The proteins recognized by AHN-17 were isolated from each of these fractions by affinity chromatography and analyzed by SDS-PAGE under reducing and non-reducing conditions. When the proteins were analyzed by SDS-PAGE under non-reducing conditions, molecules of several electrophoretic mobilities were detected (Figure 3, lanes A-D). However, only two proteins of 10 and 14 kDa were detected when the analysis was preformed under reducing conditions (Figure 3, lanes E-H). These studies show that MRP8 and MRP14 are located in the neutrophil cytosol, plasma membrane, and secondary granule, and to a lesser degree primary granule fractions.
Figure 3 Subcellular distribution of proteins recognized by antibody AHN-17. Primary granules, secondary granules, plasma membranes, and cytosol were isolated from the neutrophils by nitrogen cavitation and differential centrifugation and were suspended in extraction buffer. The antigens recognized by antibody AHN-17 were isolated from each fraction by affinity chromatography with ANH-17 and were analyzed by SDS-PAGE in a 10% gel under non-reducing (lanes A-D) and reducing conditions (lanes E-H). The amount of antigen applied to each lane was adjusted to represent the same percentage of starting material. Primary granules are shown in lanes A and E, secondary granules in lanes B and F, plasma membranes in lanes C and G, and cytosol in lanes D and H. Gels were stained with coomassie blue.
To further determine the location of the proteins in the cellular granules, primary and secondary granules were washed and disrupted by freeze-thawing and sonication and the membranes were separated from the granule contents by centrifugation as described in the methods section. Equal amounts of protein from the cytosol, plasma membranes, primary granule membranes, secondary granule membranes, and the soluble contents of the primary and secondary granules were analyzed by immunoblotting with 15H9 (Figure 4). Both MRP8 and MRP14 were detected by 15H9 in the cytosol (Figure 4, first panel, lane A) and the soluble portion of the primary and secondary granules (Figure 4, first panel, lane E and F). MRP8 was also detected on the plasma membrane fractions (Figure 4, first panel, lane D). The largest quantities of the MRP8 and MRP14 were present in cytosol and in the soluble portion of the secondary granules. In contrast, NMS did not react with any proteins of similar electrophoretic mobility (Figure 4, second panel lanes A through F).
Figure 4 Analysis of neutrophil cytosol, plasma membranes, primary granules, and secondary granules by immunoblotting with monoclonal antibody 15H9. Neutrophil plasma membranes, cytosol, primary granules and secondary granules were prepared by nitrogen cavitation and differential centrifugation. Primary and secondary granules were disrupted and the membrane and contents were separated as described in the text. Cytosol (lane A), primary granule membranes (lane B), secondary granule membranes (lane C), plasma membranes (lane D), primary granules contents (lane E), and secondary granule contents (lane F) were analyzed by SDS-PAGE in a 13% gel under reducing conditions and were immunoblotted with antibody 15H9 (left panel) or NMS (right panel). Five micrograms of protein was added to each lane except for lane D in which 2 micrograms of protein was analyzed.
These studies were repeated but the plasma membranes, primary granules and secondary granules were washed two additional times with a high ionic strength buffer containing EGTA to disrupt loose associations of MRP8 and MRP14 with plasma membranes and granules. After the primary and secondary granules were disrupted by freeze thawing and sonication, the plasma membranes, secondary granules, and soluble contents of the granules were analyzed by immunoblotting. MRP8 and MRP14 were present only in the soluble portion of the secondary granules, but not the plasma membranes or primary granules (data not shown).
The neutrophil plasma membrane, cytosol, and granule fractions were also immunoblotted with AHN-10 (anti-elastase), AHN-9 (anti-lactoferrin), AHN-11 (anti-cathepsin G), and AHN-1.1 (CD15) to confirm adequate fractionation. As expected, elastase and cathepsin G were found in the primary granule contents, and lactoferrin in the secondary granule contents. The CD15 antibody reacted with plasma membranes and secondary granule membranes as expected (data not shown).
To examine the ability of neutrophils to release MRP8/14 when activated neutrophils were stimulated with PMA or FMLP to release secondary granules and tertiary granules, the cell suspension was centrifuged and the supernatant was analyzed by immunoblotting with MoAb 15H9. A small amount of MRP8 and MRP14 was detected in the supernatant of cell incubated in HBSS, however, the quantities of MRP8 and MRP14 in the supernatant was increased significantly after stimulation with PMA and FMLP (data not shown).
Discussion
The expression of MRP8 and MRP14 in neutrophils was analyzed using MoAbs AHN-17, AHN-17.1, and 15H9. Antibody specificity was confirmed by amino acid sequencing; the N-terminal amino acid sequence of the 10 kDa protein recognized by the antibodies was found to be identical to MRP8. The N-terminus of the 14 kDa protein recognized by the antibodies was blocked, but amino acid sequencing of tryptic peptides showed that the protein was MRP14. MRP8 and MRP14 have a similar structure and other antibodies have been found to react with both proteins [8,9]. MRP8 and MRP14 are encoded by two separate genes located on chromosome 1q12-q21[26].
This study confirmed that MRP8/14 are in neutrophil cytosol and found that MRP8 and MRP14 are also in the secondary granule fraction with a much smaller amount in the primary granule fraction and on the plasma membrane. The secondary granule fraction also contains the so-called tertiary granules or gelatinase granules [25]. The association of MRP8/14 with neutrophil plasma membranes and primary granules was similar to the association of MRP8/14 with macrophage plasma membranes [2,27]. Thus while in macrophages MRP8/14 is found primarily in cytosol, its distribution of depends on the activation state of the macrophages. In resting macrophages MPR8/14 is in the cytosol, but when macrophages are stimulated, it translocates to the cell membrane and cytoskeleton. The association of MRP8/14 with macrophage membranes is calcium dependent [27]. We found that MRP8 and MRP14 had a loose calcium-dependent adherence to neutrophil primary granules and plasma membranes and they were removed from these membranes by washing with EGTA in a high ionic strength buffer. Lemarchand and coworkers also found that MRP8/14 was associated with neutrophil plasma membranes via a calcium-dependent mechanism [28].
The association for MRP8/14 with secondary granules was different than its association with plasma membranes and primary granules; MRP8/14 was within the contents of the secondary granules. It could not be removed from the secondary granules by EGTA washes and when secondary granule membranes were separated from their soluble contents, almost all of the MRP8/14 was found to be present in the soluble contents of the secondary granules.
We found that MRP8/14 is released within seconds from secondary and or tertiary granules by stimulated neutrophils. MRP8/14 was detected in the supernatant of cells incubated with buffer possibly due to the loss of MRP8/14 associated with plasma membranes. However, following the incubation of cells with stimulants known to cause the release of secondary granules and tertiary granules, MRP8/14 was released from neutrophils. The subcellular fractionation technique used in this study separated neutrophils into cytosol, primary granule, secondary granule and plasma membrane fractions. The secondary granule fraction also likely contained tertiary granules [25,29,30], and MRP8 and MRP14 may also be present in tertiary granules.
It is not certain why other studies did not detect MRP8 and MRP14 in neutrophil granules [3,10,17]. The structure of MRP8 and MRP14 in the cytosol may differ from the structure of granule MRP8 and MRP14, and it is possible that some antibodies may recognize the cytosol proteins but not the granule proteins. Alternatively, the state of neutrophil activation may have differed between the studies. While MRP8/14 is associated with plasma membranes, they are not integral membrane proteins. Neither has a membrane-anchor sequence [4]. When neutrophils are activated, the quantities of both MRP8 and MRP14 associated with plasma membranes increases [28].
The presence of MRP8/14 in neutrophil secondary granules suggests that neutophils contribute to the deposition of MRP8/MRP14 on endothelium. Since adherent neutrophils release secondary granules, it is likely that at sites of acute inflammation when neutrophils bind to endothelial cells they degranulate and release MRP8/14 which stimulates endothelium and induces thrombotic and proimflamatory changes. This suggests that MRP8/14 has an important role in neutrophil mediated inflammation.
In summary, MRP8 and MRP14 are located not only in neutrophil cytosol but also in secondary and/or tertiary granules. The release of secondary granules and MPR8/14 by activated neutrophils suggests that MRP8/14 plays an important role in neutrophil induced inflammation.
Acknowledgements
The authors thank Greg Herr, Kenneth Campbell, and Robert Spoak for their technical assistance, Greg Fields and Eric Eckelston for performing the amino acid sequencing, and Robert Milius for assistance with sequence analysis.
==== Refs
Striz I Trebichavsky I Calprotectin - a pleiotropic molecule in acute and chronic inflammation Physiol Res 2004 53 245 253 15209531
Yui S Nakatani Y Mikami M Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity Biol Pharm Bull 2003 26 753 760 12808281 10.1248/bpb.26.753
Zwadlo G Schlegel R Sorg C A monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues J Immunol 1986 137 512 518 3722815
Odink K Cerletti N Bruggen J Clerc RG Tarcsay L Zwadlo G Gerhards G Schlegel R Sorg C Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis Nature 1987 330 80 82 3313057 10.1038/330080a0
Dale I Fagerhol MK Naesgaard I Purification and partial characterization of a highly immunogenic human leukocyte protein, the L1 antigen Eur J Biochem 1983 134 1 6 6861753 10.1111/j.1432-1033.1983.tb07522.x
Wilkinson MM Busuttil A Hayward C Brock DJ Dorin JR Van HV Expression pattern of two related cystic fibrosis-associated calcium-binding proteins in normal and abnormal tissues J Cell Sci 1988 91 ( Pt 2) 221 230 3267695
Hogg N Allen C Edgeworth J Monoclonal antibody 5.5 reacts with p8,14, a myeloid molecule associated with some vascular endothelium Eur J Immunol 1989 19 1053 1061 2666142
Hessian PA Edgeworth J Hogg N MRP-8 and MRP-14, two abundant Ca(2+)-binding proteins of neutrophils and monocytes J Leukoc Biol 1993 53 197 204 8445331
Bhardwaj RS Zotz C Zwadlo-Klarwasser G Roth J Goebeler M Mahnke K Falk M Meinardus-Hager G Sorg C The calcium-binding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory lesions Eur J Immunol 1992 22 1891 1897 1378023
Edgeworth J Gorman M Bennett R Freemont P Hogg N Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells J Biol Chem 1991 266 7706 7713 2019594
Rammes A Roth J Goebeler M Klempt M Hartmann M Sorg C Myeloid-related protein (MRP) 8 and MRP14, calcium-binding proteins of the S100 family, are secreted by activated monocytes via a novel, tubulin-dependent pathway J Biol Chem 1997 272 9496 9502 9083090 10.1074/jbc.272.14.9496
Frosch M Strey A Vogl T Wulffraat NM Kuis W Sunderkotter C Harms E Sorg C Roth J Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis Arthritis Rheum 2000 43 628 637 10728757 10.1002/1529-0131(200003)43:3<628::AID-ANR20>3.0.CO;2-X
Robinson MJ Tessier P Poulsom R Hogg N The S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate glycosaminoglycans on endothelial cells J Biol Chem 2002 277 3658 3665 11723110 10.1074/jbc.M102950200
Srikrishna G Panneerselvam K Westphal V Abraham V Varki A Freeze HH Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells J Immunol 2001 166 4678 4688 11254728
Viemann D Strey A Janning A Jurk K Klimmek K Vogl T Hirono K Ichida F Foell D Kehrel B Gerke V Sorg C Roth J Myeloid-related proteins 8 and 14 induce a specific inflammatory response in human microvascular endothelial cells Blood 2005 105 2955 2962 15598812 10.1182/blood-2004-07-2520
Muller F Froland SS Aukrust P Fagerhol MK Elevated serum calprotectin levels in HIV-infected patients: the calprotectin response during ZDV treatment is associated with clinical events J Acquir Immune Defic Syndr 1994 7 931 939 7914232
Teigelkamp S Bhardwaj RS Roth J Meinardus-Hager G Karas M Sorg C Calcium-dependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14 J Biol Chem 1991 266 13462 13467 2071612
Boyum A Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g Scand J Clin Lab Invest Suppl 1968 97 77 89 4179068
Craddock PR Hammerschmidt D White JG Dalmosso AP Jacob HS Complement (C5-a)-induced granulocyte aggregation in vitro. A possible mechanism of complement-mediated leukostasis and leukopenia J Clin Invest 1977 60 260 264 874088
Skubitz KM Snook RW Monoclonal antibodies that recognize lacto-N-fucopentaose III (CD15) react with the adhesion-promoting glycoprotein family (LFA-1/HMac-1/gp 150,95) and CR1 on human neutrophils J Immunol 1987 139 1631 1639 3305706
Skubitz KM Christiansen NP Mendiola JR Preparation and characterization of monoclonal antibodies to human neutrophil cathepsin G, lactoferrin, eosinophil peroxidase, and eosinophil major basic protein J Leukoc Biol 1989 46 109 118 2746138
Stroncek DF Skubitz KM McCullough JJ Biochemical characterization of the neutrophil-specific antigen NB1 Blood 1990 75 744 755 2153425
Borregaard N Heiple JM Simons ER Clark RA Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation J Cell Biol 1983 97 52 61 6408102 10.1083/jcb.97.1.52
Skubitz KM Wehner NG Gray BH Preparation and characterization of a monoclonal antibody that inhibits human neutrophil elastase activity J Leukoc Biol 1988 44 158 165 3411232
Kjeldsen L Sengelov H Lollike K Nielsen MH Borregaard N Isolation and characterization of gelatinase granules from human neutrophils Blood 1994 83 1640 1649 8123855
Lagasse E Clerc RG Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation Mol Cell Biol 1988 8 2402 2410 3405210
Roth J Burwinkel F van den BC Goebeler M Vollmer E Sorg C MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate filaments in a calcium-dependent manner Blood 1993 82 1875 1883 8400238
Lemarchand P Vaglio M Mauel J Markert M Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation J Biol Chem 1992 267 19379 19382 1326551
Borregaard N Christensen L Bejerrum OW Birgens HS Clemmensen I Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase J Clin Invest 1990 85 408 416 2298916
Dewald B Bretz U Baggiolini M Release of gelatinase from a novel secretory compartment of human neutrophils J Clin Invest 1982 70 518 525 6286726
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-821619119910.1186/1743-422X-2-82Study ProtocolHepatitis B Virus: Inactive carriers Sharma Sanjeev Kumar [email protected] Nitin [email protected] Yogesh [email protected] Head, Department of Hepatology, PGIMER, Chandigarh, 160012 India2005 28 9 2005 2 82 82 30 12 2004 28 9 2005 Copyright © 2005 Chwla; licensee BioMed Central Ltd.2005Chwla; 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.
Inactive carriers forms the largest group in chronic HBV infected patients. Around 300 million people are inactive carriers The inactive HBsAg carrier state is diagnosed by absence of HBeAg and presence of anti-HBe, undetectable or low levels of HBV DNA in PCR-based assays, repeatedly normal ALT levels, and minimal or no necroinflammation, slight fibrosis, or even normal histology on biopsy. Inactive cirrhosis may be present in patients who had active liver disease during the replicative phase of infection. The prognosis of the inactive HBsAg carrier state is usually benign. Long-term follow- up (up to 18 years) of these carriers has indicated that the vast majority show sustained biochemical remission and very low risk of cirrhosis or hepatocellular carcinoma (HCC). Rarely, patients, even noncirrhotics, may develop liver cancer during the inactive HBsAg carrier state. In addition, approximately 20 to 30% of persons in the inactive HBsAg carrier state may undergo spontaneous reactivation of hepatitis B during follow-up. Multiple episodes of reactivation or sustained reactivation can cause progressive hepatic damage and even hepatic decompensation. Introduction
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Hepatitis B virus (HBV) infection and its sequelae are major global health problems [1]. It is estimated that 400 million people worldwide are HBV carriers [2]. The natural history of hepatitis B is complex and is influenced by many factors, including age at infection, viral factors (HBV genotype, viral mutations, level of HBV replication), host factors (gender, age, and immune status), and exogenous factors such as concurrent infection with other hepatotropic viruses or alcohol. The clinical spectrum of HBV infection ranges from subclinical to acute symptomatic hepatitis or, rarely, fulminant hepatitis during the acute phase and from the inactive hepatitis B surface antigen (HBsAg) carrier state to chronic hepatitis, cirrhosis, and its complications during the chronic phase [3,4]. Approximately 15 to 40% of people who develop chronic HBV infection are expected to progress to cirrhosis and end-stage liver disease [1]. Difficulties in defining the natural history of chronic hepatitis B include the indolent course of the disease, the lack of symptoms during the early stages, and the heterogeneity of the disease. Understanding the natural history and prognosis of hepatitis B is the basis for disease management and for designing better therapeutic strategies.
Hepatitis B Virus
HBV belongs to the family hepdnaviruses. The HBV genome is a relaxed circular, partially double stranded DNA of approximately 3,200 base pairs. There are four partially overlapping open reading frames encoding the envelope (pre-S/S), core (precore/core), polymerase, and X proteins [5]. The pre-S/S open reading frame encodes the large, middle, and small surface glycoproteins. The precore/core open reading frame is translated into precore polypeptide which is modified in to a soluble protein, the hepatitis B e antigen (HBeAg), and the nucleocapsid core protein hepatitis B core antigen (HBc Ag) [5]. The polymerase protein functions as reverse transcriptase as well as DNA polymerase. The X protein is a potent transactivator and may play role in hepatocarcinogenesis.
Prevalence
Hepatitis B is spread predominantly parenterally, through intimate personal contact, and perinatally. Individuals at risk are intravenous drug users, children of mothers with HBV, men who have sex with men, patients on hemodialysis and those exposed to blood or blood products [6,7]. Approximately 5% of the world's populations are carriers of HBV, defined as being positive for hepatitis B surface antigen. HBV is endemic in many areas of the world, such as Asia, Micronesia, and sub-Saharan Africa as well as in certain populations in Australia, New Zealand, South America, the Middle East and the Arctic. An estimated 1.25 million people in the United States are positive for hepatitis B surface antigen. Fifteen percent to forty percent of these carriers may develop hepatitis B-related sequelae in their lifetimes [8-10].
Natural History
Perinatal infection of infants from infected mothers and horizontal infection early in childhood from exposure to HBsAg-positive family members are the main routes of HBV transmission in highly endemic areas, such as Southeast Asia, Africa, the Pacific Islands, and the Arctic. In regions of low endemicity, such as Western countries, hepatitis B is primarily a disease of adolescents and adults as a result of high-risk sexual behavior and injection drug use. HBV infection is a dynamic process with replicative and nonreplicative (or low replicative) phases based on virus-host interaction. The presence of circulating HBsAg, hepatitis B e antigen (HBeAg), and high levels of serum HBV DNA characterizes the immunotolerant phase. This first phase is seen in patients with perinatal infection and often lasts for decades. During this phase patients have no symptoms, normal or slightly increased serum alanine aminotransferase (ALT) levels, and minimal histological activities, which imply that there is a lack of or a very weak immune response against the infected hepatocytes.
Experimental results in transgenic mice suggested HBeAg induces a state of immunological tolerance to HBV in neonates [11]. During the course of chronic HBV infection, for unknown reasons, the tolerogenic effect is and patients may enter the immunoactive phase, which is associated with a decrease in HBV DNA concentrations and increased ALT levels and histologic activity, reflecting immune-mediated lysis of infected hepatocytes. This second phase has a variable duration from months to years. The third low or nonreplicative phase occurs seroconversion from HBeAg to antibody to HBeAg. This phase is usually preceded by a marked reduction of serum HBV DNA to levels that are not detectable by hybridization techniques, followed by normalization of ALT levels and resolution of liver necroinflammation. In many patients, serum HBV DNA remains detectable by the sensitive technique of polymerase chain reaction (PCR). This phase is also referred as the inactive HBsAg carrier state [3,4]. The inactive carrier state may for a lifetime, but a proportion of patients may undergo subsequent spontaneous or immunosuppressioninduced reactivation of HBV replication with reappearance of high levels of HBV DNA with or without HBeAg seroreversion and a rise in ALT levels [3]. For reasons that are not yet known, replication-competent HBV variants with mutations in the precore or core promoter regions preventing or down-regulating HBeAg production may be selected during or after HBeAg seroconversion.
Patients who become HBsAg negative and develop antibody to HBsAg (anti-HBs) are diagnosed as having resolved hepatitis B [3,4]. This is an uncommon phenomenon in chronic HBV infection. During stage HBV DNA may still be detectable by PCR in serum and more often in the liver.[12] In rare cases severe immune suppression, such as cancer chemotherapy or after organ transplantation, HBV can be reactivated in patients with resolved hepatitis B [13].
Clinical spectrum
HBeAg positive Chronic Hepatitis
Patients with HBeAg-positive chronic hepatitis B usually present in the third or fourth decade of life. Men outnumber women, [14,15] liver damage ranges from mild (24 to 42%) to moderate or severe chronic hepatitis (44 to 63%) or active cirrhosis (10 to 24%) [16-20]. Chronic hepatitis B tends to be milder in children. Nevertheless, severe liver disease including cirrhosis may occur in a small proportion of patients during childhood [21,22]. A key event in the natural history of HBeAg positive chronic hepatitis is HBeAg seroconversion. Several studies have shown that seroconversion with marked reduction of HBV replication is associated with biochemical and histologic remission of inflammatory activity in the majority of patients [23-25]. Regression of fibrosis occurs gradually months to years after HBeAg seroconversion.[26] In longitudinal studies the observed probability of clearing HBeAg was about 50% and 70% within 5 and 10 years of diagnosis, respectively [23,27-29]. Most studies have found that the mean annual rate of spontaneous HBeAg seroconversion ranges from 8 to 15% in children or adults with elevated ALT [3,4,23-25,29-31,31]. Among Asian, most of whom have normal ALT, spontaneous HBeAg seroconversion occurs at a very low rate, less than 2% during the first 3 years of age and 4 to 5% in children older than 3 years [32]. Several determinants for HBeAg seroconversion have been reported, including gender, age, ALT level, and more recently HBV genotypes. Older carriers and females are more likely to clear HBeAg [33].
Frequent acute exacerbation of hepatitis, reflecting immune-mediated lysis of HBV-infected hepatocytes with ALT elevations to more than 10 times ULN and more than twice the baseline value and with HBV DNA levels rising before and falling during the flare, precede seroconversion of HBeAg to anti-HBe. These exacerbations usually last 2 to 4 months [34]. In some cases these spontaneous flares of hepatitis are not followed by subsequent HBeAg seroconversion and can be viewed as an abortive attempt at seroconversion. These flares of hepatitis are usually asymptomatic and frequently unrecognized, but some are accompanied by symptoms of acute hepatitis and rarely, primarily in patients with cirrhosis or advanced fibrosis, may lead to hepatic decompensation and even death due to massive necrosis [34].
HBsAg-negative chronic hepatitis
The diagnosis of HBeAg-negative chronic hepatitis B is based on the presence of HBsAg for more than 6 months, undetectable HBeAg, presence of anti-HBe, detectable serum HBV DNA exceeding 105 to 106 copies/mL, increased ALT levels, and hepatic necroinflammation on histology. Other causes of liver disease, such as superinfection with other hepatitis viruses, alcohol abuse, hepatotoxic drug use, and autoimmune or metabolic liver disease, should be excluded [3,4]. The atypical serological profile is related to the predominance of HBV variants, which are unable to express HBeAg. The most frequent variant has a G-to-A change at nucleotide 1896 (G1896A), which creates a stop codon in the precore region of the HBV genome and completely abolishes the production of HBeAg [35]. Other variants include changes in the start codon of the precore region or a two-nucleotide substitution (A1762T, G1764A) in the core promoter region, which reduces precore messenger RNA synthesis and HBeAg production [36].
Patient with HBeAg negative are older than patients with HBeAg-positive chronic hepatitis (median 40, range 36–45 years). Males predominate and data indicate that liver disease is more active and advanced, minimal or mild chronic hepatitis is infrequent, and severe necroinflammation is seen in more than 50% patients at diagnosis [37-39]. In reports from Mediterranean area, 29 to 38% had cirrhosis at presentation. The older age and the high rate of advanced liver damage at presentation suggest that HBeAg-negative chronic hepatitis represents a late phase in the natural history of chronic HBV infection rather than de novo infection with HBV variants that do not produce HBeAg. To further support this concept, a recent long-term study reported that HBeAg-negative chronic hepatitis accumulated over time after HBeAg seroconversion with a cumulative incidence of approximately 25% after 16 years of follow-up [40]. Thus, the increasing prevalence of HBeAg-negative. Fluctuation in level of viremia and ALT are more common and sustained response is rare. Delayed spontaneous HBsAg clearance has been estimated to occur at a low rate of 0.5% per year [40].
Inactive HBsAg Carrier State
Inactive carriers forms the largest group in chronic HBV infected patients. Around 300 million people are inactive carriers The inactive HBsAg carrier state is diagnosed by absence of HBeAg and presence of anti-HBe, undetectable or low levels of HBV DNA in PCR-based assays, repeatedly normal ALT levels, and minimal or no necroinflammation, slight fibrosis, or even normal histology on biopsy [3,4]. Inactive cirrhosis may be present in patients who had active liver disease during the replicative phase of infection. The prognosis of the inactive HBsAg carrier state is usually benign. Long-term follow- up (up to 18 years) of these carriers has indicated that the vast majority show sustained biochemical remission and very low risk of cirrhosis or hepatocellular carcinoma (HCC) [40-42]. Rarely, patients, even noncirrhotics, may develop liver cancer during the inactive HBsAg carrier state [40-43]. In addition, approximately 20 to 30% of persons in the inactive HBsAg carrier state may undergo spontaneous reactivation of hepatitis B during follow-up [29,33,34,40]. Multiple episodes of reactivation or sustained reactivation can cause progressive hepatic damage and even hepatic decompensation. HBV reactivation is usually asymptomatic but on occasion can mimic acute viral hepatitis [44]. Acute flares of hepatitis should be differentiated from superinfection with other hepatotropic viruses. As many as 20 to 30% of these acute exacerbations may be caused by superinfection with HDV, HCV, or hepatitis A virus and can be associated with an increased risk of fulminant hepatic failure [44]. Some carriers eventually become HBsAg negative and develop anti-HBs. The incidence of delayed HBsAg clearance has been estimated to be 1 to 2% per year in Western countries, where HBV infection is usually acquired in adulthood, but a lower rate from 0.05 to 0.8% per year in endemic areas, where HBV infection is mostly acquired perinatally or in early childhood. Clearance of HBsAg has been reported to be higher in women than in men and in older than younger carriers. Prognosis is improved by loss of HBsAg as liver disease is usually inactive and nonprogressive, but HBsAg clearance does not completely prevent occurrence of decompensation or HCC in patients who have already developed cirrhosis [45,46].
Change in the terminology of HBV carriers
HBV infection is termed as chronic if it continues to be HBsAg +ve for ≥6 months. Chronic HBV infection is a dynamic process with a wide spectrum of spectrum of affliction. On one hand patients are asymptomatic with no clinical evidence of liver diseases, while on other being end-stage cirrhosis and hepatocellular carcinoma. For many decades the patients were considered to have a benign, non progression infection and were designated as hepatitis B "carriers". Probably the word 'carrier' was mistakenly chosen for hepatitis B as in true sense, a carrier is an individual who (i) harbors a specific infectious agent (ii) has no discernible clinical disease and (iii) serves as a potential source of infection. For this infection the second and third points should be looked at carefully. One the basis of Asian collaborative survey the term 'carrier' was replaced by the term 'chronic hepatitis B virus infection' [47,48]. Later on for this infection the term 'Inactive HBsAg carrier' was adopted [49].
Management of Inactive HBsAg Carrier
Differentiation from chronic HBsAg negative hepatitis B, requires serial testing of ALT and HBV DNA for one year before designating carrier state [49]. In subject with inactive carrier state testing of HBV DNA and liver biopsy are not recommended. Treatment is not recommended as there is no evidence that available therapy affects HBsAg status. Family screening with HBsAg and anti-HBs, if negative vaccinate them and success of vaccination should be confirmed with anti-HBs testing. Protected sexual intercourse until partner has developed protective antibodies. The offspring need active and passive vaccination [4,47]. Use of alcohol should be avoided, possibility of reactivation or super infection by other viruses and advised if there is jaundice, malaise or increased fatigue. Regular follow-up at every 6–12 months intervals with ALT [4]. If the age of the patient is more than 50 yrs family history of HCC-AFP and ultrasonography every 6–12 monthly should be done. Universal precaution should be taken while treating these patients in the hospital. They should not be allowed to donate the blood or organ or semen. For pregnant women vaccinate the new born at birth with active and passive immunization with in 12 hours of the birth, close monitoring required if undergoing chemotherapy or immunosuppressive medication.
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Maddrey WC Hepatitis B: an important public health issue J Med Virol 2000 61 362 366 10861647 10.1002/1096-9071(200007)61:3<362::AID-JMV14>3.0.CO;2-I
Lee WM Hepatitis B virus infection N Engl J Med 1997 337 1733 1745 9392700 10.1056/NEJM199712113372406
Lok AS Heathcote EJ Hoofnagle JH Management of hepatitis B: 2000 – summary of a workshop Gastroenterology 2001 120 1828 1853 11375963
Lok ASF McMahon BJ Chronic hepatitis B Hepatology 2001 34 1225 1241 11732013 10.1053/jhep.2001.29401
Seegar C Mason WS Hepattits B virus: the major etiology of hepatocellular carcinoma Cancer 1988 61 9142 56
Margolis HS Alter MJ Hadier SC Hepatitis B: evolving epidemiology and implications for control Semin Liver Dis 1991 11 84 92 1832236
Beasley RP Hwang LY Lee GC Prevention of perinatally transmitted hepatitis B virus infections with hepatitis B immune globulin and hepatitis B vaccine Lancet 1983 2 1099 1102 6138642 10.1016/S0140-6736(83)90624-4
Lee WM Hepatitis B virus infection N Engl J Med 1997 337 1733 1745 9392700 10.1056/NEJM199712113372406
McQuillan GM Townsend TR Fields HA Carrol M Leahy M Polk BF Seroepidemiology of hepatitis B virus infection in the United States Am J Med 1989 87 5S 10S 2773982 10.1016/0002-9343(89)90523-8
CDC Immunization Practices Advisory Committee (ACIP) Hepatitis B Virus: A comprehensive strategy for limiting transmission in the United States through universal childhood vaccination MMWR Morb Mortal Wkly Rep 1991 40 1 25 1898620
Milich DR Chen MK Hughes JL Jones JE The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence J Immunol 1998 160 2013 2021 9469465
Chemin I Zoulim F Merle P High incidence of hepatitis B infections among chronic hepatitis cases of unknwon aetiology J Hepatol 2001 34 471 473 11322211 10.1016/S0168-8278(00)00100-8
Kawatani T Suou T Tajima F Incidence of hepatitis virus infection and severe liver dysfunction in patients receiving chemotherapy for hematologic malignancies Eur J Haematol 2001 67 45 50 11553266 10.1034/j.1600-0609.2001.067001045.x
Burk RD Hwang LY Ho GYF Shafritz DA Beasley RP Outcome of perinatal hepatitis B virus exposure is dependent on maternal virus load J Infect Dis 1994 170 1418 1423 7995980
Cacciola I Cerenzia G Pollicino T Genomic heterogeneity of hepatitis B virus (HBV) and outcome of perinatal HBV infection J Hepatol 2002 36 426 432 11867188 10.1016/S0168-8278(01)00295-1
Hoofnagle JH Dusheiko GM Seef LB Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis Ann Intern Med 1981 94 744 748 7235415
Fattovich G Rugge M Brollo L Clinical, virologic and histologic outcome following seroconversion from HBeAg to anti-HBe in chronic hepatitis type B Hepatology 1986 6 167 172 3957228
Moreno-Otero R Garcia-Monzòn C Garcia-Sànchez A Development of cirrhosis after chronic type B hepatitis: a clinicopathologic and follow-up study of 46 HBeAg-positiveasymptomatic patients Am J Gastroenterol 1991 86 560 564 2028945
Zarski JP Marcellin P Cohard M Comparison of anti-HBe-positive and HBe-antigen-positive chronic hepatitis B in France J Hepatol 1994 20 636 640 8071540 10.1016/S0168-8278(05)80352-6
Di Marco V Lo Iacono O Cammà C The long-term course of chronic hepatitis B Hepatology 1999 30 257 264 10385664 10.1002/hep.510300109
Chang MH Hsu HY Hsu HC The significance of spontaneous hepatitis B e antigen seroconversion in childhood: with special emphasis on the clearance of hepatitis B e antigen before 3 years of age Hepatology 1995 22 1387 1392 7590652 10.1016/0270-9139(95)90141-8
Bortolotti F Jara P Crivellaro C Outcome of chronic hepatitis B in Caucasian children during a 20-year observation period J Hepatol 1998 29 184 190 CDC. Hepatitis B virus: a comprehensive strategy for limiting transmission in the United States through universal child vaccination. Recommendation of the Immunization Practice Advisory Committee (ACIP). MMWR 1991;40:RR-13:1–25 9722198 10.1016/S0168-8278(98)80002-0
Realdi G Alberti A Rugge M Seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection Gastroenterology 1980 79 195 199 7399226
Hoofnagle JH Dusheiko GM Seef LB Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis Ann Intern Med 1981 94 744 748 7235415
Fattovich G Rugge M Brollo L Clinical, virologic and histologic outcome following seroconversion from HBeAg to anti-HBe in chronic hepatitis type B Hepatology 1986 6 167 172 3957228
Fong TL Di Bisceglie AM Gerber MA Waggoner JG Hoofnagle JH Persistence of hepatitis B virus DNA in the liver after loss of HBsAg in chronic hepatitis B Hepatology 1993 18 1313 1318 8244254 10.1016/0270-9139(93)90217-B
Yuen MF Hui CK Cheng CC Long-term follow-up of interferon alfa treatment in Chinese patients with chronic hepatitis B infection: the effect on hepatitis B e antigen seroconversion and the development of cirrhosis-related complications Hepatology 2001 34 139 145 11431745 10.1053/jhep.2001.25273
McMahon BJ Holck P Bulkow L Snowball M Serologic and clinical outcomes of 1536 Alaska natives chronically infected with hepatitis B virus Ann Intern Med 2001 135 759 768 11694101
Bortolotti F Cadrobbi P Crivellaro C Long-term outcome of chronic type B hepatitis in patients who acquire hepatitis B virus infection in childhood Gastroenterology 1990 99 805 810 2379783
Wong DKH Cheung AM O'Rourke K Effect of alpha-interferon treatment in patients with hepatitis B e antigen positive chronic hepatitis B. A meta-analysis Ann Intern Med 1993 119 312 323 8328741
Vajro P Migliaro F Fontanella A Orso G Interferon: a meta-analysis of published studies in pediatric chronic hepatitis B Acta Gastroenterol Belg 1998 61 219 223 9658614
Chang MH Sung JL Lee CY Factors affecting clearance of hepatitis B e antigen in hepatitis B surface antigen carrier children J Pediatr 1989 115 385 390 2769497
Lok ASF Lai CL Wu PC Leung EKY Lam TS Spontaneous hepatitis B e antigen to antibody seroconversion and reversion in Chinese patients with chronic hepatitis B virus infection Gastroenterology 1987 92 1839 1843 3569757
Perillo RP Acute flares in chronic hepatitis B: the natural and unnatural history of an immunologically mediated liver disease Gastroenterology 2001 120 1009 1022 11231956
Hadziyannis SJ Vassilopoulos D Hepatitis B e antigennegative chronic hepatitis B Hepatology 2001 34 617 624 11584355 10.1053/jhep.2001.27834
Chan HLY Leung NWY Hussain M Wong ML Lok ASF Hepatitis B e antigen-negative chronic hepatitis B in Hong Kong Hepatology 2000 31 763 768 10706570 10.1002/hep.510310330
Fattovich G Farci P Rugge M Randomized controlled trial of lymphoblastoid interferon alfa in patients with chronic hepatitis B who lacked hepatitis B e antigen Hepatology 1992 15 584 589 1551634
Lampertico P Del Ninno E Manzin A A randomized, controlled trial of a 24-month course of interferon alfa 2b in patients with chronic hepatitis B who had hepatitis B virus DNA without hepatitis B e antigen in serum Hepatology 1997 26 1621 1625 9398007
Tassopoulos NC Volpes R Pastore G Efficacy of lamivudine in patients with hepatitis B e antigen negative hepatitis B virus DNA-positive (precore mutant) chronic hepatitis B. Lamivudine Precore Mutant Group Hepatology 1999 29 889 896 10051494 10.1002/hep.510290321
Hsu YS Chien RN Yeh CT Long-term outcome after spontaneous HBeAg seroconversion in patients with chronic hepatitis B Hepatology 2002 35 1522 1527 12029639 10.1053/jhep.2002.33638
De Franchis R Meucci G Vecchi M The natural history of asymptomatic hepatitis B surface antigen carriers Ann Intern Med 1993 118 191 194 8417636
Bellentani S Dal Molin G Miglioli L Natural history of HBV infection: a 9 years follow-up of the Dionysos cohort J Hepatol 2002 36 228 10.1016/S0168-8278(02)80819-4
Fattovich G Giustina G Realdi G Corrocher R Schalm SW Long-term outcome of hepatitis B e antigen positive patients with compensated cirrhosis treated with interferon alfa Hepatology 1997 26 1338 1342 9362381
Tassopoulos NC Papaevangelou GJ Sjogren MH Natural history of acute hepatitis B surface antigen-positive hepatitis in Greek adults Gastroenterology 1987 92 1844 1850 3569758
Fattovich G Giustina G Sanchez-Tapias J Delayed clearance of serum HBsAg in compensated cirrhosis B: relation to interferon alpha therapy and disease prognosis Am J Gastroenterol 1998 93 896 900 9647014 10.1111/j.1572-0241.1998.00272.x
Huo TI Wu JC Lee PC Sero-clearance of hepatitis B surface antigen in chronic carriers not necessarily implies a good prognosis Hepatology 1998 28 231 236 9657117 10.1002/hep.510280130
Sarin Sk Summary and recommendations of single theme conferences on hepatitis B and C: Indian association for study of the liver (INASL) J gastro hepatol 2002 17 S197 S203 10.1046/j.1440-1746.17.s3.1.x
Sarin SK Sathpathy SK Chauhan R Hepatitis B e-antigen negative chronic hepatitis B J gastro hepatol 2002 17 S311 S321 10.1046/j.1440-1746.17.s3.20.x
EASL international consensus conference on hepatitis B Journal of Hepatology 2003 39 S3 S25 14708673
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1621246810.1371/journal.pmed.0020336Neglected DiseasesInfectious DiseasesInfectious DiseasesMedicine in Developing Countries“Rapid-Impact Interventions”: How a Policy of Integrated Control for Africa's Neglected Tropical Diseases Could Benefit the Poor Neglected DiseasesMolyneux David H Hotez Peter J *Fenwick Alan David H. Molyneux is Professor of Tropical Health Sciences and Director of the Lymphatic Filariasis Support Centre at the Liverpool School of Tropical Medicine, Liverpool, United Kingdom. Peter Hotez is Professor and Chair of the Department of Microbiology, Immunology, and Tropical Medicine of The George Washington University, Washington, District of Columbia, United States of America, and Principal Scientist of the Human Hookworm Vaccine Initiative, Sabin Vaccine Institute, Bethesda, Maryland, United States of America. Alan Fenwick is Professor of Tropical Parasitology, Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom, and Director of the Schistosomiasis Control Initiative, London, United Kingdom.
Competing Interests: DHM is supported by the UK Department for International Development and GlaxoSmithKline (London, United Kingdom) and participates in the Mectizan Expert Committee/Albendazole Coordination meetings, which are supported by Merck and Company (Whitehouse Station, New Jersey, United States of America) and GlaxoSmithKline. JH is an inventor on an international patent application (PCT/US02/33106; filed 11 November, 2002) entitled “Hookworm Vaccine.” PJH is also Co-Chair of the Scientific Advisory Council of the Sabin Vaccine Institute (New Canaan, Connecticut, United States of America) and a member of the Academic Advisory Board for the Pfizer Fellowships in Infectious Diseases. AF is Director of the Schistosomiasis Control Initiative, which is supported by the Bill and Melinda Gates Foundation (Seattle, Washington, United States of America).
*To whom correspondence should be addressed. E-mail: [email protected] 2005 11 10 2005 2 11 e336Copyright: © 2005 Molyneux 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.Controlling seven tropical infections in Africa would cost just 40 cents per person per year, and would permanently benefit hundreds of millions of people.
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Over the past two decades there have been significant achievements in the control of a handful of important human tropical infections [1]. These achievements include the substantive reductions in the prevalence and incidence of the so-called neglected diseases such as lymphatic filariasis, onchocerciasis, guinea worm, leprosy, and trachoma (Box 1) [2].
Each of these neglected diseases is a poverty-promoting and often stigmatizing condition occurring primarily in rural areas of low-income countries (Box 2) [3]. They are ancient afflictions, described in the Bible and other ancient texts, which have burdened humanity for millennia [3]. But now, as a result of aggressive regional vertical interventions, there is a possibility that some neglected tropical infections could be eventually controlled to the point of elimination in some areas of endemicity [2–8]. In the case of guinea worm infection, disease eradication might also soon be possible [9].
Box 2. Common Features of the Neglected Tropical Diseases
Ancient afflictions that have burdened humanity for centuries
Poverty-promoting conditions
Associated with stigma
Rural areas of low-income countries and fragile states
No commercial markets for products that target these diseases
Interventions, when applied, have a history of success
Neglected by Policy Makers and Donors
Somewhat surprisingly, policy makers and public health officials have largely ignored the extraordinary successes in these vertical programmes for neglected disease control and elimination. We believe that there are two main reasons for this lack of attention. The first is that the international health community, including donors, have given the highest priority to the “big three”—HIV/AIDS, tuberculosis (TB), and malaria—with the result that other infections of poverty garner less attention. The second is that donors and policy makers take a dim view of the overall value of vertical programmes that are not directed at the big three.
With regards to the big three, donors, international agencies, nongovernmental development agencies, and governments have responded through focused attention on vertical initiatives by creating UNAIDS (http://www.unaids.org) and within WHO, Stop TB (http://www.stoptb.org) and Roll Back Malaria (http://www.rollbackmalaria.org). These developments stimulated the establishment of the Global Fund to Fight AIDS, TB and Malaria (http://www.theglobalfund.org), a financing mechanism that provides up to five years of funding to projects relating to these three diseases.
However, this financing mechanism contrasts with the approach to financing health in many developing countries that is preferred by many bilateral donors, called sector-wide approaches. In a sector-wide approach, donors agree to contribute to a single basket of funds, which in turn contributes to the developing country's national plan [10,11]. There has been recognition that many of the specific problems of health cannot be resolved without effective health systems. Following the World Development Report of 1993, the World Bank promoted the concept of the need for a minimum health package that both governments and donors could afford [12]. In recent times, a large number of policy papers on strengthening health systems have appeared, many of which were presented at the Ministerial Summit in Mexico [13]. The Ministerial Summit made specific recommendations, particularly in the areas of health systems research; the mantra was that strengthening health systems is a prerequisite for improving health, and more health systems research is required to ensure cost-effectiveness of investment [13].
Neglected Diseases and the Millennium Development Goals
At the same time, there has also been increased attention on the relationship between health and poverty, especially in relation to the Millennium Development Goals (MDGs). These goals, endorsed by the international community, include the goal of reducing the number of people living in absolute poverty by 50% by 2015 (http://www.un.org/millenniumgoals). A review of the progress toward these goals—the UN Millennium Project—has recently been published, which identified sub-Saharan Africa as significantly lagging in meeting MDGs compared to other regions [14]. Several of these goals have specific health-related targets, but only the big three feature within the MDGs themselves. Other infections, including the neglected tropical diseases, which affect at least as many poor people as the big three, are relegated dismissively to the category of “other diseases.”
In parallel with changes in health financing and policy and the growing awareness of the need to strengthen health systems, there has been an explosion in the number of public–private partnerships (PPPs) created to address specific health problems. At the time this article went to press, the Initiative on Public Private Partnerships for Health listed 92 such partnerships in its “partnerships database” (http://www.ippph.org/index.cfm?page=/ippph/partnerships); several of these address parasitic diseases and are based on product donations [15,16].
The Commission for Africa (http://www.commisionforafrica.org), established by the British Prime Minister and the Chancellor of the Exchequer, proposes a Marshall plan for Africa [17]. Although the commission's report remains focused on HIV/AIDS, TB, and malaria, the neglected tropical diseases are also recognized as contributing significantly to the overall African disease burden.
The Burden of Neglected Diseases
In aggregate, the neglected tropical diseases are responsible for about 500,000 deaths annually. Using the disability-adjusted life year as a metric, the burden of neglected tropical diseases is equivalent to approximately one quarter of the disease burden from HIV/AIDS and one half that of malaria [3]. However, newer information indicates that even these high disability–adjusted-life-year figures grossly underestimate the disease burden of neglected tropical diseases [18–20].
Of the listed major neglected diseases, ten of them stand out for their high prevalence and intensity in Africa: urinary and intestinal schistosomiasis, lymphatic filariasis, onchocerciasis, the soil-transmitted helminth (STH) infections (ascariasis, trichuriasis, and hookworm infection), African trypanosomiasis, kala-azar, Buruli ulcer, and blinding trachoma. Up to 90% or more of the world's disease burden from these conditions is believed to occur in Africa (Table 1).
Table 1 Sub-Saharan Africa Has the Highest Prevalence of Nine Neglected Tropical Diseases
Evidence for the Value of Integrated Control
Of equal interest is the observation that there are currently six major PPPs working in Africa that are engaged in a vertical elimination or control programme linked to a specific neglected tropical disease (Table 2). In Africa, the six PPPs operate in parallel, using control tools comprised predominantly of one or two drugs deployed over wide areas and among large populations. In aggregate, the six PPPs are deploying four drugs—albendazole, ivermectin (Mectizan), praziquantel, and azithromycin (Zithromax) —in order to target more than 100 million Africans in around 30 countries. An added benefit of the PPP activities is their role in strengthening health systems. For example, the African Programme for Onchocerciasis Control has established a successful community-directed treatment initiative, which has provided a valuable entry point for other community-directed health interventions in regions where there is little access to traditional health services [21].
Table 2 PPPs Engaged in Vertical Programmes for Neglected Tropical Disease Control in Africa
Closer analysis of the major endemic neglected tropical diseases in Africa reveals that they exhibit considerable geographical overlap, and hence in many cases are syndemic (Figure 1) [22]. Therefore, we believe that there could be great value in exploring whether a drug employed by a vertical programme that targets one condition could also be used to simultaneously make an impact on some of the others [23]. For example, because a significant proportion of impoverished school-age children living in Africa carry multiple parasitic infections—i.e., they are polyparasitized—with three different STHs (Ascaris, Trichuris, and hookworm) and schistosomes, they could be simultaneously treated with two drugs, albendazole and praziquantel [18]. Indeed, in 2001, the 54th World Health Assembly urged its member states to undertake frequent and periodic deworming with praziquantel together with either albendazole or mebendazole as a means to control and reduce the morbidity in this paediatric age group (http://www.who.int/wormcontrol) [18].
Figure 1 Geographic Overlap of the Neglected Tropical Diseases
(Figure: Molly Brady, Emory University)
Accordingly, the Schistosomiasis Control Initiative, a PPP based in London but working in Uganda, Tanzania, Zambia, Mali, Niger, and Burkina Faso, adds albendazole to its praziquantel regimen (http://www.schisto.org). Similarly, the major drugs used for lymphatic filariasis and onchocerciasis control, ivermectin and albendazole (http://www.filariasis.org), also target the STHs in polyparasitized children as well as adults. Albendazole is the drug of choice for most of the STHs, while ivermectin also has a significant anthelmintic effect on Ascaris and Trichuris infections, and is the drug of choice for the treatment of human strongyloidiasis (http://www.themedicalletter.com/freedocs/parasitic.pdf).
More recently, selective mass treatment with ivermectin has been shown to also reduce the prevalence of ectoparasitic skin infections such as pediculosis, scabies, and tungiasis [24] as well as cutaneous larva migrans. Scabies control with ivermectin also reduces the occurrence of secondary streptococcal skin infections and even renal disease resulting from post-streptococcal glomerulonephritis [25]. There is also recent evidence that doxycycline and other antibiotics are effective in killing the adult filarial worm, Wuchereria bancrofti, because the filarial parasite depends on endosymbiotic Wolbachia rickettsia for survival and reproduction [26]. Azithromycin, which is used for the control of trachoma (http://www.trachoma.org), has also been shown to exhibit similar anti-filarial activity in vitro [27], although it is not yet clear whether this will translate into a public health impact on filaria. Widespread use of azithromycin could impact on other paediatric bacterial infections, including those caused by group A streptococci [28].
The Cost-Effectiveness of Integrated Control
Indeed, armed with four drugs (albendazole, ivermectin, azithromycin, and praziquantel), the six PPPs could integrate control of seven major neglected tropical diseases in Africa. In so doing, a rapid impact on morbidity, blindness, and skin disease could be achieved at the minimal cost of about US$0.40 per person per year [23]. For just US$200 million per year for five years, it is estimated that over 500 million individuals could benefit from preventative chemotherapy, which would rapidly contribute to poverty reduction and take steps toward seven of the eight MDGs [23]. Poverty reduction would be even more likely if the resources were allocated as a package for the control or elimination of these diseases of poverty.
In addition, the calculated economic rates of return suggest that investment in control/elimination of these diseases produces an economic rate of return of 15%–30%, and are capable of delivery on a large scale [1]. The recent publication from the Millennium Project lists under its “quick wins” (referring to situations in which simple interventions could make profound differences to survival and quality of life) regular deworming of school-aged children [14], an approach strongly advocated in a recent Lancet editorial, “Thinking beyond Deworming” [29].
The potential synergies in collateral benefits delivered using the four drugs mentioned above is appropriate, as they often have compatible approaches to delivery. Furthermore, three of these drugs are being donated by multinational pharmaceutical companies (ivermectin [Mectizan] by Merck & Co., Inc.; azithromycin [Zithromax] by Pfizer; and albendazole by GlaxoSmithKline), two of these—ivermectin and albendazole—for “as long as needed” to achieve public health goals [21].
Praziquantel for schistosomiasis is now significantly less expensive than a decade ago (US$0.07 per tablet, or US$.20 to treat a child) and so treatment to alleviate morbidity from schistosomiasis in some 166 million individuals in Africa of all ages and both sexes is now possible (http://www.schisto.org). The report of the Commission for Africa contains two statements relevant to this possibility: “donors should ensure that there is adequate funding for the treatment and prevention of parasitic diseases and micronutrient deficiency;” and “governments and global health partnerships should ensure that this [funding] is integrated into public health campaigns by 2006” (pp. 72 and 198 of [30]).
Table 3 presents the numbers requiring treatment for each of these infections, the unit drug price (if applicable), and the estimated total delivery costs of treating these chronic disabling conditions in sub-Saharan Africa. Such interventions, which are aggressively pro-poor, are based on safe, efficacious drugs that reach a high coverage of the target population, are known to be cost effective, and do not, as yet, have any associated drug resistance. They can be delivered through community-directed approaches, school health programmes, the World Food Programme school feeding programme, or the supplementary feeding and nutrition programmes of nongovernmental development organizations, usually on an annual basis (http://www.wfp.org). The estimate of US$.40 per treatment annually is equivalent to the bulk cost of about 12 condoms for prevention of HIV transmission or a quarter of the price of a single antimalarial bednet.
Table 3 Costs for Effective Chemotherapy Programmes against Parasitic and Infectious Debilitating and Blinding Diseases in Sub-Saharan Africa
Scaling Up Integrated Control
A number of issues need to be addressed before integrated control of neglected tropical diseases may be practiced on a large-scale basis in Africa. For example, the final costs of an integrated package may need to include the costs of drug use monitoring and of developing new tools for neglected disease control [18]. In some areas, neither mebendazole nor ivermectin are very effective against hookworm, the most common STH in Africa, especially when these drugs are used in a single dose [20,31,32]. Moreover, the rate of post-treatment hookworm infection is high [33], and there is additional evidence that the efficacy of benzimidazole anthelmintics diminishes even further with frequent and periodic use [32]. As a result, there are justifiable concerns about the possibility of emerging resistance, which is now common for STHs that infect livestock [34]. This has prompted efforts to develop additional new control tools including anthelmintic vaccines [20,34–36]. In addition, the widespread use of azithromycin could promote the emergence of drug-resistant pneumococcus [37]. Additional costs must therefore be considered in order to promote ongoing research and development for new neglected diseases control tools [20].
An equally important challenge will be to determine the actual feasibility of integrating six different vertical control programmes. There are currently disparities between the groups targeted for lymphatic filariasis and onchocerciasis control (treatment is excluded for children under 90 cm long and pregnant women) and the groups targeted for STH and schistosome control (control is primarily aimed at school-age children, but the World Health Organization encourages treatment of pregnant women in the second and third trimesters). Pilot studies will be necessary to identify common age groups for integrated control. There are the additional political hurdles of persuading each of the PPPs working in Africa to cooperate on disease control efforts and to fully integrate their activities.
Small Costs, Huge Impact
Overall, however, the low costs for integrated neglected disease control represent compelling figures to advocate for a pro-poor, proactive public health intervention strategy of preventative chemotherapy to be delivered to all affected populations of Africa. Such a policy would be entirely compatible with the policies advocated by the Commission for Africa and the recently published report on the progress toward the MDGs submitted to the Secretary General of the United Nations (http://www.unmillenniumproject.org). The US$0.40 per person annual cost estimate, which would bring better health to several hundred million polyparasitized and disenfranchised poor, is a fraction of the estimated treatment costs for HIV/AIDS, TB, and malaria. By comparison, the treatment for HIV/AIDS exceeds US$200 per year per person for the life of the individual [38], while TB treatment costs at least around US$200 per treatment in Africa [39], and the total costs of malaria treatment per episode are about US$7–US$10 (including indirect costs). It has been estimated that five to ten malaria episodes per year can equate to a proportion of household expenditure of about 30%–40% in the poorest households [40]. Figure 2 shows the range of treatment costs for a “rapid impact” package aimed at seven neglected diseases (schistosomiasis, trachoma, lymphatic filariasis, onchocerciasis, hookworm, trichuriasis, and ascariasis).
Figure 2 Range of Treatment Costs Per Year
Even with their high unit costs, the current curative approaches to the big three diseases are “reactive” strategies. The treatment of individuals infected with HIV/AIDS, TB, and malaria fails to significantly reduce transmission. Transmission control via bednets has a protective efficacy of around 50% against malaria fevers (although a well-documented reduction of 30%–40% in child mortality) [41], while condom use in Africa during the last occasional intercourse was reported to be 19% [42], and condom use in sex acts with a noncohabitating partner ranges from 13% in Southeast Asia to 19% in sub-Saharan Africa (p. 70 of [43]). In addition, figures published by the Joint United Nations Programme on HIV/AIDS show that there has been no increase in the prevalence of condom use during the period 1995–2000 in three out of four countries surveyed [38,44,45].
Even if the targets identified by the HIV/AIDS, TB, and malaria partnerships were to be met, transmission of all these infections would still continue at the present rate. For example if the “3 by 5” target for HIV antiretroviral treatments (treating 3 million people with antiretroviral therapy by the end of 2005) was reached, it would still leave 90% of HIV-positive individuals infected, untreated, and actively transmitting HIV infection. This would effectively ensure an ever-increasing burden of disease. Currently, the burden could only be reduced by social and/or educational interventions, which reduce prevalence in early sexually active cohorts through aggressive educational campaigns [44,45]. While recognizing the importance of doing everything possible to combat the big three diseases, we urge decision makers, policy makers, and donors to also consider supporting a programme of “rapid-impact interventions,” an approach that would bring real benefit to millions suffering disablement, poverty, and ill health. This would enable more equitable treatment of poor people, by providing such polyparasitized populations with effective and cheap interventions that would reduce stigma and disability, and also reduce morbidity and mortality, thus reaching the MDGs quickly and cost effectively.
Defining End Points for Integrated Control
Let us define end points and outcomes for integrated control. In the case of Ascaris, Trichuris, and schistosome infections, the major goal is a sustainable reduction in worm burden and control of morbidity, while for lymphatic filariasis, onchocerciasis, and trachoma, the major goals are to reduce or eliminate transmission of diseases, resulting in much-reduced morbidity in future generations [3]. The externalities of these two goals are considerable and include improved education and economic productivity. The calculated loss of US$1 billion annually from lymphatic filariasis in India [46], and US$5.3 billion from blinding trachoma [47], and substantial reductions in future wage-earning capacity as a result of chronic hookworm infection in childhood [48], illustrates the burden and costs of these diseases to poor individuals and communities. An added externality is the impact that the neglected tropical diseases have on the big three. Several recent papers highlight the immunosuppressive features of helminths (especially the STHs, schistosomes, and filariae) and their possible impact on promoting susceptibility to HIV/AIDS, TB, and malaria [49,50]. Conversely, the control of helminth infections has been suggested as a means to facilitate control of the big three [49,50], especially by reducing the frequency of malaria fevers, the frequency of severe and cerebral malaria, and the prevalence of anaemia [51–53].
We have contrasted above the unit costs of treatment for the neglected tropical diseases and compared them with the costs of HIV, TB, and malaria “control” (Figure 2). We propose the following model to illustrate some of these comparative costs for policy makers. Consider a typical sub-Saharan African country with a population of 10 million people, with a per-capita government expenditure on health of US$5 and an HIV seroprevalence of 25%. The total annual health expenditure for the country would be US$50 million. If US$200 per person treated is used to treat the HIV-positive population, the cost would be US$200 × 2.5 million = US$500 million. In other words, ten times the available health budget is being spent on antiretrovirals alone. While it is expected that substantial donor funding (e.g., via Global Fund financing) would be available for the purchase of antiretrovirals, there is also an expectation that the national health system itself would contribute to the financing of HIV services.
Conclusions
We urge policy makers and health economists to recognize that although HIV, TB, and malaria are the most serious problems facing health planners, other diseases exist that can be addressed at realistic costs with effective interventions. No discussion of disease should use the term “control” unless the interventions will permanently and gradually reduce incidence. This will be difficult to achieve with the big three due to the marginal impact of current control strategies on transmission—a direct contrast to what can be achieved for some of the “other diseases” of the MDGs affecting the poor.
There are many people in Africa who do not have HIV or TB and have survived malaria, but are nonetheless permanently polyparasitized by debilitating, disabling, and sometimes fatal conditions, which can be treated at a cost of US$0.40 per person annually. Controlling Africa's neglected diseases is one of the more convincing ways to “make poverty history” through affordable, pro-poor, effective, and tested strategies.
Citation: Molyneux DH, Hotez PJ, Fenwick A (2005) “Rapid-impact interventions”: How a policy of integrated control for Africa's neglected tropical diseases could benefit the poor. PLoS Med 2(11): e336.
Abbreviations
MDGMillennium Development Goal
PPPpublic–private partnership
STHsoil-transmitted helminth
TBtuberculosis
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References
Molyneux DH “Neglected” diseases but unrecognised successes—Challenges and opportunities for infectious disease control Lancet 2004 364 380 383 15276399
Hotez PJ Remme H Buss P Alleyne G Morel C Combating tropical communicable diseases: Workshop report of the disease control priorities project Clin Infec Dis 2004 38 871 878 14999633
Hotez PJ Ottesen E Fenwick A Molyneux DH Pollard AJ Finn A The neglected tropical diseases: The ancient afflictions of stigma and poverty and the prospects for their integrated control and elimination Hot topics in infection and immunity in children III 2006 New York Kluwer Academic/Plenum Publishers In press
Molyneux DH Zagaria N Lymphatic filariasis elimination: Progress in global programme development Ann Trop Med Parasitol 2002 96 Suppl 2 S15 S40
Mecaskey JW Knirsch CA Kumaresan JA Cook JA The possibility of eliminating blinding trachoma Lancet Infect Dis 2003 3 728 734 14592604
Lockwood DN Suneetha S Leprosy: Too complex a disease for a simple elimination paradigm Bull World Health Organ 2005 83 230 235 15798849
Levine R What Works Working Group Case 11, controlling Chagas disease in the southern cone of South America Center for Global Development. Millions saved: Proven successes in global health 2004 Washington (District of Columbia) Center for Global Development 99 104
Molyneux DH Hopkins DR Zagaria N Disease eradication, elimination and control: The need for accurate and consistent usage Trends Parasitol 2004 20 347 351 15246314
Hopkins DR Ruiz-Tiben E Diallo N Withers PC Maguire JH Dracunculiasis eradication: And now, Sudan Am J Trop Med Hyg 2002 67 415 422 12452497
Lambo E Sambo LG Health sector reform in sub-Saharan Africa: A synthesis of country experiences East Afr Med J 2003 80 Suppl 6 S1 S20 12952024
Hill PS The rhetoric of sector-wide approaches for health development Soc Sci Med 2002 54 1725 1737 12113454
World Bank World development report 1993: Investing in health 1993 New York Oxford University Press 342
World Health Organization and Government of Mexico Report from the ministerial summit on health research. Mexico City, 16–20 November 2004 2004 Available: http://www.who.int/rpc/summit/documents/summit_report_final2.pdf . Accessed 25 August 2005
Sachs JD McArthur JW The Millenium Project; A plan for meeting the Millennium Development Goals Lancet 2005 365 347 353 15664232
Walt G Buse K Partnership and fragmentation in international health: Threat or opportunity? Trop Med Int Health 2000 5 467 471 10964268
Widdus R Public-private partnerships for health: Their main targets, their diversity, and their future directions Bull World Health Organ 2001 79 713 720 11545327
The Lancet Health and poverty: A new Marshall plan? Lancet 2005 365 267 268 15664200
Hotez PJ Bundy DAP Beegle K Brooker S Drake L Helminth infections: Soil-transmitted helminth infections and schistosomiasis isease control priorities in developing countries. Second edition 2006 Oxford Oxford University Press In press
King CH Dickman K Tisch DJ Reassessment of the cost of chronic helmintic infection: Meta-analysis of disability-related outcomes in endemic schistosomiasis Lancet 2005 365 1561 1569 15866310
Hotez P Bethony J Brooker S Albonico M Eliminating neglected diseases in Africa Lancet 2005 365 1089
Levine R What Works Working Group Case 6, controlling onchocerciasis in sub-Saharan Africa Center for Global Development. Millions saved, proven successes in global health 2004 57 64
Raso G Luginbhuhl A Adjoua CA Tian-Bi NT Silue KD Multiple parasite infections and their relationship to self-reported morbidity in a community of rural Cote d'Ivoire Int J Epidemiol 2004 33 1092 1102 15256525
Fenwick A Molyneux D Nantulya V Achieving the millennium development goals Lancet 2005 365 1029 1030 15781095
Heukelbach J Winter B Wilcke T Muehlen M Albrecht S Selective mass treatment with ivermectin to control intestinal helminthiases and parasitic skin diseases in a severely infected population Bull World Health Organ 2004 82 563 571 15375445
Lawrence G Leafasia J Seridan J Hills S Wate J Control of scabies, skin sores and haematuria in children in the Solomon Islands: Another role for ivermectin Bull World Health Organ 2005 83 34 42 15682247
Taylor MJ Makunde WH McGarry HF Turner JD Mand S Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti : A double-blind, randomized placebo-controlled trial Lancet 2005 365 2116 2121 15964448
Rao R Weil GJ In vitro effects of antibiotics on Brugia malayi worm survival and reproduction J Parastiol 2002 88 605 611
Shelby-James TM Leach AJ Carapetis JR Currie BJ Matthews JD Impact of single dose azithromycin in group A streptococci in the upper respiratory tract and skin of Aboriginal children Pediatr Infect Dis 2002 21 375 380
The Lancet Thinking beyond deworming Lancet 2004 364 1993 1994 15582039
Commission for Africa Our common interest: Report for the commission for Africa 2005 Available: http://www.commissionforafrica.org/english/report/thereport/english/11-03-05_cr_report.pdf . Accessed 25 August 2005
Behnke JM Pritchard DI Wakelin D Park JR McNicholas AM Effect of ivermectin on infection with gastro-intestinal nematodes in Sierra Leone J Helminthol 1994 68 187 195 7829838
Albonico M Bickle Q Ramsan M Montresor A Savioli L Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar Bull World Health Organ 2003 81 343 352 12856052
Albonico M Smith PG Ercole E Hall A Chwaya HM Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albenadazole in a highly endemic area Trans R Soc Trop Med Hyg 1995 89 538 541 8560535
Albonico M Engels D Savioli L Monitoring drug efficacy and early detection of drug resistance in human soil-transmitted nematodes: A pressing public health agenda for helminth control Int J Parasitol 2004 34 1205 1210 15491582
Hotez PJ Bethony J Bottazzi ME Brooker S Buss P Hookworm: “The great infection of mankind” PLoS Med 2005 2 e67 10.1371/journal.pmed.0020067 15783256
Goud GN Bottazzi ME Zhan B Mendez S Deumic V Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in clinical trials Vaccine 2005 23 4754 4764 16054275
Leach AJ Shelby-James TM Mayo M Gratten M Laming AC A prospective study of the impact of community-based azithromycin treatment of trachoma on carriage and resistance of Streptococcus pneumoniae
Clin Infect Dis 1997 24 356 362 9114185
UNAIDS Report on the global HIV AIDS epidemic 2002 2002 Available: http://www.unaids.org/html/pub/global-reports/barcelona/brglobal_aids_report_en_pdf . Accessed 25 August 2005
World Health Organization, The Stop TB Partnership Final report 2001 2002 Available: http://www.stoptb.org/documents/Final_report2001.pdf . Accessed 25 August 2005
Ettling M McFarlane DD Schultz LJ Chitsulo L Economic impact of malaria in Malawian households Trop Med Parasitol 1994 45 74 79 8066390
Lengeler C Insecticide-treated bed nets and curtains for preventing malaria Cochrane Database Syst Rev 2004 CD000363 15106149
Norman LR Predictors of consistent condom use: Hierarchical analysis of adults from Kenya, Tanzania and Trinidad Int J STD AIDS 2003 14 584 590 14511493
UNAIDS 2004 report on the global AIDS epidemic 2004 Available: http://www.unaids.org/bangkok2004/report_pdf.html . Accessed 25 August 2005
Maharaj P Cleland J Condom use within marital and cohabitating partnerships in KwaZulu-Natal, South Africa Stud Fam Plann 2004 35 116 124 15260213
Hounton SH Carabin H Henderson NJ Towards an understanding of barriers to condom use in rural Benin using the Health Belief Model: A cross sectional survey BMC Public Health 2005 5 8 15663784
Ramaiah KD Das PK Michael E Guyatt H The economic burden of lymphatic filariasis in India Parasitol Today 2000 16 251 253 10827432
Frick KD Hanson CL Jacobson GA Global burden of trachoma and economics of the disease Am J Trop Med Hyg 2003 69 1 10
Bleakley H Disease and development: Evidence from the American South J Europ Econ Assoc 2003 1 376 386
Fincham JE Markus MB Adams VJ Could control of soil-transmitted helminthic infection influence the HIV/AIDS pandemic Acta Trop 2003 86 315 333 12745148
Druilhe P Tall A Sokhna C Worms can worsen malaria: Towards a new means to roll back malaria? Trends Parasitol 2005 21 359 362 15967721
Spiegel A Tall A Raphenson G Trape J-F Druilhe P Increased frequency of malaria attacks in subjects co-infected by intestinal worms and Plasmodium falciparum
Trans R Soc Trop Med Hyg 2003 97 198 199 14584377
Le Hesran JK Akiana J el Nidiaye HM Dia M Senghor P Severe malaria attack is associated with high prevalence of Ascaris lumbricoides infection among children in rural Senegal Trans R Soc Trop Med Hyg 2004 98 397 399 15138075
Christian P Khatry SK West KP Antenatal anthelminthic treatment, birthweight, and infant survival in rural Nepal Lancet 2004 364 981 983 15364190
de Silva NR Brooker S Hotez PJ Montresor A Engels D Soil-transmitted helminth infections: Updating the global picture Trends Parasitol 2003 19 547 551 The first number quoted is the one provided in the reference. However, a range is added here based on new unpublished data showing the reduction in prevalence of soil-transmitted helminth infections in China 14642761
Van der Werf MF de Vlas SJ Brooker S Looman CW Nagelkerke NJ Quantification of clinical morbidity associated with schistosome infection in sub-Saharan Africa Acta Trop 2003 86 125 139 12745133
Mariotti S New steps toward eliminating blinding trachoma N Engl J Med 2004 351 2004 2007 15525727
Zagaria N Savioli L Elimination of lymphatic filariasis: A public health challenge Ann Trop Med Parasitol 2002 96 Suppl 2 S3 S13
World Health Organization African trypanosomiasis or sleeping sickness Fact sheet No. 259. Geneva: World Health Organization 2001 March Available: http://www.who.int/mediacentre/factsheets/fs259/en/ . Accessed 1 September 2005
World Health Organization Dracunculiasis eradication Geneva: World Health Organization 2005 Available: http://www.who.int/ctd/dracun/index.html . Accessed 1 September 2005
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7074ehp0112-00164515579407ResearchReviewEnvironmental Health Disparities: A Framework Integrating Psychosocial and Environmental Concepts Gee Gilbert C. 1Payne-Sturges Devon C. 21University of Michigan School of Public Health, Department of Health Behavior and Health Education, Ann Arbor, Michigan, USA2Office of Policy, Economics and Innovation, and Office of Children’s Health Protection, U.S. Environmental Protection Agency, Washington, DC, USAAddress correspondence to G.C. Gee, University of Michigan School of Public Health, Department of Health Behavior and Health Education, 1420 Washington Heights, Room M5224, Ann Arbor, MI 48109-2029 USA. Telephone: (734) 615-7825. Fax: (734) 763-7379. E-mail:
[email protected] thank M. Zimmerman and O. Nweke for their helpful comments with previous drafts of the manuscript.
The views expressed in this document are those of the authors and do not represent official U.S. Environmental Protection Agency policy.
The authors declare they have no competing financial interests.
12 2004 16 8 2004 112 17 1645 1653 5 3 2004 16 8 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Although it is often acknowledged that social and environmental factors interact to produce racial and ethnic environmental health disparities, it is still unclear how this occurs. Despite continued controversy, the environmental justice movement has provided some insight by suggesting that disadvantaged communities face greater likelihood of exposure to ambient hazards. The exposure–disease paradigm has long suggested that differential “vulnerability” may modify the effects of toxicants on biological systems. However, relatively little work has been done to specify whether racial and ethnic minorities may have greater vulnerability than do majority populations and, further, what these vulnerabilities may be. We suggest that psychosocial stress may be the vulnerability factor that links social conditions with environmental hazards. Psychosocial stress can lead to acute and chronic changes in the functioning of body systems (e.g., immune) and also lead directly to illness. In this article we present a multidisciplinary framework integrating these ideas. We also argue that residential segregation leads to differential experiences of community stress, exposure to pollutants, and access to community resources. When not counterbalanced by resources, stressors may lead to heightened vulnerability to environmental hazards.
environmentalenvironmental justiceethnicityframeworkhealth disparitiespsychosocialracereviewstress
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The elimination of disparities in environmental health requires attention to both environmental hazards and social conditions [U.S. Environmental Protection Agency (EPA) 2003a; Institute of Medicine 1999]. However, two major challenges are implicit in this statement: first, to understand how social processes may interrelate with environmental toxicants, and second, to understand why some groups experience greater illness compared with other groups. Our purpose in this article is to provide a multidisciplinary framework that addresses both issues.
We extend the work of Sexton et al. (1993), who documented how the exposure–disease paradigm could explain variation in the health of disadvantaged populations. Implicit in their framework is the idea that disadvantaged populations encounter greater susceptibility to environmental hazards. However, it is unclear what these susceptibility factors might be.
We suggest that psychosocial stress is a key component of differential susceptibility. Stressors, when not ameliorated by resources, may directly lead to health disparities. Additionally, stressors may amplify the effects of toxicants. Residential segregation may be one important reason why communities differ in these exposures (Massey and Denton 1993).
Our framework is built on an ecological perspective, suggesting that health disparities result not only from individual factors but also from factors operating at multiple levels (Bronfenbrenner 1989; Diez-Roux 1998; Pickett and Pearl 2001; Sallis and Owen 1997). Reinvigoration in ecological approaches has paralleled the development of statistical techniques of multilevel modeling (e.g., hierarchical linear models), an appreciation that ecological factors may not necessarily lead to the ecological fallacy, and a renewed interest in the role of context in health promotion (Diez-Roux 2000; Green and Kreuter 1999).
Health Disparities and the Environment
Disparities exist for many health outcomes, including cancer, cardiovascular disease, diabetes, and mortality [U.S. Department of Health and Human Services (DHHS) 2000]. Although there has been a national decrease in disparities between 1990 and 1998 (Keppel et al. 2002), some regions have reported an increase in disparities during the same period (Margollos et al. 2004).
Environmental conditions are believed to play an important role in producing and maintaining health disparities (Lee 2002; Sexton 2000; Yen and Syme 1999). Minority neighborhoods tend to have higher rates of mortality, morbidity, and health risk factors compared with white neighborhoods, even after accounting for economic and other characteristics (Cubbin et al. 2001; Deaton and Lubotsky 2003; Geronimus et al. 2001).
The Stress–Exposure Disease Framework
The stress–exposure disease framework (Figure 1) provides a conceptual framework from which to understand the relationships among race, environmental conditions, and health. It extends the framework of Sexton et al. (1993) by a) explicitly hypothesizing that residential segregation is a major reason why “race” is important; b) incorporating an ecological or multilevel perspective; and c) arguing that racial variation in stressors may account for differences in vulnerability to health risks.
Reflecting the multilevel approach, Figure 1 emphasizes both community processes (top) and individual mechanisms (bottom). The shading reflects the exposure–disease paradigm. To simplify our presentation, we have separated individual and community processes. However, many processes are interrelated. For example, community wealth is partly a function of individual wealth (e.g., when individuals contribute to the tax base), and individual wealth is also partly determined by community wealth (e.g., when rising property values benefit individual homeowners).
The framework shows that ethnicity is highly correlated with residential location, with minorities and whites often living segregated from one another. Differential residential location comes with differential exposure to health risks. In particular, neighborhood stressors and pollution sources create adverse health conditions, which are counterbalanced by neighborhood resources. Structural factors help determine the boundaries from which health promotion is possible and partially determine the contemporary state of stressors, resources, and pollution in a community. When community stressors and pollution sources outweigh neighborhood resources, levels of community stress manifest or increase. Community stress is a state of ecological vulnerability that may translate into individual stressors, which in turn may lead to individual stress. Individual stress may then make individuals more vulnerable to illness when they are exposed to environmental hazards. Further, compromises in individual and community health may further weaken community resources, leading to a vicious cycle. Hence, we include in our framework a return loop from health back to stress.
As an example, zoning policies and tax incentives (structural factors) may encourage the entry of new pollutant industries. The increase in pollutants may lead to economic and social uncertainty (stressors) by driving down local property values, increasing the flight of jobs and fostering a climate of uncertainty and fear. Neighborhood organizations (resources) may not be able to counterbalance these effects, leading to a state of community vulnerability (community stress). Community-level vulnerability, in turn, may translate to individual vulnerability, such as when individuals lose their jobs or become anxious about perceived toxic exposures. When personal coping resources do not adequately counterbalance these external insults, individual stress and illness may result. Individual illness, in turn, may lead to further individual vulnerability, such as by reducing the ability to exercise. Additionally, individuals may affect their communities, such as when disaffected individuals cease participating in neighborhood organizations. Health disparities may arise because minorities are segregated into neighborhoods with high levels of community stress.
We do not explicitly examine the issue of genetic susceptibility in this framework for three reasons. First, we focus on factors that are amenable to policy change and social action. Second, genetic susceptibility is partly subsumed in the exposure–disease paradigm because it is presumed to partially determine one’s ability to defend against hazards. Third, although genetic factors are important in the etiology of many illnesses, it is likely that genetic factors do not explain racial health disparities (Cooper 1984; Cooper et al. 2003; Garte 2002; LaVeist 1994). It is often acknowledged that race is a social construct. What that means is that racial groups are not inherent biological taxons, but represent societally defined categories during a particular point in history and place. For example, before 1989, the child of a black father and a white mother would be classified as black, but after 1989, the same child would be classified as white (LaVeist 1994). Further, a child born in Brazil, rather than the United States, would be classified as mulatto. Thus, racial designations are the product of social consensus and public policy, rather than biology per se.
Additionally, “genetically identified” groups tend to correlate poorly with socially identified groups because there is more genetic variation within than between groups (Garte 2002; Lewontin 1982; Mountain and Cavalli-Sforza 1997). For example, genetic differences between any two Italians appear to be 5-fold greater than the difference between an Italian and a Japanese, African, or New Guinean (Mountain and Cavalli-Sforza 1997). Observations such as these have led Cooper et al. (2003) to conclude that race “has not shown to provide a useful categorization of genetic information about the response to drugs, diagnosis, or causes of disease.”
We now review the science that informs this framework, beginning with the exposure–disease paradigm.
The Exposure–Disease Paradigm
The exposure–disease paradigm is a well-known model that shows how environmental toxicants might cause disease (Lioy 1990; Lioy and Pellizzari 1995; National Research Council, 1991a, 1991b; Wagener 1987). It is a continuum that includes the emission of a contaminant from a source through human exposure to the occurrence of a health effect.
Susceptibility/vulnerability intersects the continuum, increasing or decreasing resistance to absorption and/or effect from toxicants. The term “susceptibility/vulnerability” has been used broadly to cover both biological and non-biological factors, including genetic predisposition, pre-existing health conditions, and social conditions. The exact susceptibility/ vulnerability factors and their pathways intersecting the exposure–disease paradigm are not well understood. We argue later that community and individual stress is one type of susceptibility factor.
Race and Residential Location
Segregation, the spatial separation of the residences of racial groups from one another, has persisted for many decades (Iceland et al. 2002; Massey 2001; Massey and Denton 1993). Table 1 shows the segregation of blacks, Hispanics, Native Americans, and Asians compared with whites from 1980 to 2000 for metropolitan areas, as measured with the index of dissimilarity (Logan 2003; U.S. Census Bureau 2003). Scored from 0 to 100, a given value of the index indicates the percentage of that group who would have to move to integrate the metropolitan area.
Segregation from whites is highest for African Americans, followed by Hispanics, Asian Americans, and Native Americans. In the average U.S. metropolis in the year 2000, about two-thirds of blacks (or whites) would have to move to another neighborhood in order to desegregate that metropolis.
Black–white and Native-American–white segregation has declined since the 1980s, but segregation levels for Hispanics and Asians have remained stable. Further, most of the decline in black–white segregation has occurred in metropolitan areas with the fewest numbers of blacks (Logan 2003).
The causes of segregation are still debated. Some have suggested that segregation is an artifact of broader shifts in the economy—including the decline of manufacturing jobs and suburbanization—that have left behind a cadre of the poor that are disproportionately racial minorities (Wilson 1987, 1996). Others have postulated that segregation results from personal preferences of homebuyers to cluster together (Schelling 1971). Most research has argued that segregation results from institutionalized discriminatory practices in the housing market (e.g., mortgage redlining, racialized “steering”) that persists to the current day (Massey and Denton 1993; Meyer 2000; Munnell et al. 1996; Schwartz 1998; Squires 1994; Squires and Velez 1996).
Some evidence suggests that the mechanisms for segregation vary by ethnic group and region, but most ethnic groups have encountered discriminatory treatment historically and currently (Squires 1994; Feagin and McKinney 2003; Krieger et al. 1993; Williams et al. 1997). For example, a recent audit study suggested that consistent adverse treatment in home buying was similar for Asian-American and African-American homebuyers, with one in five potential homebuyers disfavored compared with whites (Turner and Skidmore 2001). The causes of segregation notwithstanding, it is clear that neighborhoods do cluster on the basis of race and ethnicity.
Studies have reported that segregation is associated with numerous outcomes, including infant mortality [Centers for Disease Control and Prevention (CDC) 2002; LaVeist 1989, 1993], adult mortality (Hart et al. 1998; Jackson et al. 2000; Polednak 1991, 1996), tuberculosis (Acevedo-Garcia 2003), homicide (Peterson and Krivo 1993, 1999), teenage childbearing (Sucoff and Upchurch 1998), exposure to tobacco and alcohol advertising (Alaniz 1998; Luke et al. 2000; U.S. DHHS 1998), and increased exposure to air pollution (Lopez 2002).
Segregation may thus be one critical link between race and environmental health disparities because racial groups, on average, occupy different residential areas. This may lead to differential exposure to health risk factors as well as differential access to resources. Segregation is multifactorial, often conceptualized around five dimensions (Acevedo-Garcia 2000; Massey and Denton 1988, 1993): a) evenness, the inequitable distribution of groups over an area and the dimension receiving the greatest empirical study; b) isolation, the degree of potential contact between two groups within a city; c) concentration, the extent to which minority groups are confined to a compact area within the city; d) centralization, the degree to which minorities are clustered around the center of a city; and e) clustering, the extent to which minority neighborhoods are adjacent to one another. Our discussion refers to the general principle of segregation, although it will be an important research endeavor to examine which specific dimensions of segregation are related to environmental health disparities.
Having established a link between race and residence, we now turn to the proximal mechanisms that may account for the relationship between environmental conditions and racial health disparities.
Environmental Hazards and Pollutants
Briefly, environmentally relevant disparities are evident in a variety of outcomes, including asthma, cancer, and chemical poisoning (Institute of Medicine 1999). Although debated, the main hypothesis explaining these disparities is that disadvantaged communities encounter greater exposure to environmental toxicants such as air pollution, pesticides, and lead (Burger et al. 2001; Calderon et al. 1993; Corburn 2002; Fitzgerald et al. 1998; Institute of Medicine 1999; Morello-Frosch 2001; Moses et al. 1993; Northridge et al. 2003; Perera 2003; Pirkle et al. 1998; Woodruff et al. 2003). Mediators of the relationship between toxic exposure and disadvantaged status include the siting of pollution sources (e.g., waste incinerators), illegal dumping, poor enforcement of environmental regulations, and inadequate response to community complaints (Anderton et al. 1994, 1997; Bullard 1983, 1990; Bullard and Wright 1993; Goldman and Fitton 1994; Institute of Medicine 1999; Maantay 2001, 2002; Mohai and Bryant 1992; Perlin et al. 1999, 2001; United Church of Christ 1987; U.S. General Accounting Office 1983).
Structural Factors
Structural factors refer to the historically evolving infrastructure that provides boundaries for health promotion. That is, structural factors are constraints that shape how new conditions emerge as “salutogens” (factors that support health) or pathogens in a community. The local economy, for example, is a structural factor that will help determine a community’s ability to mobilize resources in order to reject undesirable changes (e.g., introduction of a waste facility) or develop desirable ones (e.g., construction of a park). Structural factors that may be especially pertinent to environmental health disparities include the local and national economy, neighborhood physical conditions, land use patterns, and health infrastructure. This is not an exhaustive list, but rather is meant to be illustrative.
One primary effect of residential segregation may be to concentrate disadvantage (Massey and Denton 1993). Compared with whites, minorities are overrepresented in neighborhoods with diminishing and constrained economic opportunities (Jargowsky 1997; Wilson 1987). For example, in Los Angeles, California, in 1990, only 4.9% of blacks lived in high-job-growth areas, compared with 52.3% of whites (Pastor 2001). Cutler and Glaeser (1997) reported that a decrease in segregation by one standard deviation (13%) would eliminate one-third of the black–white differences in education and employment. Thus, segregation not only may concentrate poverty but also may be partly responsible for the production of poverty among racial minorities (Massey and Denton 1993; Williams and Collins 2001).
There is a clear association between socioeconomic position and health, such that individuals of higher social standing tend to have improved health compared with those of lower standing (Evans and Kantrowitz 2002; Kaplan et al. 2001; Krieger and Fee 1994; Marmot et al. 1987, 1998; O’Neill et al. 2003; Williams and Collins 1995). Further, the relationship between socioeconomic position and health holds not only at the individual level but also at the community level (Haan et al. 1987; Kaplan 1996). That is, persons living in poor neighborhoods, even after accounting for their individual socioeconomic characteristics, tend to have worse health outcomes (Diez-Roux et al. 1997, 2001; Merkin et al. 2002; Waitzman and Smith 1998; Winkleby and Cubbin 2003).
Neighborhood economic deprivation may compromise health-promoting resources (Diez-Roux et al. 2001). For example, poor and minority neighborhoods tend to have fewer grocery stores with healthy foods (Morland et al. 2002) and fewer pharmacies with needed medications (Morrison et al. 2000). Poor nutrition can increase susceptibility to environmental pollutants by compromising immune function (Beck and Weinstock 1988; Rios et al. 1993). Additionally, disadvantaged neighborhoods are also exposed to greater health hazards, including tobacco and alcohol advertisements, toxic waste incinerators, and air pollution (Morello-Frosch et al. 2002). Finally, economic stress within a community may exacerbate tensions between social groups, magnify workplace stressors, and induce “maladaptive” coping behaviors, such as smoking and alcohol use (Brenner 1995). Tobacco and alcohol use can increase susceptibility to environmental toxicants that are normally metabolized by impairing host defense (Rios et al. 1993).
In general, racial minorities have lower socioeconomic position compared with whites. Although it is intuitive to hypothesize that disparities in health arise because of socioeconomic differences between racial groups, studies often find that racial disparities persist even after accounting for socioeconomics factors (Hayward et al. 2000; Sorlie et al. 1995; Williams 1999).
Although socioeconomic differences do not completely explain racial disparities, it is often argued that social class is an important mediator. That is, it is hypothesized that race determines one’s economic resources, which in turn determine health (Williams and Collins 1995). Thus, although socioeconomic conditions do not fully account for health disparities, they are a necessary part of the equation.
Neighborhood physical conditions present another structural factor that may contribute to health disparities (Cohen et al. 2003). Minorities are more likely to live in areas with building code violations and neighborhoods with deteriorated housing (Perera et al. 2002; Rosenbaum et al., unpublished data). In 1999, 3.4% of blacks, 3.8% of Hispanics, and 1.7% of Asian Americans and Pacific Islanders reported living in housing units with severe problems with heating, plumbing, electricity, public areas, or maintenance, compared with 1.5% of whites (U.S. Census Bureau 2000). Substandard housing may contribute to a variety of problems, including exposure to toxicants, increased risk of injuries from falls and fires, and illness due to ineffective waste disposal and presence of disease vectors (Bashir 2002; Jacobs et al. 2002; Krieger and Higgins 2002; Northridge et al. 2003).
Urban minorities tend to fare worse than their counterparts in rural areas (Geronimus et al. 1999; Geronimus et al. 2001). This may be due in part to land use patterns in urban areas. In Detroit, many minority neighborhoods exist next to highways that expose residents to hazards (Schulz et al. 2002). Sugrue (1996) argues that
Detroit’s highway planners were careful to ensure that construction of new … expressways would only minimally disrupt middle-class residential areas, but they had little such concern for black neighborhoods.
Similarly, New York City rezoned its neighborhoods between 1961 to 1998 so as to increase manufacturing zones in areas with higher minority populations and to decrease those zones in areas with fewer minorities (Maantay 2001). Those rezoning efforts led to a higher concentration of industrial burden within manufacturing-designated areas. Further, some policies that appear neutral prima facie may result in adverse impacts on already disadvantaged communities, as in the example of emissions trading systems and their potential to create pollution “hot spots” (Schmidt 2001; Soloman and Lee 2000).
Health infrastructure may also be associated with race. Minorities tend to reside in areas with a lower physician-per-population ratio and lower medication supply (Morrison et al. 2000; Rosenbaum et al., unpublished data; Schulz et al. 2002). Community hospitals are more likely to close in urban minority communities (Whiteis 1992). These findings suggest that segregated communities face structural disadvantages in the provision of health services.
Because so many different structural forces appear to confer disadvantage among minority communities, some scholars have suggested that they continue a history of institutionalized discrimination against minorities (Feagin and McKinney 2003; Gee 2002; Jones, 2000; Krieger et al. 1993; Massey and Denton, 1993; Squires 1994; Williams and Collins 2001). This discrimination may not have a purposeful intent but still may confer adverse impact.
Community Stressors
Community stress theory derives from a century of research on the stress process among individuals (Aneshensel 1992; Lazarus and Folkman 1984; McEwen 1998; Selye 1936; Steptoe and Feldman 2001). “Stress” is a state of activation of physical and psychological readiness to act in order to help an organism survive external threats. “Stressors” are the factors that produce stress and include such phenomena as crime (Morenoff 2003), noise (Babisch et al. 2001; Ouis 2001), traffic (Gee and Takeuchi 2004), and litter, density, and residential crowding (Fleming et al. 1987; Evans and Lepore 1993). Stressors can result directly from environmental hazards, including technological and natural disasters (Baum et al. 1983; Brown 2002).
Health effects of stress.
Stressors can trigger the sympathoadrenal system, whose hallmark is rapid release of adrenalin and noradrenalin, which leads to various “fight or flight” responses, including arousal, bronchodilation, tachycardia, and increased blood pressure. The hypothalamic–pituitary–adrenal system is also activated, signified by release of corticotrophin-releasing factor, adrenocorticotropic hormone, and cortisol. These glucocorticoids have several metabolic and psychological effects, including the mobilization of energy reserves, suppression of the immune system, and heightened vigilance. Chronic activation of the stress system is believed to lead to allostatic load, which is the “wear and tear” on organ systems resulting from stress (McEwen 1998). A full discussion of the biology of stress is beyond the scope of this article but can be found in several publications (Brunner 2000; Hadley 1992; McEwen 1998).
The key point is that stressors can cause illness by weakening the body’s ability to defend against external challenges. As an example, Cohen et al. (1991) asked volunteers to self-rate their levels of stress and then randomized them to receive nasal drops containing either placebo or respiratory viruses. Rates of respiratory infection and clinically diagnosed colds followed a positive dose response with level of psychological stress. Findings from this controlled experiment were unaffected by controlling for a variety of factors (e.g., allergic status).
Intriguingly, some evidence suggests that stress may influence the internal dose of a given toxicant. This is because stress may a) increase the absorption of toxicants into the body through increased respiration, perspiration, and consumption (Gordon 2003); b) compromise host defense systems (McEwen 1998); and c) directly cause illness, which in turn may lead to an amplification loop whereby sick individuals are less likely to cope with environmental toxicants (Rios et al. 1993; U.S. EPA 2003b). Stress may induce or unmask a latent effect of a toxicant, possibly altering basal levels of neurofunctioning and shifting the threshold for neurotoxicity [Agency for Toxic Substances and Disease Registry (ATSDR) 1995].
Two factors are purported to determine individual response to stress: how one appraises the situation, and their general state of physical health (Lazarus and Folkman 1984; McEwen 1998). Coping resources, such as social support, help determine the extent to which a stressor is perceived as a threat and subsequent health responses (Israel et al. 2002). For example, workers with high levels of job strain and low levels of co-worker support have higher risk of cardiovascular disease than do those with similar levels of strain and more support (Johnson et al. 1996). Additionally, physical illness will impair an individual’s ability to respond to stressors. Individual stress and coping have macro-level analogs, community stressors and neighborhood resources.
Types of community stressors.
Community stressors can be categorized into two major types, physical and psychosocial. Physical conditions, including noise, temperature, humidity, barometric/water pressure, visible light, geomagnetism, radiation, and particulate matter, may contribute to stress (Gordon 2003). These stressors can induce a physiological response that makes the body more susceptible to illness. Heat stress, for example, induces sweating and increased skin blood flow, which in turn can facilitate the transcutaneous absorption of pesticides (Chang et al. 1994; Funckes et al. 1963; Wester et al. 1996). Individuals subject to ambient noise have higher levels of noradrenalin, a stress biomarker (Babisch et al. 2001). In a natural experiment, Evans et al. (1998) found that the chronic exposure to aircraft noise elevated resting blood pressure, norepinephrine, and epinephrine biomarker levels and decreased self-reported quality of life over a 2-year period.
Psychosocial conditions—including crowding, social disorganization, racial discrimination, fear, and economic deprivation—may also be sources of stress (Krieger and Higgins 2002; Macintyre et al. 2002). One stressor that has received extensive attention is fear of crime (Morenoff 2003; Warr and Ellison 2000). Minority neighborhoods tend to have higher crime rates, which may contribute to health disparities. Perceptions of crime and disorder within an individual’s community has been associated with numerous outcomes, including anxiety depression, posttraumatic stress disorder, and substance use (Aneshensel and Sucoff 1996; Cutrona et al. 2000; Fick and Thomas 1995; Geis and Ross 1998; Ross et al. 2000; Ross and Jang 2000). Morenoff (2003) found that the neighborhood violent crime rate was one of the “most robust” environmental predictors of infant birth weight, after controlling for both individual (e.g., smoking during pregnancy) and neighborhood (e.g. percentage of poor families) characteristics.
Physical and psychosocial stressors may interact with one another, as seen with natural and technological disasters (Ginexi et al. 2000; Kaniasty and Norris 2000). For example, the trauma of the Love Canal incident in New York resulted from both the chemical hazards and public perceptions (Edelstein and Wandersman 1987; Gibbs 1983; Holden 1980). Further, the relationship between environmental and subjective stressors occurs not only for highly salient events but also for everyday events. Gee and Takeuchi (2004), using multilevel models, reported that persons perceiving stress due to automobile traffic had greater psychological distress and lowered general health status than did those perceiving less stress. However, these outcomes were worst for persons perceiving high stress and living in high traffic areas.
Racial disparities in exposure to stressors.
There are racial disparities in the burden of stressors that accumulate over the life course (Geronimus et al. 2001; Holland et al. 2000; Jones 2000; Krieger et al. 1993; Williams et al. 1997). Some have called this racially differential burden of cumulative stress the “weathering hypothesis” (Astone et al. 2002; Geronimus 1996). One of the most prominent stressors may be racial discrimination (Gee 2002; Krieger and Sidney 1996; LaVeist et al. 2000; Williams and Neighbors 2001; Williams et al. 1997). Because racial discrimination has profoundly shaped the experiences of racial groups, discrimination may be among the factors that shape health disparities. Evidence suggests that racial discrimination still occurs in the present day, especially in structurally important domains such as housing, education, and employment (Essed 1992; Feagin 1991, 2000). Audit studies send a white and a minority prospective tester with identical portfolios (e.g., similar income and job titles) to assess a given housing market. These audits have consistently found that whites are favored over minorities. Hispanics, for example, are more likely to be quoted a higher rent for a given unit than are their white counterparts (Turner and Skidmore 2001). Other studies have shown that minorities are more likely to face discrimination in applying for a job (Kirschenman and Neckerman 1991) or shopping (Lee 2000).
Further, discriminatory treatment within the health care system also might contribute to disparities (Krieger 1999). Minorities appear to have longer waiting times for kidney transplants (Eggers 1995; Klassen et al. 2002) and liver transplants (Kjellstrand 1988; Young and Gaston 2000) and report less satisfaction with their medical visits (Cooper-Patrick et al. 1999; Saha et al. 2003). A review by the Institute of Medicine (2002) concluded:
Racial and ethnic minorities tend to receive a lower quality of healthcare than non-minorities, even when access-related factors, such as patients’ insurance status and income are controlled.… [T]he study committee found evidence that stereotyping, biases, and uncertainty on the part of healthcare providers can all contribute to unequal treatment.
Stress from discrimination may lead to illness. Kessler et al. (1999) have suggested that
The conjunction of high prevalence and strong impact would mean that discrimination is among the most important of all the stressful experiences that have been implicated as causes of mental health problems.
Studies have reported that stress due to racial discrimination is associated with high blood pressure (Krieger and Sidney 1996), mental health (Dion et al. 1992; Gee 2002; Kessler et al. 1999; Kuo 1976; Williams et al. 1997), and alcohol consumption (Yen et al. 1999).
Neighborhood Resources
Although a common argument is that segregation is harmful to the health of minorities, there is some indication that segregation may have a counterbalancing effect by concentrating social resources, such as black political power (LaVeist 1993). Others have reported that the clustering of ethnic groups may build a sense of collective identity that helps mitigate trauma (Mazumdar et al. 2000). Thus, supportive social relationships within minority communities may help promote health and well-being and ameliorate the effects of community risks. Our view is that segregation concentrates both risks and resources. It is not a matter of whether segregation is either “bad” or “good,” but to what degree the negative effects of segregation outweigh positive effects.
Neighborhood resources buffer community stressors (Israel et al. 1998; Kretzman and McKnight 1993). Generally, these resources have been conceptualized in terms of relationships among residents, including social cohesion, social capital, psychological sense of community, informal social control, and community empowerment (Berkman and Clark 2003; Kawachi et al. 1999; Ross and Jang 2000; Sampson et al. 1997). “Social cohesion” is the “extent of connectedness and solidarity among groups in society” (Kawachi and Berkman 2000). Essentially, a community with a high degree of social cohesion has strong social ties between members and minimal conflict. “Social capital” can be considered a type of resource that emerges from socially cohesive groups that facilitates collective action. These resources include norms of reciprocity, aid, and interpersonal trust.
Collective efficacy, defined as “mutual trust and willingness to intervene for the common good” (Sampson et al. 1997), may mediate the adverse effects of concentrated disadvantage and fear (Ross and Jang 2000). Pastor et al. (2001) suggested that social capital was stronger in communities with less “ethnic churning,” referring to the replacement of one minority group with another within a community. They argued that ethnic churning may “weaken the usual social bonds constituted by race and make an area more susceptible to siting of noxious land uses.” Their data indicated that ethnic churning in Los Angeles was associated with the siting of hazardous waste storage and disposal facilities over a two-decade period, after adjusting for economic factors.
Another potential resource is residents’ ability to control their environment, which may mitigate community problems in two ways. First, empowered communities may be able to protect themselves from the instruction of new hazards and eliminate extant ones (Bullard and Wright 1993; Lee 1993; Morello-Frosch et al. 2002; Phoenix 1993; Rich et al. 1995; Zimmerman 2000). These communities may also be able to control the political arena that shapes their health beyond the effect of environmental pollutants. Black political participation, defined by the presence of African-American legislators, has been associated with lower mortality rates in African-American communities (LaVeist 1993). This is possibly due to a higher preponderance among African-American communities to provide a wider range of social services compared with white communities (Schneider and Logan 1982). Second, control per se may be an important factor determining stress and health. Workers with greater control over their work process have lower risk of cardiovascular disease than do workers with less control (Karasek and Theorell 1990; Kuper and Marmot 2003; Landsbergis et al. 1997). Further, collective control by workers and their unions may also provide health benefits (Johnson 1989; Sorensen et al. 2004).
Community Stress
The cumulation of environmental pollutants, structural process, community stressors, and neighborhood resources is community stress. Community stress is a state of ecological vulnerability. Community resources help buffer community stressors and protect against environmental exposure, but when resources are inadequate, community stress arises. Structural factors constrain the limits of resources and stressors.
Although several factors cross the threshold from “community” to “individual,” we focus on the intersection between community stress and individual stress. In particular, community stress may itself lead to individual stressors. These individual stressors may in turn lead to individual stress and subsequent illness. The terrorist attacks of 11 September 2001 provide an extreme example of how community stress can translate to individuals. The attack was a threat to the American “community.” Although most citizens were not close to the epicenter, many individuals across the United States felt some measure of distress from the attack (Schlenger et al. 2002; Schuster et al. 2001).
Future Directions
Our stress–exposure–disease framework is meant to stimulate dialogue between environmental and social scientists. Several avenues for future work are suggested. First and foremost, although several components within the framework have undergone extensive study, such as between individual stress and health, relatively little work has attempted to integrate the elements as a whole. Studies are just beginning to consider the connections among factors at multiple levels, such as among community stress, individual stress, and health. Future work should continue to test the components of the framework and incorporate multilevel modeling (Raudenbush and Bryk 2002). Longitudinal studies will be necessary to establish the temporal ordering between variables.
Second, public health should more seriously consider the role that residential segregation plays in the production of health disparities. Several lines of inquiry are possible regarding segregation alone. For example, what role might environmental risk perception play in maintaining segregation? Are certain dimensions of segregation more important than others? Are the mechanisms linking segregation to health all negative, or might there be some health-promoting pathways, such as in the clustering of cultural resources? What are the forms of segregation outside of the United States, and are the mechanisms similar? Does the relationship between segregation and health generalize to all ethnic groups?
Third, we hope that this framework will encourage the environmental justice movement to expand the notion of “environmental hazards” to include community stressors. Are minority communities more likely to receive the siting of workplaces with high job strain (Karasek and Theorell 1990)? Do differences in community stress lead to the “weathering” (Geronimus 1996) of minority communities compared with whites? This means not only examining the main effects of stress and toxicants, but also examining whether psychosocial stress may potentiate (i.e., amplify) the effects of toxicants on the body.
Fourth, research should not only examine the relationship between minority communities and exposures, but also study how the structural conditions of communities may confer additional vulnerability. Disadvantaged communities may be more vulnerable to exposure to environmental hazards because structural conditions, such as substandard housing, may render them more likely to be exposed than are counterparts in more advantaged communities equally distant from these hazards. That is, do minority communities have less protection against a given level of exposure, and do these disparities in protection result from differential social policy?
Conclusions
Our work has implications for environmental justice by suggesting that exposure to physical and chemical hazards is only one route whereby neighborhoods affect the health of racial minorities. Health promotion may require policies and interventions aimed at eliminating environmental toxicants, fostering community resources, and reducing social stressors. Reduction of the gap in health between advantaged and disadvantaged groups, however, may require interventions targeted at eliminating the gap in advantages themselves.
We emphasize racial differences in exposure to stress, rather than racial differences in response to stress. The former conceptualization emphasizes interventions on macro-level social policy (e.g., housing policy), whereas the latter perspective emphasizes interventions at the micro level (e.g., psychological counseling or pharmacological agents). Although micro-level approaches are useful, one disadvantage is that individual interventions require tremendous resources in order to manifest outcomes at the population level (and hence reduce group differences) and, further, are less efficient because interventions must be reapplied to each new birth cohort. However, policy-level changes that target socially produced stressors may prove a promising way to improve the public’s health.
Figure 1 Exposure–disease–stress model for environmental health disparities.
Table 1 Segregation of ethnic minorities compared with whites, United States, 1980–2000.
1980 1990 2000
Native Americans 37.3 36.8 33.3
African Americans 72.7 67.8 64.0
Asian Americans and Pacific Islanders 40.5 41.2 41.1
Hispanics 50.2 50.0 50.9
Segregation was determined using the index of dissimilarity, which measures the evenness of groups over space and can be interpreted as the percentage of a particular group who would have to move in order integrate the two groups over the region as a whole. For example, in the year 2000, 64% of all African Americans (or whites) would have to move to another census tract in order to integrate all metropolitan areas nationwide. Data are adapted from the U.S. Census Bureau (2003).
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References
Acevedo-Garcia D 2000 Residential segregation and the epidemiology of infectious diseases Soc Sci Med 51 1143 1161 11037206
Acevedo-Garcia D 2003 Future directions in residential segregation and health research: a multilevel approach Am J Public Health 93 215 221 12554572
Alaniz ML 1998 Alcohol availability and targeted advertising in racial/ethnic minority communities Alcohol Health Res World 22 286 289 15706757
Anderton DL Anderson AB Oakes JM Fraser MR 1994 Environmental equity: the demographics of dumping Demography 31 229 248 7926187
Anderton DL Oakes JM Egan KL 1997 Environmental equity in Superfund. Demographics of the discovery and prioritization of abandoned toxic sites Eval Rev 21 3 26 10183267
Aneshensel CS 1992 Social stress: theory and research Annu Rev Soc 18 15 38
Aneshensel CS Sucoff CA 1996 The neighborhood context of adolescent mental health J Health Soc Behav 37 293 310 8997886
Arata K Picou JS Johnson GD McNally TS 2000 Coping with technological disaster: an application of the conservation of resources model to the Exxon Valdez oil spill J Traum Stress 13 28 39
Astone NM Ensminger ME Juon HS 2002 Early adult characteristics and mortality among inner-city African American women Am J Public Health 92 640 645 11919065
ATSDR 1995. Report of the Expert Panel Workshop on the Psychological Responses to Hazardous Substances. Atlanta, GA:Agency for Toxic Substances and Disease Registry.
Babisch W Fromme H Beyer A Ising H 2001 Increased catecholamine levels in urine in subjects exposed to road traffic noise: the role of stress hormones in noise research Environ Int 26 475 481 11485215
Bashir SA 2002 Home is where the harm is: inadequate housing as a public health crisis Am J Public Health 92 733 738 11988437
Baum A Fleming R Singer J 1983 Coping with victimization by technological disaster J Soc Issues 39 117 138
Beck BD Weinstock S 1988. Age and nutrition. In: Variations in Susceptibility to Inhaled Pollutants: Identification, Mechanisms, and Policy Implications (Brian JD, ed). Baltimore, MD:Johns Hopkins University Press, 104–126.
Berkman LF Glass T 2000. Social integration, social networks, social support, and health. In: Social Epidemilology (Berkman LF, Kawachi I, eds). New York:Oxford University Press, 137–173.
Brenner MH 1995. Political economy and health. In: Society and Health (Amick BC, Levine S, Tarlov AR, Chapman Walsh D, eds). New York:Oxford University Press, 211–246.
Bronfenbrenner U 1989 Ecological system theories Ann Child Dev 6 187 251
Brown JS Jr 2002. Environmental and chemical toxins and psychiatric illness. Washington, DC:American Psychiatric Publishing.
Brunner EJ 2000. Toward a new social biology. In: Social Epidemiology (Berkman LF, Kawachi I, eds). New York:Oxford University Press, 306–331.
Bullard RD 1983 Solid waste sites and the black Houston community Sociol Inq 53 273 288 11635985
Bullard RD 1990. Dumping in Dixie: Race, Class and Environmental Quality. Boulder, CO:Westview Press.
Bullard RD Wright BH 1993 Environmental justice for all: community perspectives on health and research needs Toxicol Ind Health 9 821 841 8184445
Burger J Gaines KF Gochfeld M 2001 Ethnic differences in risk from mercury among Savannah River fishermen Risk Anal 21 533 544 11572431
Calderon RL Johnson CC Jr Craun GF Dufour AP Karlin RJ Sinks T 1993 Health risks from contaminated water: do class and race matter? Toxicol Ind Health 9 879 900 8184447
CDC (Centers for Disease Control and Prevention) 2002. Racial and ethnic disparities in infant mortality rates—60 largest U.S. cities, 1995–1998. MMWR Morb Mortal Wkly Rep 51:329–332, 343.
Chang SK Brownie C Riviere JE 1994 Percutaneous absorption of topical parathion through porcine skin: in vitro studies on the effect of environmental pertubations J Vet Pharmacol Ther 17 434 439 7707488
Cohen DA Mason K Bedimo A Scribner R Basolo V Farley TA 2003 Neighborhood physical conditions and health Am J Public Health 93 467 471 12604497
Cohen S Tyrrell DA Smith AP 1991 Psychological stress and susceptibility to the common cold N Engl J Med 325 606 612 1713648
Cooper RS 1984 A note on the biological concept of race and its application in epidemiologic research Am Heart J 108 715 723 6382997
Cooper RS Kaufman JS Ward R 2003 Race and genomics N Engl J Med 348 1166 1170 12646675
Cooper-Patrick L Gallo JJ Gonzales JJ 1999 Race, gender, and partnership in the patient-physician relationship JAMA 282 583 589 10450723
Corburn J 2002 Combining community-based research and local knowledge to confront asthma and subsistence-fishing hazards in Greenpoint/Williamsburg, Brooklyn, New York Environ Health Perspect 110 suppl 2 241 248 11929734
Cubbin C Hadden WC Winkleby MA 2001 Neighborhood context and cardiovascular disease risk factors: the contribution of material deprivation Ethn Dis 11 687 700 11763293
Cubbin C LeClere FB Smith GS 2000 Socioeconomic status and injury mortality: individual and neighbourhood determinants J Epidemiol Community Health 54 517 524 10846194
Cutler E Glaeser E 1997 Are ghettos good or bad? Q J Econ 112 827 872
Cutrona CE Russell DW Hessling RM Brown PA Murry V 2000 Personality processes and individual differences-direct and moderating effects of community context on the psychological well-being of African American women J Pers Soc Psychol Psychology 79 1088 1101
Deaton A Lubotsky D 2003 Mortality, inequality and race in American cities and states Soc Sci Med 56 1139 1153 12600354
Diez-Roux AV 1997 Neighborhood environments and coronary heart disease: a multilevel analysis Am J Epidemiol 146 48 63 9215223
Diez-Roux AV 1998 Bringing context back into epidemiology: variables and fallacies in multilevel analysis Am J Public Health 88 216 222 9491010
Diez-Roux AV 2000 Multilevel analysis in public health research Annu Rev Public Health 21 171 192 10884951
Diez-Roux AV Merkin SS Arnett D Chambless L Massing M Nieto FJ 2001 Neighborhood of residence and incidence of coronary heart disease N Engl J Med 345 99 106 11450679
Dion KL Dion KK Pappas G 1992 Personality-based hardiness as a buffer for discrimination-related stress in members of Toronto’s Chinese community Can J Behav Sci 24 517 536
Edelstein MR Wandersman A 1987. Community dynamics in coping with toxic contaminants. In: Neighborhood and Community Environments (Altman I, Wandersman A, eds). New York:Plenum, 69–112.
Eggers PW 1995 Racial differences in access to kidney transplantation in the Medicare ESRD population Health Care Financ Rev 17 89 103 10157383
Essed P 1992. Understanding Everyday Racism. Newbury Park, CA:Sage.
Evans GW Lepore SJ 1993 Household crowding and social support: a quasiexperimental analysis J Pers Soc Psychol 65 308 316 8366422
Evans GW Bullinger M Hygge S 1998 Chronic noise exposure and physiological response: a prospective study of children living under environmental stress Psychol Sci 9 75 77
Evans GW Kantrowitz E 2002 Socioeconomic status and health; the potential role of environmental risk exposure Annu Rev Pub Health 23 303 331 11910065
Feagin JR 1991 The continuing significance of race: antiblack discrimination in public places Am Sociol Rev 56 101 116
Feagin JR 2000. Racist America: Roots, Current Realities, and Future Reparations. New York:Routledge.
Feagin JR McKinney KD 2003. The Many Costs of Racism. Lanham, MD:Rowman and Littlefield.
Fick AC Thomas SM 1995 Growing up in a violent environment: relationship to health-related beliefs and behaviors Youth Soc 27 136 147
Fleming I Baum A Weiss L 1987 Social density and perceived control as mediators of crowding stress in high-density residential neighborhoods J Pers Social Psychol 52 899 906
Funckes AJ Hayes GR Jr Hartwell WV 1963 Urinary excretion of paranitrophenol by volunteers following dermal exposure to parathion at different ambient temperatures J Agric Food Chem 11 455 457
Garte S 2002 The racial genetic paradox in biomedical research and public health Public Health Rep 117 421 425 12500957
Gee GC 2002 A multilevel analysis of the relationship between institutional racial discrimination and health status Am J Public Health 92 615 623 11919062
Gee GC Takeuchi DT 2004 Traffic stress, vehicular burden and well-being: a multilevel analysis Soc Sci Med 59 405 414 15110429
Geis KJ Ross CE 1998 A new look at urban alienation: the effect of neighborhood disorder on perceived powerlessness Soc Psychol Q 61 232 246
Geronimus AT 1996 Black/white differences in the relationship of maternal age to birthweight: a population-based test of the weathering hypothesis Soc Sci Med 42 589 597 8643983
Geronimus AT Bound J Waidmann TA 1999 Poverty, time, and place: variation in excess mortality across selected U.S. populations, 1980–1990 J Epidemiol Community Health 53 325 334 10396478
Geronimus AT Bound J Waidmann TA Colen CG Steffick D 2001 Inequality in life expectancy, functional status, and active life expectancy across selected black and white population in the United States Demography 38 227 251 11392910
Gibbs LM 1983 Community response to an emergency situation: psychological destruction and the Love Canal Am J Community Psychol 11 116 125
Ginexi EM Weihs K Simmens SJ Hoyt DR 2000 Natural disaster and depression: a prospective investigation of reactions to the 1993 midwest floods Am J Community Psychol 28 495 518 10965388
Goldman BA Fitton L 1994. Toxic Wastes and Race Revisited: An Update of the 1987 Report on the Racial and Socioeconomic Characteristics of Communities with Hazardous Waste Sites. Washington, DC:Center for Policy Alternatives.
Gordon CJ 2003 Role of environmental stress in the physiological response to chemical toxins Environ Res 92 1 7 12706749
Green LW Kreuter MW 1999. Health Promotion Planning: An Educational and Ecological Approach. Mountain View, CA:Mayfield.
Haan M Kaplan GA Camacho T 1987 Poverty and health: prospective evidence from the Alameda County Study Am J Epidemiol 125 989 998 3578257
Hadley ME 1992. Endocrinology. Englewood Cliffs, NJ:Prentice Hall.
Hart KD Kunitz SJ Sell RR Mukamel DB 1998 Metropolitan governance, residential segregation, and mortality among African Americans Am J Public Health 88 434 438 9518976
Hayward MD Crimmins EM Miles TP Yang Y 2000 The significance of socioeconomic status in explaining the race gap in chronic health conditions Am Sociol Rev 65 910 930
Holden C 1980 Love Canal residents under stress Science 208 1242 1244 7375935
Holland P Berney L Blane D Davey-Smith G Gunnell DJ Montgomery SM 2000 Life course accumulation of disadvantage: childhood health and hazard exposure during adulthood Soc Sci Med 50 1285 1295 10728848
Iceland J Weinberg DH Steinmetz E 2002. Racial and Ethnic Residential Segregation in the United States: 1980–2000. U.S. Census Bureau, Series CENSR-3. Washington, DC:U.S. Government Printing Office.
Institute of Medicine 1999. Toward Environmental Justice: Research, Education, and Health Policy Needs. Washington, DC:National Academy Press.
Institute of Medicine 2002. Unequal Treatment: Confronting Racial and Ethnic Disparities in Health Care. Washington, DC:National Academies Press.
Israel BA Farquhar S James SA Schulz AM Parker EA 2002 The relationship between social support, stress, and health among women on Detroit’s East Side Health Educ Behav 29 342 360 12038743
Israel BA Schulz AJ Parker EA Becker AB 1998 Review of community-based research: assessing partnership approaches to improve public health Annu Rev Public Health 19 173 202 9611617
Jackson SA Anderson RT Johnson NJ Sorlie PD 2000 The relation of residential segregation to all-cause mortality: a study in black and white Am J Public Health 90 615 617 10754978
Jacobs DE Clickner RP Zhou JY Viet SM Marker DA Rogers JW 2002 The prevalence of lead-based paint hazards in U.S. housing Environ Health Perspect 110 599 606
Jargowsky P 1997. Poverty and Place: Ghettos, Barrios, and the American City. New York:Russell Sage Foundation.
Johnson JV 1989 Collective control: strategies for survival in the workplace Int J Health Serv 19 469 480 2753580
Johnson JV Stewart W Hall EM Fredlund P Theorell T 1996 Long-term psychosocial work environment and cardiovascular mortality among Swedish men Am J Public Health 86 324 331 8604756
Jones CP 2000 Levels of racism: a theoretical framework and gardener’s tale Am J Public Health 90 1212 1215 10936998
Kaniasty K Norris FH 2000 Help-seeking comfort and receiving social support: the role of ethnicity and context of need Am J Community Psychol 28 545 581 10965390
Kaplan GA 1996 People and places: contrasting perspectives on the association between social class and health Int J Health Serv 26 507 519 8840199
Kaplan GA Turrell G Lynch JW Everson SA Helkala EL Salonen JT 2001 Socioeconomic position and cognitive function in adutlhood Int J Epidemiol 30 256 263 11369724
Karasek R Theorell T 1990. Healthy Work: Stress, Productivity, and the Reconstruction of Working Life. New York:Basic Books.
Kawachi I Berkman LF 2000. Social cohesion, social capital, and health. In: Social Epidemiology (Berkman LF, Kawachi I, eds). New York:Oxford University Press, 174–190.
Kawachi I Kennedy BP Glass R 1999 Social capital and self-rated health: a contextual analysis Am J Public Health 89 1187 1193 10432904
Keppel KG Pearcy JN Wagener D 2002. Trends in racial and ethnic-specific rates for the United States indicators: United States, 1990–1998. In: Healthy People Statistical Notes No. 23. Hyattsville, MD:National Center for Health Statistics.
Kessler RC Michelson KD Williams DR 1999 The prevalence, distribution and mental health correlates of perceived discrimination in the United States J Health Soc Behav 40 208 230 10513145
Kirschenman J Neckerman KM 1991. “We’d love to hire them, but …”: The meaning of race for employers. In: The Urban Underclass (Jencks C, Peterson P, eds). Washington, DC:Brookings Institution, 203–232.
Kjellstrand CM 1988 Age, sex, and race inequality in renal transplantation Arch Int Med 148 1305 1309 3288159
Klassen AC Hall AG Saksvig B Curbow B Klassen DK 2002 Relationship between patients’ perceptions of disadvantage and discrimination and listing for kidney transplantation Am J Public Health 92 811 817 11988452
Kretzman JP McKnight JL 1993. Building Communities from the Inside Out. Evanston, IL:Northwestern University.
Krieger J Higgins DL 2002 Housing and health: time again for public health action Am J Public Health 92 758 768 11988443
Krieger N 1999 Embodying inequality: a review of concepts, measures, and methods for studying health consequences of discrimination Int J Health Serv 29 295 352 10379455
Krieger N Fee E 1994 Social class: the missing link in U.S. health data Int J Health Serv 24 25 44 8150566
Krieger N Rowley DL Herman AA Avery B Phillips MT 1993 Racism, sexism, and social class: implications for studies of health, disease, and well-being Am J Prev Med 9 82 122 8123288
Krieger N Sidney S 1996 Racial discrimination and blood pressure: the CARDIA study of young black and white adults Am J Public Health 86A 1370 1378 8876504
Kuo W 1976 Theories of migration and mental health: an empirical testing on Chinese-Americans Soc Sci Med 10 297 306 793020
Kuper H Marmot MG 2003 Job strain, job demands, decision latitude, and risk of coronary heart disease within the Whitehall II study J Epidemiol Community Health 57 147 153 12540692
Landsbergis PA Schnall PL Dietz DK Warren K Pickering TG 1997 Job strain and health behaviors: results from a prospective study Am J Health Promot 12 237 245 10178616
LaVeist TA 1989 Linking residential segregation to the infant-mortality race disparity in U.S. cities Soc Sci Res 73 94
LaVeist TA 1993 Segregation, poverty, and empowerment: health consequences for African Americans Milbank Q 71 41 64 8450822
LaVeist TA 1994 Beyond dummy variables and sample selection: what health services researchers ought to know about race as a variable Health Serv Res 29 1 16 8163376
LaVeist TA Nickerson KJ Bowie JV 2000 Attitudes about racism, medical mistrust, and satisfaction with care among African American and white cardiac patients Med Care Res Rev 27 146 161 11092161
Lazarus RS Folkman S 1984. Stress, Appraisal and Coping. New York:Springer.
Lee C 1993. Beyond toxic wastes and race. In: Confronting Environmental Racism: Voices from the Grassroots (Bullard RD, Chavis B, eds). Boston:South End Press, 41–52.
Lee C 2002 Environmental justice: building a unified vision of health and the environment Environ Health Perspect 110 suppl 2 141 144 11929721
Lee J 2000 The salience of race in everyday life: black customers’ shopping experiences in black and white neighborhoods Work Occup 27 353 376
Lewontin RC 1982. Human Diversity. Redding, CT:Scientific American/Freeman.
Lioy PJ 1990 Assessing total human exposure to contaminants Environ Sci Technol 24 938 945
Logan JR 2003. Ethnic diversity grows, neighborhood integration lags. In: Redefining Urban and Suburban America (Katz B, Lang RE, eds). Washington, DC:Brookings Institution, 235–256.
Lopez R 2002 Segregation and black/white differences in exposure to air toxics in 1990 Environ Health Perspect 110 suppl 2 289 295 11929740
Luke D Esmundo E Bloom Y 2000 Smoke signs: patterns of tobacco billboard advertising in a metropolitan region Tobacco Control 9 16 23 10691754
Maantay J 2001 Zoning, equity, and public health Am J Public Health 91 1033 1041 11441726
Maantay J 2002 Mapping environmental injustices: pitfalls and potential of geographic information systems in assessing environmental health and equity Environ Health Perspect 110 suppl 2 161 171 11929725
Macintyre S Ellaway A Cummins S 2002 Place effects on health: how can we conceptualise, operationalise, and measure them? Soc Sci Med 55 125 139 12137182
Margellos H Silva A Whitman S 2004 Comparison of health status indicators in Chicago: are black-white disparities worsening? Am J Public Health 94 116 121 14713708
Marmot MG Fuhrer R Ettner S Marks NF Bumpass LL Ryff CD 1998 Contribution of psychosocial factors to socioeconomic differences in health Milbank Q 76 403 440 9738169
Marmot MG Kogevinas M Elaston MA 1987 Social/economic status and disease Annu Rev Public Health 8 111 135 3555518
Massey D 2001. Residential segregation and neighborhood conditions in U.S. metropolitan areas. In: America Becoming: Racial Trends and Their Consequences (Smelser NJ, Wilson WJ, Mitchell F, eds). Washington, DC:National Academy Press, 391–434.
Massey D Denton NA 1988 The dimensions of residential segregation Soc Forces 67 281 315
Massey D Denton NA 1993. American Apartheid: Segregation and the Making of the Underclass. Cambridge, MA:Harvard University Press.
Mazumdar S Mazumdar S Docuyanan F McLaughlin CM 2000 Creating a sense of place: the Vietnamese-Americans and Little Saigon J Environ Psychol 20 319 333
McEwen BS 1998 Protective and damaging effects of stress mediators New Engl J Med 338 171 179 9428819
Merkin SS Stevenson L Powe N 2002 Geographic socioeconomic status, race, and advanced-stage breast cancer in New York City Am J Public Health 92 64 70 11772763
Meyer SG 2000. As Long as They Don’t Move Next Door: Segregation and Racial Conflict in American Neighborhoods. Oxford:Rowman & Littlefield.
Mohai P Bryant B 1992. Environmental racism: reviewing the evidence. In: Race and the Incidence of Environmental Hazards: A Time for Discourse (Bryant B, Mohai P, eds). Boulder, CO:Westview, 163–176.
Morello-Frosch R Pastor M Jr Porras C Sadd J 2002 Environmental justice and regional inequality in southern California: implications for future research Environ Health Perspect 110 suppl 2 149 154 11929723
Morello-Frosch R Pastor M Jr Sadd J 2001 Environmental justice and southern California’s “riskscape”: the distribution of air toxics expsoures and health risks among diverse communities Urban Affairs Rev 36 551 578
Morenoff JD 2003 Neighborhood mechanisms and the spatial dynamics of birth weight Am J Sociol 108 976 1017 14560732
Morenoff JD Sampson RJ Raudenbush S 2001 Neighborhood inequality, collective efficacy and the spatial dynamics of homicide Criminology 39 3 517 560
Morland K Wing S Diez-Roux AV Poole C 2002 Neighborhood characteristics associated with the location of food stores and food service places Am J Prev Med 22 23 29 11777675
Morrison RS Wallenstein S Natale DK Senzel RS Huang LL 2000 “We don’t carry that”—failure of pharmacies in predominantly nonwhite neighborhoods to stock opioid analgesics N Engl J Med 3426 1023 1026 10749965
Moses M Johnson ES Anger WK Burse VW Horstman SW Jackson RJ 1993 Environmental equity and pesticide exposure Toxicol Ind Health 9 913 59 8184449
Mountain JL Cavalli-Sforza LL 1997 Multilocus genotypes, a tree of individuals, and human evolutionary history Am J Hum Genet 61 705 718 9326336
Munnell AH Tootell GMB Browne LE McEneaney J 1996 Mortgage lending in Boston: interpreting HMDA data Am Econ Rev 86 25 53
National Research Council 1991a. Human Exposure Assessment for Airborne Pollutants: Advances and Opportunities. Washington, DC:National Academy Press.
National Research Council 1991b. Environmental Epidemiology, Vol 1. Public Health and Hazardous Wastes. Washington, DC:National Academy Press.
Northridge ME Shepard PM 1997 Environmental racism and public health Am J Public Health 87 730 732 9184496
Northridge ME Stover GN Rosenthal JE Sherard D 2003 Environmental equity and health: understanding complexity and moving forward Am J Public Health 93 209 214 12554571
O’Neill MS Jerrett M Kawachi I Levy JI Cohen AJ Gouveia N 2003 Health, wealth, and air pollution: advancing theory and methods Environ Health Perspect 111 1861 1870 14644658
Ouis D 2001 Annoyance from road traffic noise: a review J Environ Psychol 21 101 120
Pastor M 2001. Geography and opportunity. In: America Becoming: Racial Trends and Their Consequences (Smelser N, Wilson WJ, Mitchell F, eds). Washington, DC:National Academy Press, 435–468.
Pastor M Sadd J Hipp J 2001 Which came first? Toxic facilities, minority move-in, and environmental justice J Urban Affairs 23 1 21
Perera FP Illman SM Kinney PL Whyatt RM Kelvin EA Shepard P 2002 The challenge of preventing environmentally related disease in young children: community-based research in New York City Environ Health Perspect 110 197 204 11836150
Perera FP Rauh V Tsai W-Y Kinney P Camann D Barr D 2003 Effects of transplacental exposure to environmental pollutants on birth outcomes in a multiethnic population Environ Health Perspect 111 201 205 12573906
Perlin S Setzer RW Creason J Sexton K 1995 Distribution of industrial air emissions by income and race in the United States: an approach using the Toxics Release Inventory Environ Sci Technol 29 69 80 22200202
Perlin SA Sexton K Wong DW 1999 An examination of race and poverty for populations living near industrial sources of air pollution J Expo Anal Environ Epidemiol 9 29 48 10189625
Peterson RD Krivo LJ 1993 Racial segregation and black urban homicide Soc Forces 71 1001 1026
Peterson RD Krivo LJ 1999 Racial segregation, the concentration of disadvantage, and black and white homicide victimization Sociol Forum 14 465 493
Phoenix J 1993. Getting the lead out of the community. In: Confronting Environmental Racism (Bullard RD, ed). Boston:South End Press, 77–92.
Pickett KE Pearl M 2001 Multilevel analyses of neighbourhood socioeconomic context and health outcomes: a critical review J Epidemiol Community Health 55 111 122 11154250
Pirkle JL Kaufmann RB Brody DJ Hickman T Gunter EW Paschal DC 1998 Exposure of the U.S. population to lead, 1991–1994 Environ Health Perspect 106 745 750 9799191
Polednak AP 1991 Black-white differences in infant mortality in 38 standard metropolitan statistical areas Am J Public Health 81 1480 1482 1951808
Polednak AP 1996 Segregation, discrimination and mortality in U.S. blacks Ethn Dis 6 99 108 8882839
Pope CA Verrier RL Lovett EG Larson AC Raizenne ME Kanner RE 1999 Heart rate variability associated with particulate air pollution Am Heart J 138 890 899 10539820
Raudenbush SW Bryk AS 2002. Hierarchical Linear Models: Applications and Data Analysis Methods. Thousand Oaks, CA:Sage Publications.
Rich RC Edelstein M Hallman WK Wandersman AH 1995 Citizen participation and empowerment: the case of local environmental hazards Am J Community Psychol 23 657 676 8851344
Rios R Poje GV Detels R 1993 Susceptibility to environmental pollutants among minorities Toxicol Ind Health 9 797 820 8184444
Ross CE Jang SJ 2000 Neighborhood disorder, fear, and mistrust: the buffering role of social ties with neighbors Am J Community Psychol 28 401 420 10965384
Ross CE Reynolds JL Geis KJ 2000 The contingent meaning of neighborhood stability for residents’ psychological well-being Am Sociol Rev 65 581 597
Saha S Arbelaez JJ Cooper LA 2003 Patient–physician relationships and racial disparities in the quality of health care Am J Public Health 98 1713 1719 14534227
Sallis JF Owen N 1997. Ecological models. In: Health Behavior and Health Education: Theory, Research, and Practice (Glanz K, Lewis FM, Rimer BK, eds). San Francisco:Jossey-Bass, 403–424.
Sampson RJ Raudenbush SW Earls F 1997 Neighborhoods and violent crime: a multilevel study of collective efficacy Science 277 918 924 9252316
Schelling TC 1971 Dynamic models of segregation J Math Sociol 1 143 186
Schlenger WE Caddell JM Ebert L Jordan BK Rourke KM Wilson D 2002 Psychological reactions to terrorist attacks: findings from the national study of Americans’ reactions to September 11 JAMA 288 581 588 12150669
Schmidt CW 2001 The market for pollution Environ Health Perspect 109 A379 A381
Schneider M Logan JR 1982 Suburban racial segregation and black access to local public resources Social Sci Q 63 762 770
Schulz A Israel B Williams D Parker E Becker A James S 2000 Social inequalities, stressors and self reported health status among African American and white women in the Detroit metropolitan area Soc Sci Med 51 1639 1653 11072884
Schulz AJ Williams DR Israel BA Lempert LB 2002 Racial and spatial relations as fundamental determinants of health in Detroit Milbank Q 80 677 707 12532644
Schuster MA Stein BD Jaycox LH Collins RL Marshall GN Elliott MN 2001 A national survey of stress reactions after the September 11, 2001 terrorist attacks N Engl J Med 345 1507 1512 11794216
Schwartz A 1998 Bank lending to minority and low-income households and neighborhoods: do community reinvestment agreements make a difference? J Urban Affairs 20 269 301
Selye H 1936 Syndrome produced by diverse nocuous agents [Letter] Nature 138 32
Sexton K 2000 Socioeconomic and racial disparities in environmental health: is risk assessment part of the problem or part of the solution? Hum Ecol Risk Assess 6 561 574
Sexton K Olden K Johnson BL 1993 “Environmental justice”: the central role of research in establishing a credible scientific foundation for informed decision making Toxicol Ind Health 9 685 727 8184441
Shepard PM Northridge ME Prakash S Stover G 2002 Preface: advancing environmental justice through community-based participatory research Environ Health Perspect 110 suppl 2 139 140 11836141
Soloman BD Lee R 2000 Emissions trading systems and environmental justice Environment 42 34 46
Sorensen G Barbeau E Hunt MK Emmons K 2004 Reducing disparities in tobacco use: a social-contextual model for reducing tobacco use among blue-collar workers Am J Public Health 94 230 239 14759932
Sorlie PD Backlund E Keller JB 1995 U.S. mortality by economic, demographic, and social characteristics: the National Longitudinal Mortality Study Am J Public Health 85 949 956 7604919
Squires GD 1994. Capital and Communities in Black and White. Albany:State University of New York Press.
Squires GD Velez W 1996 Mortgage lending and race: is discrimination still a factor? Environ Plan 28 1199 1208
Steptoe A Feldman PA 2001 Neighborhood problems as sources of chronic stress: development of a measure of neighborhood problems and associations with socioeconomic status and health Ann Behav Med 23 177 185 11495218
Sucoff CA Upchurch DA 1998 Neighborhood context and the risk of childbearing among metropolitan-area black adolescents Am Sociol Rev 63 571 585
Sugrue TJ 1998. The Origins of the Urban Crisis: Race and Inequality in Postwar Detroit. Princeton, NJ:Princeton University Press.
Turner MA Skidmore F 2001. Mortgage Lending Discrimination: A Review of Existing Evidence. Washington, DC:Urban Institute.
United Church of Christ 1987. Toxic Wastes and Race in the United States: A National Report on the Racial and SocioEconomic Characteristics of Communities Surrounding Hazardous Waste Sites. New York:United Church of Christ, Commission for Racial Justice.
U.S. Census Bureau 2000. The Population Profile of the United States: 2000 (Internet Release). Washington, DC:U.S. Census Bureau. Available: http://www.census.gov/population/www/pop-profile/profile2000.html [accessed 21 October 2004].
U.S. Census Bureau 2003. Housing Patterns - Table 7-1. Available: http://www.census.gov/hhes/www/housing/resseg/tab7-1.html [accessed 14 October 2004].
U.S. DHHS 1998. Tobacco Use among U.S. Racial/Ethnic Minority Groups—African Americans, American Indians and Alaska Natives, Asian Americans and Pacific Islanders, and Hispanics: A Report of the Surgeon General. Atlanta, Georgia: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. Available: http://www.cdc.gov/tobacco/sgr/sgr_1998/sgr-min-sgr.htm [accessed 7 October 2004].
U.S. DHHS 2000. Healthy People 2010. 2nd ed. Washington, DC:U.S. Department of Health and Human Services. Available: http://www.healthypeople.gov/publications/ [accessed 7 October 2004].
U.S. EPA 2003a. Framework for Cumulative Risk Assessment. EPA/600/P-02/001F. Washington, DC:U.S. Environmental Protection Agency, Office of Research and Development.
U.S. EPA 2003b. Supplemental Guidance for Assessing Cancer Susceptibility from Early-Life Exposure to Carcinogens. (External Review Draft). EPA/630/R-03/003. 2003. Washington, DC:U.S. Environmental Protection Agency.
U.S. General Accounting Office 1983. Siting of Hazardous Waste Landfills and Their Correlation with Racial and Economic Status of Surrounding Communities. Washington, DC:General Accounting Office.
Wagener DK ed 1987 The Role of Biomarkers in Reproductive and Developmental Toxicology Environ Health Perspect 74 3 199 3691432
Waitzman NJ Smith KR 1998 Separate but lethal: the effects of economic segregation on mortality in metropolitan America Milbank Q 76 341 373 9738167
Warr M Ellison CG 2000 Rethinking social reactions to crime: personal and altruistic fear in family households Am J Soc 106 551 578
Wester RC Quan D Maibach HI 1996 In vitro percutaneous absorption of model compounds glyphosate and malathion from cotton fabric into and through human skin Food Chem Toxicol 34 731 735 8883475
Whiteis DG 1992 Hospital and community characteristics in closures of urban hospitals, 1980–87 Public Health Rep 107 409 416 1641437
Williams DR Collins CA 1995 U.S. socioeconomic and racial differences in health: patterns and explanations Annu Rev Sociol 21 349 386
Williams DR Collins C 2001 Racial residential segregation: a fundamental cause of racial disparities in health Public Health Rep 116 404 416 12042604
Williams DR Neighbors H 2001 Racism, discrimination and hypertension: evidence and needed research Ethn Dis 11 800 816 11763305
Williams DR Yu Y Jackson JS Anderson NB 1997 Racial differences in physical and mental health: socioeconomic status, stress, and discrimination J Health Psychol 2 335 351 22013026
Williams RW 1999 Environmental injustice in America and its politics of scale Political Geography 18 49 73
Wilson WJ 1987. The Truly Disadvantaged. Chicago:University of Chicago Press.
Wilson WJ 1996. When Work Disappears: The World of the New Urban Poor. New York:Alfred A. Knopf.
Winkleby MA Cubbin C 2003 Influence of individual and neighborhood socioeconomic status on mortality among black, Mexican-American, and white women and men in the United States J Epidemiol Community Health 57 444 452 12775792
Woodruff TJ Parker JD Kyle AD Schoendorf KC 2003 Disparities in exposure to air pollution during pregnancy Environ Health Perspect 111 942 946 12782496
Yen IH Syme SL 1999 The social environment and health: a discussion of the epidemiologic literature Annu Rev Public Health 20 287 308 10352860
Yen IH Ragland DR Greiner BA Fisher JM 1999 Workplace discrimination and alcohol consumption: findings from the San Francisco Muni Health and Safety Study Ethn Dis 9 70 80 10355476
Young CJ Gaston RS 2000 Renal transplantation in black Americans New Engl J Med 343 1545 1552 11087885
Zimmerman MA 2000. Empowerment theory: psychological, organizational and community levels of analysis. In: Handbook of Community Psychology (Rappaport J, Seidman E, eds). New York:Kluwer Academic/Plenum Publishers, 43–63.
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7134ehp0112-00165415579408ResearchArticlesSex and Ceruloplasmin Modulate the Response to Copper Exposure in Healthy Individuals Méndez Marco A. Araya Magdalena Olivares Manuel Pizarro Fernando González Mauricio Institute of Nutrition and Food Technology, University of Chile, Santiago, ChileAddress correspondence to M.A. Méndez, Institute of Nutrition and Food Technology, University of Chile, Macul 5540, Santiago 11, Chile. Telephone: 56-2-678-1545. Fax: 56-2-221-4030. E-mail:
[email protected] investigation was funded by the International Copper Association in the form of an unrestricted research grant.
The authors declare they have no competing financial interests.
12 2004 17 8 2004 112 17 1654 1657 26 3 2004 16 8 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Previous studies indicated that sex might influence the response to copper exposure. Ceruloplasmin (Cp) is an indicator of Cu status, but it is not clear whether and how it reflects changes of Cu status among healthy individuals. In this study, 82 apparently healthy women and men were chosen from 800 individuals because their Cp values belonged to the higher and lower 10% of the group Cp distribution curve. Before and after receiving a supplement of 10 mg Cu/day (upper limit of daily intake) for 2 months, we performed blood and urinary biochemical measurement of potential Cu markers. We used principal component analysis and linear discriminant analysis to identify blood and/or urinary Cu indicators that showed a differential response to copper. Results showed that Cp values in serum represent a reliable indicator to differentiate subgroups within the normal population in their response to Cu exposure. The response depends on Cp values and on sex, such that women with higher and men with lower Cp values exhibit the greatest response.
copper exposurediscriminant analysishealthy individualsprincipal component analysis
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Copper is required for the function of several cuproenzymes; therefore, its presence is essential for different physiological functions (Linder 1991). Cu, however, is able to generate free radicals and oxidize cellular components through its redox activity (Aust et al. 1985). These conflicting properties demand a close regulation of the metal at the organism level. Effects associated with severe lack or excess Cu are well described in genetic conditions, such as Menkes disease (Chelly et al. 1993; Mercer et al. 1993; Tanzi et al. 1993; Vulpe et al. 1993) and Wilson’s disease (Bull et al. 1993; Tanzi et al. 1996). In contrast, much less is known about relevant biological effects associated with situations when no excess or deficit of Cu is present (Davis 2003; Hambidge 2003). It is well known that serum ceruloplasmin (Cp) and Cu values are higher in young children and increase during even mild inflammatory/infectious processes, but their relationships to Cu intake and markers of Cu status are not clear, and available data suggest that they modify only when exposure changes by several orders of magnitude (Araya et al. 2003b, 2003c). It is still not clear whether marginal or moderate changes in Cu exposure may result in adverse effects to human health because there are no sensitive indicators of marginal changes in Cu status and because early functionally relevant responses are not well defined.
With the aim of improving our understanding about the early effects of Cu on human health, we have conducted a series of studies on asymptomatic adults undergoing controlled Cu exposure. This varied between approximately 3 and 10 times the customary dietary intake. Clinical trials showed that nausea is the earliest and most frequent response, and we used the generated data to calculate the dose–response curve to acute Cu exposure (Araya et al. 2003a, 2003b, 2003c; Olivares et al. 2001; Pizarro et al. 2001). A community survey in which participants ingested between 0.9 and 10 mg Cu/day for 2 months [the concentration defined as tolerable daily intake (TDI) of Cu ingestion for humans; Araya et al. (2003c)] allowed us to describe the full range of responses to Cu exposure, showing that there are more gastrointestinal responses (mainly nausea) with increasing Cu exposure (Cu concentration in water) and that these responses diminish with time, suggesting adaptation (Araya et al. 2004). In all of these studies, the statistical analyses suggested that the variable sex influenced the results.
No changes in biochemical blood parameters were detected in a previous study in which healthy participants were exposed to up to 6 mg Cu/L water, which represented as much as 14 mg Cu/day on occasional days depending on the volume of fluids ingested (Araya et al. 2003c). To further assess the homeostatic responses to Cu exposure, in a recent study we assessed the effects of exposing asymptomatic adults grouped by their Cp values to the TDI for 2 months, administered as a single daily supplement. A series of biochemical responses of blood and urinary potential Cu indicators were measured, and their detailed analysis will be reported elsewhere (Araya et al. unpublished data). The existing evidence is insufficient to determine the appropriateness of the different indicators proposed to assess Cu status among apparently normal individuals; in this article we present a multivariate strategy to evaluate a series of proposed indicators for their capacity to identify differences within the apparently healthy group. We performed our analysis as a function of sex, including the relative importance of each measurement before and after a Cu supplementation period.
Materials and Methods
The study was a prospective controlled trial in healthy adults. Participants were 18–50 years of age, and approximately 50% were younger and older than 30 years of age. One-half of them were women who were not pregnant and did not get pregnant throughout the study. The need for volunteers was advertised in the southeastern area of Santiago; potential participants received detailed information about the protocol, and those who agreed to participate signed an informed consent before we formed the study groups. The Committee on Ethics for Human Research, Institute of Nutrition and Food Technology, University of Chile, approved the protocol. All individuals received 10 mg Cu/day administered under direct supervision as two gelatin-coated 5-mg Cu tablets (as Cu sulfate). This dose was chosen based on two criteria. First, Pratt et al. (1985) showed no significant changes in hematocrit, triglycerides, serum glutamic oxaloacetic transaminase (GOT), gamma glutamyltransferase (GGT), lactate dehydrogenase, cholesterol, or alkaline phosphatase in adult humans after administering 10 mg Cu/day as Cu gluconate or placebo capsules for 12 weeks. Second, the upper limit (10 mg Cu/day) is defined as the maximum intake from food, water, and supplements that is unlikely to pose risk of adverse health effects from excess Cu in almost all (97.5%) apparently healthy individuals, in an age- and sex-specific population group (Institute of Medicine 2001). We hypothesized that individuals may have a differential response to Cu supplementation depending on their position in the serum Cp distribution curve, considering a lower value as an index of long-term low intake. Dietary surveys assessing total daily Cu intake from food and water in Chile have revealed that 16.4% of men and 33.3% of women between 20 and 60 years of age are below the estimated average requirements (Olivares et al. 2004). Accordingly, 800 apparently healthy individuals were screened for their serum Cp protein, and 82 individuals that represented the 10% higher (high-Cp group) and 10% lower (low-Cp group) values in the Cp distribution curve were assessed (n = 41 for each group). Inclusion criteria were a) being free of acute infectious/inflammatory processes (C-reactive protein < 0.8, as indicated by the kit manufacturer; b) white cell count in a hemogram < 12,000 cells/mL, the lower limit of the normal range (Dallman 1977) for chronic illnesses and for chronic multimedication that may interfere with the study.
Before and after the 2-month Cu supplementation, we performed blood and urine studies. These studies included 18 potential markers of Cu status chosen from published data and our experience. Studies in blood included measurement of Cu in serum by atomic absorption spectrometry (model 2280; Perkin Elmer, Norwalk, CT, USA) and in peripheral mononuclear cells (MNCs; model SIMAA 6100; Perkin Elmer); Cp protein measured by nephelometry (array protein system; Beckman Instruments Inc., Brea, CA, USA); liver enzymes [serum GOT, serum glutamic-pyruvic transaminase (GPT), and GGT] determined using a commercial kit (Boehringer Mannheim, Mannheim, Germany); homocysteine values determined using an IMX system homocysteine kit (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA); zinc–Cu superoxide dismutase (SOD) activity in erythrocytes measured by a Bioxytech SOD-525 Assay (OXIS International Inc, Portland, OR, USA); and glutathion measured in peripheral MNCs using a glutathione assay kit (Calbiochem; Cayman Chemical Company, Ann Arbor, MI, USA). Studies in urine included measurement of urinary Cu excretion after a chelator challenge with 300 mg 2,3-dimercaptopropane-1-sulfonate (DMPS or Dimaval; Heyl Laboratory, Berlin, Germany), within 3 days before and after supplementation beginning and end. Calculating sample size using α at 5% and power at 80%, we needed 35–45 individuals per group to detect a delta of 0.5 standard deviations in the biochemical measurements that were planned.
Data were analyzed using SYSTAT 5.0 (SYSTAT, Inc., Evanston, IL, USA; Wilkinson 1996). All data were log transformed in order to meet the assumptions of normality of data. Because study groups were formed on the basis of individuals’ serum Cp values (high-Cp and low-Cp), data were first assessed by univariate analysis at the beginning and end of the supplementation period to determine whether groups formed by individuals’ Cp values were significantly different. The integral (multivariate) response to Cu exposure (defined as the response of all biochemical measurements at the same time) was explored by multivariate analysis using principal component analysis (PCA) and linear discriminant analysis (LDA). PCA is a multivariate statistical tool that simplifies complex data sets by transforming the original variables into new independent and uncorrelated variables named principal components, which explain the observed variability in decreasing order. Thus, the first components concentrate maximal information (variance explained) about the analysis; additionally, for each component there is an eigenvalue with an associated variance value (explained variance). On the other hand, we used LDA as a classification function to calculate scores for each variable in the different groups; LDA permits evaluating whether there are significant relationships between qualitative variables or classes (in this case, sex and low-Cp and high-Cp groups) and quantitative predictor variables (in our case, eigenvalues of each biochemical variable). Because we knew the classes, we built a linear discriminant function to estimate the goodness of this classification within each class. A matrix originated by PCA served as the basis for LDA input data; in all analyses, we added eigenfactors until obtaining close to 80% of variability in the model. Thus, the LDA output allowed assessing a) whether sex and Cp levels (low-Cp and high-Cp) differences are associated with responses to copper; and b) the integral (multivariate) response of individuals to Cu exposure. Additionally, because the discriminant function was applied to the same sample used to derive it, we used both cross-validation and jackknife procedures to obtain unbiased estimates (Hair et al. 1992). Using these procedures, we obtained a classification matrix that allowed evaluating the performance of the defined classes (sex and low-Cp and high-Cp groups), verifying which individuals had high values of correct classifications. As a control, we also assessed whether the analyses performed using both cross-validation and jackknife procedures yielded the same values of correct classifications; because no differences were found, we only show the results of the jackknife matrix. All the LDA data showed normal multivariate distribution, which was evaluated using the Sen and Puri test (Sen and Puri 1968).
Results
Univariate analysis.
As expected, Cp values of individuals grouped by Cp group (low-Cp and high-Cp) and sex showed significant differences at the beginning [analysis of variance (ANOVA), Cp group: F = 103.99, df = 1, p < 0.0001; sex: F = 22.256, df = 1, p < 0.0001; interaction Cp group × sex: F = 10.591, df = 80, p < 0.002; Figure 1], and end of treatment (ANOVA, Cp group: F = 126.710, df = 1, p < 0.0001; sex: F = 22.256, df = 1, p < 0.0001; interaction Cp group × sex: F = 10.591, df = 80, p < 0.008; Figure 1).
Multivariate analysis.
As a first step, we explored whether differences existed when all biochemical measurements were considered at the same time (integral response), assorting individuals both by sex and Cp groups. PCA showed that the first four components explained 68.62% of the variability; in the first component, ferritin, GGT, and GPT obtained the highest loading value (Table 1), whereas serum Zn and Cu and Cu in MNCs obtained the lowest values. LDA, using the matrix obtained from the first six PCA components (which explained > 80.8% of the variability observed) and using sex as a classification variable, revealed statistically significant differences between sexes (Wilks’s lambda = 0.423; F = 17.043; df = 6, 75; p < 0.0001). A discriminant classification matrix showed high values of correct classifications (80% for men, 88% for women), indicating that there were differences related to sex associated with the parameters evaluated. In view of these results, the next analyses were performed separately on women and men, before and after Cu supplementation.
Before Cu supplementation.
In women, PCA showed that the first four components explained 70.6% of the variability. In the first component, ferritin, GGT, and GPT obtained the highest loading value (Table 2). Among men, PCA showed that the first four components explained 70.38% of the variability. Compared with women, the relative importance of each element in the first component somewhat differed: GPT and GGT were included, but not ferritin, and DMPS (1–4 hr) was among the variables with the highest loading value (Table 2). The LDA analysis performed using high- and low-Cp group as classificatory variables revealed that these groups were statistically different (Wilks’s lambda = 0.5136; F = 5.3673; df = 6, 34; p < 0.0005), showing high values of correct classifications in the classification matrix (low-Cp group, 80%; high-Cp group, 81%). This analysis also shows that although differences between the Cp groups were significant (Wilks’s lambda = 0.660; F = 2.919; df = 6, 34; p < 0.020), the values of correct classifications were lower in men than in women (low-Cp group, 70%; high-Cp group, 67%).
After Cu supplementation.
In women, PCA showed that the first four components explained 73.27% of the variability. In the first component, as before, Cu supplementation, GGT, ferritin, and GPT obtained the highest loading value (Table 2). The LDA showed significant differences between the Cp groups (Wilks’s lambda = 0.574; F = 4.074; df = 6, 33; p < 0.0003) and a classification matrix with values of correct classifications > 50% (low-Cp group, 76%; high-Cp group, 68%). Among men, PCA showed that the first components explained 65.91% of the variability. In the first component, GGT, GPT, and GOT showed the highest loading value, showing that in comparison with analysis before Cu supplementation, GOT replaced DMPS (1–4 hr; Table 2). LDA in men also showed differences between Cp groups (Wilks’s lambda = 0.584; F = 2.496; df = 8, 32; p < 0.016), and as in the case of women, values of correct classifications were > 50% (low-Cp group, 60%; high-Cp group, 67%). Both in women and in men, the percentages of correct classification were lower in comparison with the figures obtained before Cu supplementation.
Discussion
It is well known that serum Cp and Cu vary responding to rather minor inflammatory and infectious events; at the same time, these indicators are used to assess changes in Cu status in pathological situations. To what extent they may reflect mild yet relevant changes of Cu status among apparently healthy individuals is still a matter of debate (Araya et al. 2003b, 2003c; Davis 2003; Hambidge 2003; Kehoe et al. 2000). In this study, participants were healthy and remained clinically healthy during the 2-month controlled Cu exposure. Serum activities of aminotranferases are the traditional biochemical blood measurements used clinically to assess liver function. Participants received a daily Cu dose defined as the upper safe limit for human consumption, so toxic responses were not expected; liver aminotranferases were evaluated to satisfy ethical considerations. We detected no responses that may represent toxic effects of the Cu dose used.
Both the univariate and bivariate analyses support the idea that Cp values in serum represented a reliable indicator of Cu status responding to chronic Cu exposure and that sex was indeed a factor that modulated the response. It is interesting that both in women and in men and both before and after Cu supplementation, GGT and GPT were always included in the first component with high loading values; ferritin was included only among women, whereas urinary Cu excretion after DMPS challenge was included among men. Because it is difficult to interpret these differences with the present data and there is little experience with DMPS challenge among normal populations, these findings deserve further research. The LDA also showed, both in women and in men, that the biochemical indicators measured were significantly associated with the Cp values; the classification matrix showed that the correct assignment values were higher among women (~ 80%), whereas among men they reached values of about 70%, suggesting that the Cp value is a good indicator to separate the groups, and a reliable descriptor of the integral response to Cu exposure, but it may be more sensitive for women that for men.
By the end of the Cu exposure period, in both sexes the first four PCA components explained a lower proportion of variance (73.3% in women and 65.9% in men), suggesting that variability increased in the groups after Cu supplementation. It is intriguing that, among women, ferritin decreased its loading value whereas GGT became the most relevant variable (Table 2). Among men, GGT is also the main variable explaining the variance, and the three transaminases as a whole (GGT, GPT, and GOT) are the factors with the highest loading values. This is a relevant finding because these enzymes classically change in hepatic diseases, but it is not clear that they respond to subclinical situations (Cashman et al. 2001; Jones et al. 1997; Lowe et al. 2002; Nayak et al. 2003; Olusi et al. 2003); even accepting that they may respond to other illnesses, our results support their use to monitor potential adverse effects of Cu in the liver.
Conclusion
We conclude that Cp values in serum represent a reliable indicator of Cu status and of the host response to Cu exposure. This information is relevant to risk assessment studies of Cu effects in human health and environmental epidemiology. This response depends on sex and also on the Cp value, such that women with higher Cp values and men with lower Cp values exhibit the greatest response. Why women respond differently than men and why apparently healthy individuals respond differently depending on their Cp values is not clear; ongoing studies are currently exploring these aspects.
Figure 1 Cp concentration (media ± SE) in men and women before and after Cu supplementation grouped by Cp concentrations of individuals.
Table 1 Values (loadings) in the first four PCAs for each of the 18 variables assessed in adult individuals before Cu supplementation.
Variables PC1 PC2 PC3 PC4
Hemoglobin 0.104 0.049 −0.184 −0.018
Ferritin 1.347 0.077 −2.606 1.396
Homocysteine 0.127 −0.104 −0.177 −0.719
Serum Cu −0.003 0.418 0.917 −0.300
Serum Fe 0.169 0.578 −0.365 −0.397
Serum Zn −0.003 0.002 −0.086 0.247
Cu in MNC −0.005 −0.066 0.130 0.075
Fe in MNC −0.013 −0.143 0.128 −0.294
Zn in MNC 0.017 −0.099 −0.023 −0.105
GOT 0.542 −0.437 1.328 0.929
GPT 0.945 −1.187 1.111 1.372
GGT 1.680 0.659 1.342 −1.571
SOD 0.223 0.264 0.395 1.491
DMPS 0.261 2.124 −0.240 0.025
DMPS 0–4 hr −0.333 0.502 0.782 2.621
DMPS 5–24 hr −0.052 0.830 0.619 −0.217
DMPS 0–24 hr −0.318 1.199 0.443 1.185
Urinary Cu 0.058 0.648 0.038 0.297
Percent variance explained 29.08 18.11 12.8 9.650
Value for DMPS is Cu excreted during the indicated time period after administration of 300 mg DMPS.
Table 2 Loadings from the first PCAs studied before and after Cu supplementation.
Before
After
Variables Women Men Women Men
Hemoglobin 0.059 0.006 0.021 −0.029
Ferritin 1.161a −0.346 1.553a 0.428
Homocysteine −0.034 −0.147 0.147 0.293
Serum Cu 0.263 0.038 0.295 0.132
Serum Fe 0.132 0.093 0.130 −0.106
Serum Zn 0.006 0.058 −0.033 −0.125
Cu in MNC 0.001 −0.074 0.116 0.090
Fe in MNC −0.005 −0.112 0.076 0.102
Zn in MNC 0.018 −0.094 0.072 0.143
GOT 0.679 −0.993a 0.722 0.870a
GPT 0.848a −1.640a 0.781a 0.885a
GGT 1.656a −1.197a 2.175a 2.540a
SOD 0.326 0.012 −0.152 −0.321
DMPS 0.716 0.908 0.512 0.844
DMPS 0–4 hr 0.165 0.675
DMPS 5–24 hr 0.104 0.206
DMPS 0–24 hr −0.229 0.512 0.472 −0.441
Urinary Cu 0.100 0.279 0.497 0.337
Percent variance explained 31.58 29.58 29.57 26.19
Values for DMPS are Cu excreted during the indicated time period after administration of 300 mg DMPS.
a Variables with highest loading in the first component.
==== Refs
References
Araya M Chen B Klevay LM Strain JJ Johnson L Robson P 2003a Confirmation of an acute no-observed-adverse-effect level (NOAEL) and low-observed-adverse-effect level (LOAEL) for copper in bottled drinking water in a multi-site international study Regul Toxicol Pharmacol 38 389 399 14623488
Araya M Olivares M Pizarro F González M Speisky H Uauy R 2003b Copper exposure and potential biomarkers of copper metabolism Biometals 16 199 204 12572679
Araya M Olivares M Pizarro F Gonzalez M Speisky H Uauy R 2003c Gastrointestinal symptoms and blood indicators of copper load in apparently healthy adults undergoing controlled copper exposure Am J Clin Nutr 77 3 646 650 12600855
Araya M Olivares M Pizarro F Llanos A Figueroa G Uauy R 2004 Community-based randomized double-blind study of gastrointestinal effects and copper exposure in drinking water Environ Health Perspect 112 1068 1073 15238279
Aust SD Morehouse LA Thomas CE 1985 Role of metals in oxygen radical reactions J Free Radic Biol Med 1 1 3 25 3013969
Bull PC Thomas GR Rommens JM Forbes JR Cox DW 1993 The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene Nat Genet 5 327 337 8298639
Cashman KD Baker A Ginty F Flyn A Strain JJ Bonham MP 2001 No effect of copper supplementation on biochemical markers of bone metabolism in healthy young adult females despite apparently improved copper status Eur J Clin Nutr 55 525 531 11464225
Chelly J Tümer Z Tønneson T Petterson A Ishikawa-Brush Y Tommerup N 1993 Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein Nat Genet 3 14 19 8490646
Dallman PR 1977. White blood cells. In: Pediatrics (Rudolph AM, ed). 16th ed. New York:Appleton Century-Crafts, 1176–1179.
Davis CD 2003 Low dietary copper increases fecal free radical production, fecal water alkaline phosphatase activity and cytotoxicity in healthy men J Nutr 133 2 522 527 12566494
Hair J Anderson R Tatham R Black W 1992. Multivariate Data Analysis with Readings. London:Macmillan.
Hambidge M 2003 Biomarkers of trace mineral intake and status J Nutr 133 suppl 3 948S 955S 12612181
Institute of Medicine 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC:National Academy Press.
Jones AA DiSilvestro RA Coleman M Wagner TL 1997 Copper supplementation of adult men: effects on blood copper enzyme activities and indicators of cardiovascular disease risk Metabolism 46 1380 1383 9439530
Kehoe CA Turley E Bonham MP O’Connor M McKeown A Faughan MS 2000 Response of putative indices of copper status to copper supplementation in human subjects Br J Nutr 84 151 156 11029965
Linder MC 1991. Biochemistry of Copper. New York:Plenum Press.
Lowe NM Fraser WD Jackson MJ 2002 Is there a potential therapeutic value of copper and zinc for osteoporosis? Proc Nutr Soc 61 181 185 12133199
Mercer JFB Livingston J Hall B Paynter JA Begy C Chandrasekharappa S 1993 Isolation of a partial candidate gene for Menkes disease by positional cloning Nat Genet 3 20 25 8490647
Nayak SB Bhat VR Upadhyay D Udupa SL 2003 Copper and ceruloplasmin status in serum of prostate and colon cancer patients Indian J Physiol Pharmacol 47 1 108 110 12708132
Olivares M Araya M Pizarro F Uauy R 2001 Nausea threshold in apparently healthy individuals who drink fluids containing graded concentrations of copper Regul Toxicol Pharmacol 33 271 275 11407930
Olivares M Pizarro F de Pablo S Araya M Uauy R 2004 Iron, zinc and copper: contents in common Chilean foods and daily intakes in Santiago City, Chile Nutrition 20 205 212 14962688
Olusi S Al-Awadhi A Abiaka C Abraham M George S 2003 Serum copper levels and not zinc are positively associated with serum leptin concentrations in the healthy adult population Biol Trace Elem Res 91 2 137 144 12719608
Pizarro F Olivares M Araya M Gidi V Uauy R 2001 Gastrointestinal effects associated with soluble and insoluble copper in drinking water Environ Health Perspect 109 949 952 11673125
Pratt WB Omdahl JL Sorenson JR 1985 Lack of effects of copper gluconate supplementation Am J Clin Nutr 42 4 681 682 2931973
Sen PK Puri ML 1968 On a class of multivariate multisample rank order tests. II. Tests for homogeneity of dispersion matrices Sankhya 30 1 22
Tanzi RE Petrukhin K Chernov I Pellequer JL Wasco W Ross B 1993 The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene Nat Genet 5 344 350 8298641
Tanzi RE Petrukhin K Chernov I Pellequer JL Wasco W Ross B 1996 Biochemical characterization and intracellular localization of the Menkes disease protein Proc Natl Acad Sci USA 93 14030 14035 8943055
Vulpe C Levinson B Whitney S Packman S Gitschier J 1993 Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase Nat Genet 3 7 13 8490659
Wilkinson L 1996. SYSTAT 5: The System for Statistics. Evanston, IL:SYSTAT Inc.
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7168ehp0112-00165815579409ResearchArticlesSynergistic Embryotoxicity of Polycyclic Aromatic Hydrocarbon Aryl Hydrocarbon Receptor Agonists with Cytochrome P4501A Inhibitors in Fundulus heteroclitus Wassenberg Deena M. Di Giulio Richard T. Nicholas School of the Environment and Earth Sciences, Integrated Toxicology Program, Duke University, Durham, North Carolina, USAAddress correspondence to D.M. Wassenberg, Room 449 Jones Building, Research Dr., Duke University Medical Center, Durham, NC 27710 USA. Telephone: (919) 684-6744. Fax: (919) 668-1799. E-mail:
[email protected] thank L. Barber for help with fish care, M. Rocca for help with statistics, and J. Meyer for thoughtful review of the manuscript.
This research was supported by the Superfund Basic Research Programs grant P42 ES10356 and a U.S. Environmental Protection Agency STAR fellowship awarded to D.M.W.
The authors declare they have no competing financial interests.
12 2004 18 8 2004 112 17 1658 1664 12 4 2004 18 8 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Widespread contamination of aquatic systems with polycyclic aromatic hydrocarbons (PAHs) has led to concern about effects of PAHs on aquatic life. Some PAHs have been shown to cause deformities in early life stages of fish that resemble those elicited by planar halogenated aromatic hydrocarbons (pHAHs) that are agonists for the aryl hydrocarbon receptor (AHR). Previous studies have suggested that activity of cytochrome P4501A, a member of the AHR gene battery, is important to the toxicity of pHAHs, and inhibition of CYP1A can reduce the early-life-stage toxicity of pHAHs. In light of the effects of CYP1A inhibition on pHAH-derived toxicity, we explored the impact of both model and environmentally relevant CYP1A inhibitors on PAH-derived embryotoxicity. We exposed Fundulus heteroclitus embryos to two PAH-type AHR agonists, β-naphthoflavone and benzo(a)pyrene, and one pHAH-type AHR agonist, 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), alone and in combination with several CYP1A inhibitors. In agreement with previous studies, coexposure of embryos to PCB-126 with the AHR antagonist and CYP1A inhibitor α-naphthoflavone decreased frequency and severity of deformities compared with embryos exposed to PCB-126 alone. In contrast, embryos coexposed to the PAHs with each of the CYP1A inhibitors tested were deformed with increased severity and frequency compared with embryos dosed with PAH alone. The mechanism by which inhibition of CYP1A increased embryotoxicity of the PAHs tested is not understood, but these results may be helpful in elucidating mechanisms by which PAHs are embryotoxic. Additionally, these results call into question additive models of PAH embryotoxicity for environmental PAH mixtures that contain both AHR agonists and CYP1A inhibitors.
α-naphthoflavonearyl hydrocarbon receptorbenzo(a)pyreneβ-naphthoflavonecytochrome P4501AdeformityfluorantheneFundulus heteroclituspolychlorinated biphenylspolycyclic aromatic hydrocarbons
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Polycyclic aromatic hydrocarbons (PAHs) are important environmental contaminants that are generated by the incomplete combustion of organic compounds. PAHs enter the environment through natural sources such as forest fires and seeps in ocean floors and through anthropogenic activities, including combustion of fossil fuels and wood and petroleum refining (Douben 2003; Latimer and Zheng 2003). PAH contamination in estuarine settings originates from point sources such as municipal wastewater discharges, industrial outfalls, and oil shipping and refinery operations, and from non-point sources such as urban runoff and dry and wet depositions of atmospheric PAHs (Latimer and Zheng 2003). The ubiquity of PAH contamination at U.S. national priority sites (Superfund sites), along with their known and suspected human toxicity, has led to the listing of PAHs as eighth on the Agency for Toxic Substances and Disease Registry’s (ATSDR) priority list; 15 individual PAHs are also listed throughout the priority list of 275 entries (ATSDR 2003). Furthermore, environmental contamination by PAHs has steadily increased in recent years (Van Metre et al. 2000).
Some PAHs have impacts on early life stages of fish, including reduced growth, cranial–facial malformations, yolk sac and pericardial edema, and subcutaneous hemorrhaging (Billiard et al. 1999; Carls et al. 1999; Hawkins et al. 2002). These deformities closely resemble the “blue sac syndrome” that has been described in several fish species, including rainbow trout (Oncorhynchus mykiss), zebrafish (Danio rerio), medaka (Oryzias latipes), and killifish (Fundulus heteroclitus), exposed to certain halogenated aromatic compounds that are agonists for the aryl hydrocarbon receptor (AHR) (Chen and Cooper 1999; Elonen et al. 1998; Helder 1981; Toomey et al. 2001; Walker and Peterson 1991; Wannemacher et al. 1992). These compounds include coplanar polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), collectively referred to here as planar halogenated aromatic hydrocarbons (pHAHs). Some of the PAHs that induce these deformities are, like TCDD and coplanar PCBs, agonists for the AHR (Billiard et al. 2002).
The AHR is a cytoplasmic receptor whose activation initiates the transcription of a battery of genes, including the monooxygenase cytochrome P4501A (here generally referred to as CYP1A, although two CYP1As exist in mammals as well as in rainbow trout; mammalian CYP1As are referred to as CYP1A1 and CYP1A2; Hankinson 1995). The AHR pathway is similar between mammals and nonmammalian vertebrates, including fish, reptiles, and birds (Hahn 1998); however, two AHRs (AHR1 and AHR2) have been identified and characterized in several fish species, including killifish and zebrafish (Andreasen et al. 2002; Hahn et al. 1997; Karchner et al. 1999). The mechanism for the toxicity of pHAHs has been widely studied, and there are well-established positive relationships among compounds’ affinity for the AHR, their potency for CYP1A induction, and their toxicity (Guiney et al. 1997; Heid et al. 2001; Safe 1990, 1993).
The critical role of AHR in pHAH toxicity has been demonstrated by AHR knockout studies in which AHR knockout mice do not show typical dioxin-induced toxicity compared with their AHR-expressing littermates (Fernandez-Salguero et al. 1996). There is evidence that some of the toxicity of these pHAHs may be directly due to CYP1A activity; for example, CYP1A2 knockout mice are resistant to liver damage and uroporphyria when exposed to TCDD (Smith et al. 2001). And male CYP1A1 knockout mice are protected against TCDD-mediated lethality and wasting syndrome (Uno et al. 2004b). Furthermore, Cantrell et al. (1996) were able to reduce TCDD-induced DNA degradation and damage to the medial yolk vein in medaka by cotreating the embryos with the P450 inhibitor piperonyl butoxide (PBO). Dong et al. (2002) found that cotreatment of zebrafish embryos with the partial AHR antagonist and CYP1A inhibitor α-naphthoflavone (ANF) or the P450 inhibitors SKF525A or miconazole reversed the reduction of blood flow in the mesencephalic vein and midbrain apoptosis caused by TCDD. Another study by Teraoka et al. (2003) showed that a morpholino knockdown of CYP1A and AHR2 in zebrafish prevented the pericardial edema and trunk circulation failure caused by TCDD.
Although there is a strong, positive relationship between the ability of PAHs to bind the AHR and their induction of CYP1A (Billiard et al. 2002), conclusions regarding the role of the AHR pathway and CYP1A activity in the toxicity of PAHs have been less clear. In a mammalian study, homozygous CYP1A1 knockout mice showed less liver damage and survived the acute effects of injection of the PAH benzo(a)pyrene (BaP) for 3 days longer than did those that were heterozygous for CYP1A1 (Uno et al. 2001). However, these CYP1A1 knockout mice also showed 4-fold higher levels of BaP–DNA adducts than did those heterozygous for CYP1A1. This study suggests that acute lethality of BaP was reduced by lack of CYP1A1 but that genotoxicity was actually increased by the lack of CYP1A1 (Uno et al. 2001). In a recent study by this group, BaP administered in the diet caused lethality in CYP1A1 knockout mice at a dose that was not lethal to CYP1A1-expressing mice (Uno et al. 2004a). These authors suggested that rather than CYP1A1 activity enhancing the toxicity of BaP, as has been previously suggested, CYP1A1 is critical for the detoxication of orally administered BaP in mice.
Billiard (2002) compared a variety of PAHs with various affinities for the AHR and potencies for CYP1A induction in juvenile rainbow trout; chemicals ranged from the strong CYP1A inducer benzo(k)fluoranthene, to the relatively weak, alkylated inducer retene and the noninducer phenanthrene. Billiard (2002) found that the rank order for CYP1A induction in these fish did not predict the rank order for the induction of blue-sac-like symptoms; in fact, the only PAHs that caused blue-sac-like symptoms were retene and phenanthrene, the low- and non-inducing PAHs used in that study. Hawkins et al. (2002) observed apparent additive toxicity in juvenile and larval rainbow trout coexposed to one of two PAHs, the alkylated AHR agonist retene or the non-AHR-agonist phenanthrene, with the P450 inhibitor PBO. In contrast, another study found that cotreatment with the partial AHR antagonist and CYP1A inhibitor ANF prevented the reduction of circulation in the dorsal midbrain of zebrafish caused by the PAH-type AHR agonist β-naphthoflavone (BNF; Dong et al. 2002). From these studies, it is clear that the relationship between CYP1A activity and PAH toxicity is complex and that reduced CYP1A activity is sometimes, but not always, protective of PAH toxicity.
In an attempt to a) clarify the role of CYP1A activity in the toxicity of PAHs and b) explore the possible effects of co-occurring PAH-type CYP1A inducers and inhibitors, we cotreated Fundulus heteroclitus (killifish) embryos with three different AHR agonists [the pHAH 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126) and the PAHs BNF and BaP] and four CYP1A inhibitors that work by various mechanisms (Table 1). The compounds here collectively referred to as CYP1A inhibitors have all been shown to inhibit CYP1A activity (see references in Table 1); however, the specificities of these CYP1A inhibitors for CYP1A over other P450s in our system are not known. These inhibitors included the aforementioned model compounds ANF and PBO and the environmentally relevant hydrocarbons fluoranthene (FL) and 2-aminoanthracene (AA) (Watson et al. 1995; Willett et al. 1998, 2001). We then observed embryos for in ovo CYP1A activity, as measured by ethoxyresorufin-O-deethylase (EROD) activity, and for deformities, including pericardial edema, heart elongation, cranial–facial malformations, and tail abnormalities. In these experiments, we used a wide range of concentrations of AHR agonists to elicit a range of EROD inductions with and without inducing deformities; concentrations of inhibitors were selected with the goal of eliciting the maximal inhibition of EROD without inducing deformities. Our results indicate that coexposure to PAH-type AHR agonists and CYP1A inhibitors consistently enhanced embryotoxicity beyond levels predicted by an additive toxicity model.
Materials and Methods
Reagents.
BaP, BNF, ANF, FL, AA, PBO, and ethoxyresorufin were purchased from Sigma Aldrich (Saint Louis, MO). PCB-126 was purchased from Chem Service (West Chester, PA). Dimethyl sulfoxide (DMSO) and acetone were purchased from Mallinckrodt Baker (Phillipsburg, NJ).
Fish care.
Adult killifish were captured with minnow traps from King’s Creek, Virginia (a well-characterized reference site with low sediment PAH levels; Mulvey et al. 2002) and transported to the Ecotoxicology Laboratory of Duke University. Fish were maintained in 70-L or 100-L aquaria at 24°C with a 16-hr light/8-hr dark cycle and were fed TetraMin flakes (Tetra Sales, Blacksburg, VA) ad libitum. Fish were held in laboratory conditions for at least 3 weeks before embryo acquisition. Embryos were obtained from in vitro fertilization of pooled oocytes stripped from 9–12 females with pooled milt from 4–5 males.
In ovo EROD.
We used an in ovo EROD method, modified slightly from the method described by Nacci et al. (1998, in press), to measure the CYP1A activity of embryos. Several hours after fertilization, embryos with dividing cells were selected and placed individually in 20-mL scintillation vials with 10 mL artificial seawater (20 parts per thousand; Instant Ocean, Mentor, OH) containing 21 μg/L ethoxyresorufin with or without an EROD inducer (BNF, BaP, or PCB-126) and/or an EROD inhibitor (ANF, AA, FL, or PBO). We used either acetone or DMSO as the solvent, and solvent concentrations were < 0.015% for all treatments except the high doses in the ANF-alone dose group (Figure 1), in which solvent concentrations were ≤0.1%. Embryos were in dosing solution for 7 days, during which resorufin, the fluorescent product of CYP1A metabolism of ethoxyresorufin, accumulated in the embryos’ bi-lobed urinary bladders. On day 7 of development, embryos were placed in clean artificial seawater, and embryo bladders were visualized by fluorescent microscopy (50× magnification using rhodamine red filter set; Axioskop; Zeiss, Thornwood, NY). EROD activity was measured as intensity of the bladder fluorescence and was quantified digitally by IPLab software (Scanalytics Inc., Fairfax, VA). In ovo EROD values were expressed as a percentage of control intensity. Individuals with deformed bladders or with fluorescence in areas other than the bladder (e.g., the pericardial sac in some embryos with severe pericardial edema) were excluded from in ovo EROD measurement. Although ethoxyresorufin has been shown to be nondetrimental to embryos (Nacci et al. 1998), coexposures of ANF and BNF were done with and without ethoxyresorufin to rule out a possible interactive effect of the ethoxyresorufin. No differences were observed between the deformities of embryos with or without ethoxyresorufin (data not shown).
Deformity assessment.
Embryos were scored blind for heart elongation (tube heart), pericardial edema, tail shortening, and hemorrhaging on day 10 of development. Heart deformities were found to be the most sensitive end point scored, so this end point was used for further analysis. Heart elongation severity was ranked between 0 and 5, and a deformity index for each treatment was calculated as sum of scores for individuals in that treatment group divided by the maximum score possible (the number of individuals multiplied by 5). This quotient was then multiplied by 100.
Experimental approach.
Embryos were exposed to nominal concentrations of one of three AHR agonists alone and in combination with nominal concentrations of one of four CYP1A inhibitors. We used the AHR agonists PCB-126, BNF, and BaP (Table 1). BNF and BaP were chosen as model PAH-type AHR agonists. BNF is a synthetic compound, commonly used as a model AHR agonist in studies, whereas BaP is a naturally occurring PAH, commonly found in environmental mixtures. We chose PCB-126 as a model pHAH-type AHR agonist.
We used the inhibitors ANF, PBO, FL, and AA in this study; their mechanisms of actions are listed in Table 1. We chose ANF because it is well characterized for its activities as both a partial AHR antagonist (Merchant et al. 1990, 1992) and a competitive CYP1A inhibitor (Goujon et al. 1972; Testa and Jenner 1981). BNF and ANF dose–response curves were first established using a range of concentrations and scoring for deformities and in ovo EROD (Figure 1). Subsequently, coexposures were performed using a range of BNF concentrations that spanned concentrations found to induce EROD, but not deformities, to concentrations that caused both EROD induction and deformities, with a concentration of ANF (100 μg/L) that dramatically lowered in ovo EROD measurements but did not by itself cause deformities (Figure 2).
In order to distinguish between the effects of AHR antagonism and CYP1A inhibitory effects, both of which occur with ANF exposure, we also used the P450 inhibitor PBO. PBO is a quasi-irreversible P450 inhibitor that acts by forming a metabolic intermediate complex with the heme group of P450 enzymes, thereby preventing the redox cycling of the enzyme (Hodgson and Philpot 1974; Testa and Jenner 1981). We cotreated embryos with a range of BNF concentrations (1–100 μg/L) and either 1 or 9 mg/L PBO (Figure 3).
To test the effects of EROD inhibition on embryos coexposed to an environmentally relevant AHR agonist, BaP and ANF coexposures were conducted. In this experiment the ANF concentration was 100 μg/L, a concentration previously established as effective at lowering in ovo EROD without inducing deformities. BaP concentrations ranged from 1 to 100 μg/L (Figure 4).
To test the effectiveness of environmentally relevant PAHs at inhibition of in ovo EROD and to determine how inhibition by these compounds affected deformities, embryos were exposed to a range of FL and AA concentrations alone and with 1 μg/L BNF (Figure 5).
In order to assess interactions between a representative pHAH and a CYP1A inhibitor in killifish, embryos were exposed to concentrations of PCB-126 that spanned from concentrations known to induce EROD that cause low-deformity indices, to concentrations that induce severe deformities, with and without 100 μg/L ANF (Figure 6).
Data analysis and representation.
Data were analyzed using Statview for Windows (Version 5.0.1; SAS Institute Inc., Cary, NC). EROD values were analyzed by one- and two-way analysis of variance (ANOVA). When ANOVA yielded significance (p < 0.05), Fisher’s protected least-significant differences was used as a post hoc test. Deformity data were ordinal in nature and were therefore assessed using rank order tests—the Mann-Whitney U-test for analyses with two variables and the Kruskal-Wallis test for analyses with three or more variables. p-Values corrected for ties in rank are reported for these analyses as “tied p-values.” Each graph represents a separate experiment. Although deformities were analyzed statistically using individual severity rankings, deformity data are shown as a deformity index for clarity. Interactions were characterized as synergistic based on significance of a one-group chi square analysis comparing the observed frequencies of deformities with frequencies predicted by an additive interaction (calculated as a sum of the deformity frequency for each treatment; predicted frequency had minimum value of 1 for this analysis because chi square calculation requires predicted frequency in the denominator of an equation).
Results
Embryos dosed with BNF alone showed in ovo EROD induction at all concentrations tested (p ≤0.0002) that was maximal at the 10 μg/L concentration (Figure 1A). At 50 and 100 μg/L, EROD activities declined to below the maximal level (p = 0.0001 and 0.0003, respectively). Coincident with this decline, embryos exposed to 50 and 100 μg/L BNF exhibited elevated deformity indices (effect of BNF on deformities, tied p < 0.0001). Embryos exposed to 10, 100, and 500 μg/L ANF alone displayed lower EROD activities than controls (Figure 1B; p < 0.0001). Embryos exposed to ANF levels > 500 μg/L were too deformed to allow for measurement of in ovo EROD. Embryos exposed to 10 μg/L ANF and 100 μg/L ANF exhibited no deformities, whereas those exposed to ≥500 μg/L ANF exhibited high deformity indices (effect of ANF on deformities, tied p < 0.0001).
In a separate experiment designed to explore the interaction between ANF and BNF coexposures, embryos were dosed with a range of BNF concentrations with or without 100 μg/L ANF (Figure 2), the dose of ANF shown to be most effective in inhibiting EROD without causing deformities by itself (Figure 1). Embryos exposed to BNF alone exhibited significant EROD induction at all concentrations (p < 0.0001). Cotreatment with ANF significantly inhibited in ovo EROD activities (p < 0.0001). Embryos cotreated with ANF and 110 μg/L BNF were too deformed for in ovo EROD measurements. In embryos treated with BNF alone, deformities were noted only at the 110 μg/L concentration (effect of BNF alone on deformities, tied p = 0.0011). However, ANF-cotreated embryos were deformed at all BNF concentrations. That is, embryos were deformed at BNF concentrations three orders of magnitude lower when BNF treatment was combined with 100 μg/L ANF than when treated with BNF alone (overall effect of BNF and ANF on deformities, tied p < 0.0001 for each).
In an experiment exploring the effect of cotreatment of embryos with BNF and PBO (Figure 3), all BNF concentrations significantly induced in ovo EROD activities (p < 0.0001). Cotreatment with both concentrations of PBO (1 and 9 mg/L) lowered in ovo EROD across all BNF concentrations (p < 0.0001). Embryos exposed to PBO at the low concentration had very low deformities that were not statistically different from controls (tied p = 0.3173). Embryos exposed to the high concentration of PBO had an elevated deformity index (effect of PBO alone on deformities, tied p = 0.0448). Coexposures to BNF and PBO caused increased deformity indices over those seen in embryos dosed with BNF alone or PBO alone at all BNF concentrations (overall effect of BNF and PBO on deformities, tied p < 0.0001 and = 0.0021 respectively).
We also examined a range of concentrations (1–100 μg/L) of BaP, an environmentally relevant PAH, with and without coexposure to 100 μg/L ANF (Figure 4). BaP alone significantly induced EROD at all doses tested (p < 0.0001), and ANF cotreatment lowered the in ovo EROD activity (p < 0.0001). Embryos dosed with BaP alone exhibited low deformity indices that were not statistically different from controls (effect of BaP alone on deformities, p = 0.1856), whereas those dosed with BaP in combination with 100 μg/L ANF had elevated deformity indices at all BaP concentrations tested (overall effect of ANF on deformities, tied p < 0.0001).
Exposure to environmentally relevant CYP1A inhibitor FL by itself caused in ovo EROD activities below control levels (p < 0.0001; Figure 5); however, when embryos were coexposed to FL with 1 μg/L of the inducer BNF, EROD activities were induced (p < 0.0001) and there was an FL-dose–dependent decrease in in ovo EROD activities (p < 0.0001). Embryos exposed to FL alone did not exhibit elevated deformity indices (tied p = 0.3764); BNF at this concentration also did not cause an elevated deformity index (effect of BNF alone on deformities, tied p = 0.1681). However, when FL exposure was combined with 1 μg/L BNF, high deformity indices were observed at FL levels of ≥50 μg/L (overall effect of FL on deformities, tied p = 0.0002; overall effect of BNF on deformities, tied p < 0.0001).
Exposure to AA alone elicited slight EROD induction at the 10 and 50 μg/L concentrations (p < 0.0001 and p = 0.0163, respectively; Figure 5); however, when embryos were coexposed to 1 μg/L BNF, the BNF-mediated EROD induction was inhibited in a dose-dependent fashion by increasing AA concentrations (p < 0.0001). Embryos dosed with AA alone exhibited low deformity indices (not significant, tied p = 0.6609), but when embryos were coexposed to AA with 1 μg/L BNF, deformity indices were elevated in cotreatments of BNF with AA concentrations of ≥50 μg/L (overall effect of AA and BNF on deformities, tied p < 0.0001 for each).
The pHAH PCB-126 significantly induced in ovo EROD over controls at all doses tested (p < 0.0001; Figure 6). Concentrations of 300 and 600 ng/L induced EROD levels less than the maximal levels achieved by 30 and 100 ng/L (p < 0.0001 for each). Deformity indices were elevated in embryos exposed to PCB-126 concentrations of ≥100 ng/L (effect of PCB-126 alone on deformities, tied p > 0.0001). In the case of PCB-126, however, coexposure with 100 μg/L ANF dramatically decreased the deformity indices of PCB-126 treatment groups (overall effect of PCB-126 and ANF on deformities, tied p < 0.0001 and = 0.0003, respectively).
Synergistic interactions, determined by one-group chi square analyses, yielded deformity frequencies greater than predicted additive values for cotreatments with BNF + ANF, BaP + ANF, BNF + FL, and BNF + AA (p < 0.001 for each; Figures 2, 4, and 5, respectively). The interaction for BNF + 1 mg/L PBO cotreatment approached significance (p = 0.051); however, BNF + 9 mg/L PBO cotreatment was not synergistic (Figure 3).
Discussion
The results of this study demonstrate that the embryotoxicity of the pHAH PCB-126 was decreased with coexposure to the CYP1A inhibitor and AHR antagonist ANF. This result is in general agreement with other studies showing the reduction of early-life-stage toxicity of pHAHs when CYP1A activity or AHR-mediated signaling was decreased (Cantrell et al. 1996; Dong et al. 2002; Teraoka et al. 2003). In contrast, in the present study the embryotoxicities of two PAH-type AHR agonists were increased when CYP1A was inhibited by chemicals that act by various modes of action. The data for the interactions between the PAH-type inducers and inhibitors clearly indicate a synergistic effect on embryotoxicity for coexposures to BNF + ANF, BaP + ANF, BNF + FL, and BNF + AA. The BNF + 1 mg/L PBO dose was nearly significant for synergism (p = 0.051).
The various inhibitors used in this study caused similar increases in PAH toxicity, although these inhibitors varied in structure and mechanism of inhibition. This suggests that the increased toxicity of PAHs by CYP1A inhibitors is due to the shared characteristic of CYP1A inhibition and is not specific for a particular structure or mechanism of inhibition. The PAH interactions with CYP1A inhibitors observed in this study are in general agreement with those found in a previous study in which we showed that an extract from a site highly contaminated with PAHs was more toxic when coexposed with several CYP1A inhibitors (Wassenberg and Di Giulio 2004).
Although the pHAH PCB-126 and the PAHs BNF and BaP share the characteristic of being AHR agonists, the difference between the effect of CYP1A inhibition in the pHAH-versus the PAH-dosed embryos is striking. This difference may be due to the fundamentally different chemistries and somewhat different toxicities of these two classes of compounds. PCBs and other halogenated compounds are relatively stable, long-lived compounds. Although pHAHs induce mono-oxygenases such as CYP1A, metabolism of these compounds is relatively slow (White et al. 1997). The half-life of PCB-126 administered to juvenile rainbow trout in their diet was found to be between 82 and 180 days (Brown et al. 2002). In contrast, PAHs are rapidly metabolized. Half-lives of nine PAHs orally administered to adult rainbow trout were estimated to be ≤9 days (Niimi and Palazzo 1986). In vitro metabolism of BaP was found to be 2,000–4,000 times faster than metabolism of the coplanar pHAH PCB-77 in induced scup (Stenotomus chrysops) microsomes (Stegeman et al. 1981; White et al. 1997). This rapid metabolism of PAHs allows for more rapid excretion of the compound but can also activate PAHs into more reactive intermediates that can bind to and damage cellular constituents. Studies of PAH metabolism by fish embryos are very limited. However, Fong et al. (1993) demonstrated extensive phase 1 and phase 2 metabolism of 7,12-dimethylbenz(a)anthracene by rainbow trout embryos. Additionally, the presence and inducibility of CYP1A in killifish embryos observed in this and previous studies (Meyer et al. 2002; Nacci et al. 1998; Toomey et al. 2001) support the hypothesis that PAH metabolism is occurring in embryos in the present study. Therefore, it is possible that inhibition of CYP1A in the PAH-treated embryos extended the half-life of the PAH, causing prolonged AHR agonism, similar to AHR agonism in pHAH-treated animals.
Some PAHs act through a narcotic mechanism in which the compounds accumulate in tissues to a level at which they physically interfere with membranes (McCarty and Mackay 1993). The inclusion of a CYP1A inhibitor with PAHs would be likely to slow metabolism of the PAHs. However, it is not likely that narcosis is responsible for the synergy observed in these experiments. First, even if the total amount of compound to which the embryos were exposed in deformed treatment groups accumulated within the embryo, the concentration of PAH would not reach the 2–8 mmol/kg threshold for acute narcosis (McCarty and Mackay 1993). Second, narcotic modes of action are, by definition, additive, and an additive model of toxicity does not fit our data.
It has been suggested that the toxicity of pHAHs is at least in part tied to an oxidative stress mode of damage (Nebert et al. 2000; Stohs 1990). The pHAHs fit into the active site for CYP1A but are poor substrates for CYP1A metabolism, causing an uncoupling of electron flow between the enzyme and the substrate. This uncoupling, together with increased expression of CYP1A via the AHR, is believed to lead to the production of reactive oxygen and oxidative damage (Schlezinger and Stegeman 2001; Schlezinger et al. 1999; Shertzer et al. 2004). We included PBO as an inhibitor in our studies because it binds to the heme group of P450s, thereby inhibiting electron flow from the enzyme and preventing this uncoupling. Because PBO enhanced toxicity in PAH-cotreated embryos, P450 uncoupling is not supported as the mechanism underlying the interactive toxicity of PAHs and CYP1A inhibitors observed in this study.
However, other mechanisms of oxidative stress may play a role in PAH-driven toxicity. An oxidative stress mechanism for the toxicity of the alkylated PAH retene has been proposed based on reduced ratios of glutathione to glutathione disulfide (GSH:GSSG) in rainbow trout larvae at retene exposures that exhibited blue-sac-like symptoms (Billiard 2002). Many PAHs (including BaP) can be metabolized to quinones (Bolton et al. 2000). These reactive metabolic intermediates are capable of further AHR agonism, redox cycling, and generation of reactive oxygen species, which can then perturb cellular redox status and damage macromolecules and are cytotoxic and mutagenic (Bolton et al. 2000; Burezynski and Penning 2000). The metabolism of PAHs to reactive compounds is clearly associated with their genotoxicity and carcinogenicity (Levin et al. 1982; Sjögren et al. 1996). Inhibition of CYP1A would likely alter the metabolism of PAHs, possibly generating more embryotoxic intermediates. However, the extent to which altered metabolism affected the PAH toxicity observed in this study is not known. Current studies are addressing mechanisms underlying the interactive toxicities reported herein.
Importance of findings.
PAH contamination levels are increasing in aquatic systems across the United States (Van Metre et al. 2000). Sites with PAH mixtures generally contain agonists for the AHR that can induce CYP1A activity, such as BaP, chrysene, and benzo(k)fluoranthene. These mixtures may also contain compounds that can act as CYP1A inhibitors. The noncompetitive CYP1A inhibitor FL, for example, is one of the more prevalent PAHs found in marine sediments, lakes, and rainwater (Latimer and Zheng 2003; Van Metre et al. 2000). Aminoanthracenes are components in coal liquefaction products (Pelroy and Wilson 1981; Wilson 1980) and may also be found in environmental mixtures. It is possible that other compounds found in environmental mixtures may also be as yet uncharacterized CYP1A inhibitors. The synergisms found in this study indicate that compounds such as BaP, FL, and AA, which can be commonly found in environmental mixtures, may be substantially more toxic in their mixtures than an additive approach to PAH toxicity would predict, and that additive models currently used to estimate PAH toxicity (e.g., Barron et al. 2004; Di Toro et al. 2000) may underestimate the toxicity of PAH mixtures. Additionally, the observed end point for this synergy was cardiovascular development during early development, a sensitive life stage for vertebrates in general.
Figure 1 Dose–response curves showing percent control in ovo EROD induction and deformity index in embryos exposed to (A) BNF or (B) ANF. EROD values are missing for the 1,000, 5,000, and 10,000 μg/L concentrations because embryos from these treatment groups were too deformed to score for in ovo EROD. For the BNF control group, n = 20; for all other BNF treatments, n = 9 or 10. For each ANF treatment group, n = 8–10. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.
Figure 2 Effects of BNF with and without 100 μg/L ANF cotreatment on in ovo EROD and deformity index. The EROD value is missing for the 110 μg/L BNF + ANF treatment group because embryos in this treatment group were too deformed to score for in ovo EROD; n = 8 or 9 for each treatment group, except for EROD measurement in the 1.1 μg/L BNF + ANF (n = 6) and 11 μg/L BNF + ANF (n = 2) treatment groups, because the remainder of embryos were too deformed to score for in ovo EROD. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.
Figure 3 Effects of BNF with and without 1 or 9 mg/L PBO cotreatment on in ovo EROD and deformity index; n = 7–10 for each treatment group, except for EROD measurements in the 50 μg/L BNF + 9 mg/L PBO (n = 5) and 100 μg/L BNF + PBO (n = 6) treatment groups, because the remainder of embryos were too deformed to score for in ovo EROD. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.
Figure 4 Effects of BaP with and without 100 μg/L ANF cotreatment on in ovo EROD and deformity index; n = 9 or 10 for each treatment group, except for EROD measurement in the 5 μg/L BaP + ANF (n = 7), 10 μg/L BaP + ANF (n = 7), and 100 μg/L BaP + ANF (n = 3) treatment groups, because the remainder of embryos were too deformed to score for in ovo EROD. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.
Figure 5 Effects of FL (A) and AA (B) with and without 1 μg/L BNF cotreatment on in ovo EROD and deformity index. For (A), n = 9 or 10 for each treatment group, except for EROD measurement in the 1 μg/L BNF + 1,000 μg/L FL treatment group (n = 4), because the remainder of embryos were too deformed to score for in ovo EROD. For (B), n = 16 and 19 for control and BNF-alone treatment groups, respectively; for other treatment groups, n = 6–10. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.
Figure 6 Effects of PCB-126 with and without 100 μg/L ANF cotreatment on in ovo EROD and deformity index; n = 9 or 10 for all treatment groups. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.
Table 1 AHR agonists and CYP1A inhibitors used in this study.
Compound Type Structure Mechanism of action Sample references
AHR agonists
BNF Synthetically derived model PAH Matsuda et al. 1995; Ronisz and Förlin 1998
BaP Environmentally relevant PAH Chaloupka et al. 1993; Fent and Bätscher 2000; Van Veld et al. 1997
PCB-126 Environmentally relevant pHAH Abnet et al. 1999; Dabrowska et al. 2000
CYP1A inhibitors
ANF Synthetically derived model PAH Partial AHR antagonist and competitive inhibitor of CYP1A Goujon et al. 1972; Lu et al. 1996; Merchant et al. 1990, 1992, 1993; Merchant and Safe 1995; Miranda et al.1998; Testa and Jenner 1981
PBO Methlenedioxybenzene derivative P450 inhibitor; forms a metabolic intermediate with heme group of P450 Hodgson and Philpot 1974; Miranda et al. 1998; Murray and Reidy 1990; Testa and Jenner 1981
FL Environmentally relevant PAH Competitive inhibitor of CYP1A in vitro; modestly lowers CYP1A protein expression in vivo Willett et al. 1998, 2001
AA Environmentally relevant aromatic amine Mechanism-based CYP1A inhibitor; binds to CYP1A and causes its degradation Watson et al. 1995
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References
Abnet CC Tanguay RL Heideman W Peterson RE 1999 Transactivation activity of human, zebrafish and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-p -dioxin, dibenzofuran and biphenyl congeners Toxicol Appl Pharmacol 159 41 51 10448124
Andreasen EA Hahn ME Heideman W Peterson RE Tanguay RL 2002 The zebrafish (Danio rerio ) aryl hydrocarbon receptor type 1 is a novel vertebrate receptor Mol Pharmacol 62 234 249 12130674
ATSDR 2003. 2003 CERCLA Priority List. Atlanta, GA:U.S. Agency for Toxic Substances and Disease Registry. Available: http://www.atsdr.cdc.gov/clist.html [accessed 15 May 2004].
Barron MG Carls MG Heintz R Rice SD 2004 Evaluation of fish early life stage toxicity models of chronic embryonic exposures to complex polycyclic aromatic hydrocarbon mixtures Toxicol Sci 78 60 67 14691206
Billiard SM 2002. Potency of Polycyclic Aromatic Hydrocarbons (PAHs) for Causing Early Life Stage Toxicity in Fish: A Quantitative Structure Activity (QSAR) Approach [PhD Thesis]. Kingston, Ontario, Canada:Queens University.
Billiard SM Hahn ME Franks DG Peterson RE Bols NC Hodson PV 2002 Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs) Comp Biochem Physiol B Comp Biochem 133 55 68
Billiard SM Querbach K Hodson PV 1999 Toxicity of retene to early life stages of two freshwater fish species Environ Toxicol Chem 18 2070 2077
Bolton JL Trush MA Penning TM Dryhurst G Monks TJ 2000 Role of quinones in toxicology Chem Res Toxicol 13 135 160 10725110
Brown SB Fisk AT Brown M Villella M Muir DCG Evans RE 2002 Dietary accumulation and biochemical responses of juvenile rainbow trout (Oncorhynchus mykiss ) to 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126) Aquat Toxicol 59 139 152 12127732
Burezynski ME Penning TM 2000 Genotoxic polycyclic aromatic hydrocarbon ortho -quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor Cancer Res 60 908 915 10706104
Cantrell SM Lutz LH Tillitt DE Hannink M 1996 Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD): the embryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in medaka (Orizias latipes ) Toxicol Appl Pharmacol 141 23 34 8917672
Carls MG Rice SD Hose JE 1999 Sensitivity of fish embryos to weathered crude oil: part I. Low-level exposure during incubation causes malformations, genetic damage, and mortality in larval pacific herring (Clupea pallasi ) Environ Toxicol Chem 18 481 493
Chaloupka K Harper N Krishnan V Santostefano M Rodriguez LV Safe S 1993 Synergistic activity of poly-nuclear aromatic hydrocarbon mixtures as aryl hydrocarbon (Ah) receptor agonists Chem Biol Interact 89 141 158 8269543
Chen CM Cooper KR 1999 Developmental toxicity and EROD induction in the Japanese medaka treated with dioxin congeners Bull Environ Contam Toxicol 63 423 429 10501717
Dabrowska H Fisher SW Ciereszko R Dabrowski K Woodin BR Stegeman JJ 2000 Hepatic P4501A activity, plasma sex steroids, and gonadal steroidogenesis in vitro in yellow perch exposed to 3,3’,4,4’,5-pentachlorobiphenyl Environ Toxicol Chem 19 3052 3060
Di Toro DM McGrath JA Hansen DJ 2000 Technical basis for narcotic chemicals and polycyclic aromatic hydrocarbon criteria. I. Water and tissue Environ Toxicol Chem 19 1951 1970
Dong W Teraoka H Yamazaki K Tsukiyama S Imani S Imagawa T 2002 2,3,7,8-Tetrachlorodibenzo-p -dioxin toxicity in the zebrafish embryo: local circulation failure in the dorsal midbrain is associated with increased apoptosis Toxicol Sci 69 191 201 12215674
Douben PET 2003. Introduction. In: PAHs: An Ecotoxicological Perspective (Douben PET, ed). West Sussex, UK:John Wiley & Sons, 3–6.
Elonen GE Spehar RL Holcombe GW Johnson RD Fernandez JD Erickson RJ 1998 Comparitive toxicity of 2,3,7,8-tetrachlorodibenzo-p -dioxin to seven freshwater fish species during early life-stage development Environ Toxicol Chem 17 472 483
Fent K Bätscher R 2000 Cytochrome P4501A induction potencies of polycyclic aromatic hydrocarbons in a fish hepatoma cell line: demonstration of additive interactions Environ Toxicol Chem 19 2047 2058
Fernandez-Salguero PM Hilbert DM Rudikoff S Ward JM Gonzalez FJ 1996 Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p -dioxin-induced toxicity Toxicol Appl Pharmacol 140 173 179 8806883
Fong AT Dashwood RH Cheng R Mathews C Ford B Hendricks JD 1993 Carcinogeneicity, metabolism and Ki-ras proto-oncogene activation by 7,12-dimethyl-benz[a]anthracene in rainbow trout embryos Carcinogenesis 14 629 635 8472326
Goujon FM Nebert DW Gielen JE 1972 Genetic expression of aryl hydrocarbon hydroxylase induction. IV. Interaction of various compounds with different forms of cytochrome P-450 and the effect on benzo[a]pyrene metabolism in vitro Mol Pharmacol 8 667 680 4118365
Guiney PD Smolowitz RM Peterson RE Stegeman JJ 1997 Correlation of 2,3,7,8-tetrachlorodibenzo-p -dioxin induction of cytochrome P4501A in vascular endothelium with toxicity in early life stages of lake trout Toxicol Appl Pharmacol 143 256 273 9144443
Hahn ME 1998 The aryl hydrocarbon receptor: a comparative perspective Comp Biochem Physiol 121 23 53
Hahn ME Karchner SI Shapiro MA Perera SA 1997 Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family Proc Natl Acad Sci USA 94 13743 13748 9391097
Hankinson O 1995 The aryl hydrocarbon receptor complex Annu Rev Pharmacol Toxicol 35 307 340 7598497
Hawkins SA Billiard SM Tabash SP Brown RS Hodson PV 2002 Altering cytochrome P4501A activity affects polycyclic aromatic hydrocarbon metabolism and toxicity in rainbow trout (Oncorhynchus mykiss ) Environ Toxicol Chem 21 1845 1853 12206424
Heid SE Walker MK Swanson HI 2001 Correlation of cardio-toxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation Toxicol Sci 61 187 196 11294989
Helder T 1981 Effects of 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) on early life stages of rainbow trout (Salmo gairdneri , Richardson) Toxicology 19 101 112 6267736
Hodgson E Philpot RM 1974 Interaction of methylene-dioxyphenyl (1,3-benzodioxole) compounds with enzymes and their effects on mammals Drug Metab Rev 3 231 301 4423562
Karchner SI Powell WH Hahn ME 1999 Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the teleost Fundulus heteroclitus J Biol Chem 274 33814 33824 10559277
Latimer JS Zheng J 2003. The sources, transport, and fate of PAHs in the marine environment. In: PAHs: An Ecotoxicological Perspective (Douben PET, ed). West Sussex, UK:John Wiley & Sons, 9–33.
Levin W Wood A Chang R Ryan D Thomas P Yagi H 1982 Oxidative metabolism of polycyclic aromatic hydrocarbons to ultimate carcinogens Drug Metab Rev 13 555 580 6290166
Lu Y-F Santostefano M Cunningham BDM Threadgill MD Safe S 1996 Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists Biochem Pharmacol 51 1077 1087 8866830
Matsuda T Imaoka S Funae Y Otori K Fukushima S 1995 Induction of CYP isoenzymes in various organs of rats by 3-methylcholanthrene or β-naphthoflavone Cancer Lett 97 137 143 7497454
McCarty LS Mackay D 1993 Enhancing ecotoxicological modeling and assessment: body residues and modes of toxic action Environ SciTechnol 27 1718 1728
Merchant M Arellano L Safe S 1990 The mechanism of action of α-naphthoflavone as an inhibitor of 2,3,7,8-tetra-chlorodibenzo-p -dioxin-induced CYP1A1 gene expression Arch Biochem Biophys 281 84 89 2166479
Merchant M Krishnan V Safe S 1993 Mechanism of action of α-naphthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells Toxicol Appl Pharmacol 120 179 185 8390116
Merchant M Morrison V Santostefano M Safe S 1992 Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p -dioxin-induced CYP1A1 gene expression Arch Biochem Biophys 298 389 394 1329656
Merchant M Safe S 1995 In vitro inhibition of 2,3,7,8-tetra-chlorodibenzo-p -dioxin-induced activity by α-naphthoflavone and 6-methyl-1,3,8-trichlorodibenzofuran using an aryl hydrocarbon (Ah)-responsive construct Biochem Pharmacol 50 663 668 7669069
Meyer JN Nacci DE Di Giulio RT 2002 Cytochrome P4501A (CYP1A) in killifish (Fundulus heteroclitus ): heritability of altered expression and relationship to survival in contaminated sediments Toxicol Sci 68 69 81 12075112
Miranda CL Henderson MC Buhler DR 1998 Evaluation of chemicals as inhibitors of trout cytochrome P450s Toxicol Appl Pharmacol 148 237 244 9473531
Mulvey M Newman MC Vogelbein W Unger MA 2002 Genetic structure of Fundulus heteroclitus from PAH-contaminated and neighboring sites in the Elizabeth and York rivers Aquat Toxicol 61 195 209 12359390
Murray M Reidy GF 1990 Selectivity in the inhibition of mammalian cytochromes P-450 by chemical agents Pharmacol Rev 42 85 101 2198606
Nacci D Coiro L Kuhn A Champlin D Munns W Jr Specker J 1998 Nondestructive indicator of ethoxyresorufin-O -deethylase activity in embryonic fish Environ Toxicol Chem 17 2481 2486
Nacci DE Coiro L Wassenberg DM Di Giulio RT In press. A non-destructive technique to measure cytochrome P4501A enzyme activity in living embryos of the estuarine fish Fundulus heteroclitus In: Techniques in Aquatic Toxicology, Vol 2 (Ostrander GK, ed). Boca Raton, FL:Lewis Publishers/CRC Press, 209–225.
Nebert DW Roe AL Dieter MZ Solis WA Yang Y Dalton TP 2000 Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis Biochem Pharmacol 59 65 85 10605936
Niimi AJ Palazzo V 1986 Biological half-lives of eight polycyclic aromatic hydrocarbons (PAHs) in rainbow trout (Salmo gairdneri ) Water Res 20 503 507
Pelroy RA Wilson BW 1981 Relative concentrations of poly-aromatic primary amines and azaarenes in mutagenically active nitrogen fractions from a coal liquid Mutat Res 90 321 335 6174860
Ronisz D Förlin L 1998 Interaction of isosafrole, β-naphthoflavone and other CYP1A inducers in liver of rainbow trout (Oncorhynchus mykiss ) and eelpout (Zoarces viviparus ) Comp Biochem Physiol C Toxicol Pharmacol 121 289 296
Safe S 1990 Polychlorinated biphenyls (PCBs), dibenzo-p -dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs) CRC Crit Rev Toxicol 21 51 88
Safe S 1993 Development of bioassays and approaches for the risk assessment of 2,3,7,8-tetrachlorodibenzo-p -dioxin and related compounds Environ Health Perspect 101 suppl 3 317 325 8143638
Schlezinger JJ Stegeman JJ 2001 Induction and suppression of cytochrome P450 1A by 3,3’,4,4’,5-pentachlorobiphenyl and its relationship to oxidative stress in the marine fish scup (Stenotomus chrysops ) Aquat Toxicol 52 101 115 11164533
Schlezinger JJ White RD Stegeman JJ 1999 Oxidative inactivation of cytochrome P-450 1A (CYP1A) stimulated by 3,3′,4,4′-tetrachlorobiphenyl: production of reactive oxygen by vertebrate CYP1As Mol Pharmacol 56 588 597 10462547
Shertzer HG Clay CD Genter MB Chames MC Schneider SN Oakley GG 2004 Uncoupling-mediated generation of reactive oxygen by halogenated aromatic hydrocarbons in mouse liver microsomes Free Radic Biol Med 36 618 631 14980705
Sjögren M Ehrenberg L Rannug U 1996 Relevance of different biological assays in assessing initiating and promoting properties of polycyclic aromatic hydrocarbons with respect to carcinogenic potency Mutat Res 358 97 112 8921980
Smith AG Clothier B Cathew P Childs NL Sinclair PR Nebert DW 2001 Protection of the CYP1a2 null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p -dioxin Toxicol Appl Pharmacol 173 89 98 11384210
Stegeman JJ Klotz AV Woodin BR Pajor AM 1981 Induction of hepatic cytochrome p-450 in fish and the indication of environmental induction in scup (Stenotomus chrysops ) Aquat Toxicol 1 197 212
Stohs SJ 1990 Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) Free Radic Biol Med 9 79 90 2210442
Teraoka H Dong W Tsujimoto Y Iwasa H Endoh D Ueno N 2003 Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p -dioxin in zebrafish Biochem Biophys Res Commun 304 223 228 12711302
Testa B Jenner P 1981 Inhibitors of cytochrome P-450s and their mechanism of action Drug Metab Rev 12 1 117 7028434
Toomey BH Bello S Hahn ME Cantrell S Wright P Tillitt DE 2001 2,3,7,8-Tetrachlorodibenzo-p -dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos Aquat Toxicol 53 127 138 11311389
Uno S Dalton TP Derkenne S Curran CP Miller ML Shertzer HG 2004a Oral exposure to benzo[a ]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation Mol Pharmacol 65 1225 1237 15102951
Uno S Dalton TP Shertzer HG Genter MB Warshawsky D Talaska G 2001 Benzo[a ]pyrene-induced toxicity: paradoxical protection in CYP1A1 (–/–) knockout mice having increased hepatic BaP-DNA adduct levels Biochem Biophys Res Commun 289 1049 1056 11741297
Uno S Dalton TP Sinclair PR Gorman N Wang B Smith AG CYP1a1(–/–) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria Toxicol Appl Pharmacol 196 410 421 15094312
Van Metre PC Mahler BJ Furlong ET 2000 Urban sprawl leaves its PAH signature Environ Sci Technol 34 4064 4070
Van Veld PA Vogelbein WK Cochran MK Goksøyr A Stegeman JJ 1997 Route-specific cellular expression of cytochrome P4501A (CYP1A) in fish (Fundulus heteroclitus ) following exposure to aqueous and dietary benzo[a ]pyrene Toxicol Appl Pharmacol 142 348 359 9070358
Walker MK Peterson RE 1991 Potencies of polychlorinated dibenzo-p -dioxin, dibenzofuran, and biphenyl congeners, relative to 2,3,7,8-tetrachlorodibenzo-p -dioxin, for producing early life stage mortality in rainbow trout (Oncorhynchus mykiss ) Aquat Toxicol 21 219 238
Wannemacher R Rebstock A Kulzer E Schrenk D Bock KW 1992 Effects of 2,3,7,8-tetrachlorodibenzo-p -dioxin on reproduction and oogenesis in zebrafish (Branchydanio rerio ) Chemosphere 24 1361 1368
Wassenberg DM Di Giulio RT 2004 Teratogenesis in Fundulus heteroclitus embryos exposed to a creosote-contaminated sediment extract and CYP1A inhibitors Mar Environ Res 58 163 168 15178029
Watson DE Ménard L Stegeman JJ Di Giulio RT 1995 Aminoanthracene is a mechanism-based inactivator of CYP1A in channel catfish hepatic tissue Toxicol Appl Pharmacol 135 208 215 8545829
White RD Shea D Stegeman JJ 1997 Metabolism of the aryl hydrocarbon receptor agonist 3,3’4,4’-tetrachlorobiphenyl by the marine fish scup (Stenotomus chrysops ) in vivo and in vitro Drug Metab Dispos 25 564 572 9152595
Willett KL Randerath K Zhou G-D Safe SH 1998 Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene Biochem Pharmacol 55 831 839 9586956
Willett KL Wassenberg DM Lienesch L Reichert W Di Giulio RT 2001 In vivo and in vitro inhibition of CYP1A-dependent activity in Fundulus heteroclitus by the polynuclear aromatic hydrocarbon fluoranthene Toxicol Appl Pharmacol 177 264 271 11749126
Wilson BW 1980 Identification of primary aromatic amines in mutagenically active subfractions from coal liquefacation materials Mutat Res 79 193 202 7012601
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7234ehp0112-00166515579410ResearchArticlesThe Relationship of Urinary Metabolites of Carbaryl/Naphthalene and Chlorpyrifos with Human Semen Quality Meeker John D. 1Ryan Louise 2Barr Dana B. 3Herrick Robert F. 1Bennett Deborah H. 1Bravo Roberto 3Hauser Russ 11Department of Environmental Health and2Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts, USA3Centers for Disease Control and Prevention, Atlanta, Georgia, USAAddress correspondence to R. Hauser, Department of Environmental Health, Occupational Health Program, Harvard School of Public Health, Building 1, Room 1405, 665 Huntington Ave., Boston, MA 02115 USA. Telephone: (617) 432-3326. Fax: (617) 432-0219. E-mail:
[email protected] thank L. Godfrey-Bailey who recruited the study patients and collected the biological specimens; J. Frelich who was responsible for data management; and A. Trisini and R. Dadd, who assisted with manuscript preparation and literature reviews.
This work was supported by grants ES09718 and ES00002 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH).
Contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS or the NIH.
The authors declare they have no competing financial interests.
12 2004 7 9 2004 112 17 1665 1670 6 5 2004 7 9 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Most of the general population is exposed to carbaryl and other contemporary-use insecticides at low levels. Studies of laboratory animals, in addition to limited human data, show an association between carbaryl exposure and decreased semen quality. In the present study we explored whether environmental exposures to 1-naphthol (1N), a metabolite of carbaryl and naphthalene, and 3,5,6-trichloro-2-pyridinol (TCPY), a metabolite of chlorpyrifos and chlorpyrifos-methyl, are associated with decreased semen quality in humans. Subjects (n = 272) were recruited through a Massachusetts infertility clinic. Individual exposures were measured as spot urinary concentrations of 1N and TCPY adjusted using specific gravity. Semen quality was assessed as sperm concentration, percent motile sperm, and percent sperm with normal morphology, along with sperm motion parameters (straight-line velocity, curvilinear velocity, and linearity). Median TCPY and 1N concentrations were 3.22 and 3.19 μg/L, respectively. For increasing 1N tertiles, adjusted odds ratios (ORs) were significantly elevated for below-reference sperm concentration (OR for low, medium, and high tertiles = 1.0, 4.2, 4.2, respectively; p-value for trend = 0.01) and percent motile sperm (1.0, 2.5, 2.4; p-value for trend = 0.01). The sperm motion parameter most strongly associated with 1N was straight-line velocity. There were suggestive, borderline-significant associations for TCPY with sperm concentration and motility, whereas sperm morphology was weakly and nonsignificantly associated with both TCPY and 1N. The observed associations between altered semen quality and 1N are consistent with previous studies of carbaryl exposure, although suggestive associations with TCPY are difficult to interpret because human and animal data are currently limited.
biological markersenvironmenthumanpesticidessemen
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Despite the ubiquitous use of insecticides and subsequent exposure among the general population [Centers for Disease Control and Prevention (CDC) 2003; Hill et al. 1995; MacIntosh et al. 1999], there are limited human studies investigating associations between exposure to contemporary-use insecticides at environmental levels and male reproductive health. Human and animal data suggest a potential association between exposures to some commonly used insecticides and decreased semen quality. A study of workers that packaged carbaryl found an increased proportion of oligozoospermic (< 20 million sperm/mL) and teratospermic (> 60% abnormal sperm morphology) men compared with a reference group of chemical workers (Whorton et al. 1979; Wyrobek et al. 1981). Further support for carbaryl’s testicular toxicity comes from studies in laboratory rats that showed associations between carbaryl exposure and sperm shape abnormalities and chromosomal aberrations (Luca and Balan 1987), as well as dose–response relationships between carbaryl exposure and a decline in epididymal sperm count and motility and increased abnormal sperm morphology (Pant et al. 1995, 1996; Rybakova 1966; Shtenberg and Rybakova 1968). Carbaryl was also found to disrupt endocrine regulation of gonadal function in fish (Ghosh and Bhattacharya 1990). Chlorpyrifos, a frequently used insecticide until being banned for residential use (Lewis 2000), is less studied than is carbaryl for its testicular toxicity but has been found to disrupt endocrine regulation in ewes (Rawlings et al. 1998). Recently, the CDC reported measurable levels of urinary 3,5,6-trichloro-2-pyridinol (TCPY), a metabolite of chlorpyrifos and chlorpyrifos-methyl, and 1-naphthol (1N), a metabolite of carbaryl and naphthalene, in > 90% and 75% of males in the United States, respectively (CDC 2003).
The present study was designed to investigate the association between environmental exposure to the nonpersistent insecticides chlorpyrifos and carbaryl and altered semen quality among adult men. Insecticide metabolite levels in urine were used as biomarkers of chlorpyrifos and carbaryl exposure.
Materials and Methods
Study subjects were men who were partners in subfertile couples seeking infertility diagnosis from the Vincent Burnham Andrology lab at Massachusetts General Hospital (Boston, MA) between January 2000 and April 2003. The study was approved by the human studies institutional review boards of the Massachusetts General Hospital and the Harvard School of Public Health. After the study procedures were explained and all questions answered, subjects signed informed consent forms. Details of subject recruitment have been previously described (Hauser et al. 2003). Briefly, consecutive eligible men were recruited to participate. Of those approached, 65% consented. Most men who declined to participate in the study cited lack of time on the day of their clinic visit as the reason for not participating. Men with a medical history of risk factors for infertility (e.g., varicocele or orchidopexy) were a priori excluded from the study analyses. None of the men reported occupational exposure to pesticides or other agents suspected to be associated with semen quality. A single spot urine sample was collected from each subject on the same day as the semen sample. Urine samples were frozen at −20°C and mailed on dry ice to the CDC, where TCPY and 1N were measured as previously described by Hill et al. (1995). Briefly, samples were fortified with stable isotope analogs of the target analytes, and glucuronide or sulfate-bound metabolites were liberated using an enzyme hydrolysis. TCPY and 1N were isolated using liquid–liquid extraction, chemically derivatized, and measured using gas chromatography–chemical ionization–tandem mass spectrometry.
Although creatinine concentrations are commonly used to adjust for variable urine dilution in spot samples when measuring pesticide metabolites, creatinine adjustment may not be appropriate for compounds that undergo active tubular secretion, which includes organic compounds such as TCPY and 1N that can be conjugated by the liver in the form of glucuronides or sulfates (Boeniger et al. 1993). Creatinine levels also vary by sex, age, muscle mass, race, diet, activity, and time of day. Therefore, adjusting urine insecticide metabolite concentrations using specific gravity (SG) may be more appropriate; thus, SG was used as the primary method for dilution adjustment in the present study. However, in addition to SG-adjusted results, volume-based (unadjusted) and creatinine-adjusted TCPY and 1N concentrations were also determined to allow for comparisons with exposure distributions from other studies. Samples with creatinine concentrations > 300 mg/dL or < 30 mg/dL, or with SG > 1.03 or < 1.01, were considered too concentrated or too dilute, respectively, to provide valid results (Teass et al. 1998) and were excluded. Creatinine was measured photo-metrically using kinetic colorimetric assay technology with a Hitachi 911 automated chemistry analyzer (Roche Diagnostics, Indianapolis, IN). SG was measured using a handheld refractometer (National Instrument Company Inc., Baltimore, MD).
Measurement of the semen parameters (sperm concentration, motility, and morphology) has been described previously (Hauser et al. 2003). Briefly, we measured sperm count and motility by computer-aided semen analysis (CASA) using the Hamilton Thorne IVOS 10 Analyzer (Hamilton-Thorne Research, Beverly, MA). To assess sperm morphology, we evaluated 200 sperm using the Tygerberg Strict Criteria (Kruger et al. 1988). In addition, seven CASA motion parameters were measured. Measurement of these parameters has been previously described (Duty et al. 2004). Briefly, CASA outcomes included a mathematically smoothed velocity (designated VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) that corresponds to the mean width of the head oscillation as the cell swims, and beat cross frequency (BCF), which measures the frequency with which the cell track crosses the cell path in either direction. VAP, VSL, straightness (STR = VSL/VAP × 100), and linearity (LIN = VSL/VCL × 100) are indicators of sperm progression, whereas VCL, ALH, and BCF are indicators of sperm vigor. We also used STR and LIN to describe sperm swimming pattern. Some of the CASA parameters were strongly correlated with each other because they describe different aspects of the same movement. Measures of progression, VAP and VSL, were highly correlated, which indicated they were likely measuring a similar characteristic of sperm movement. We chose VSL over VAP as a measure of progression because it is a direct measurement as opposed to a mathematically smoothed value. VCL was chosen as a measure of vigor and was strongly and positively correlated with ALH but not correlated with BCF. The two measures of swimming pattern (LIN and STR) were strongly correlated, indicating they were likely measuring a similar characteristic of sperm movement. We chose LIN as a measure of swimming pattern because the other parameters chosen for this study (VSL and VCL) are components of LIN and not of STR. Therefore, we chose measure of progression (VSL), vigor (VCL), and swimming pattern (LIN) for statistical analyses. These three measures are also not as heavily dependent on the type of CASA instrument used, allowing for some comparison with results from other studies.
Statistical analysis.
Statistical analyses were performed using semen parameters both as a continuous measure and dichotomized using World Health Organization (WHO) reference values for sperm concentration (< 20 million sperm/mL) and motility (< 50% motile sperm; WHO 1999). We used the Tygerberg Strict Criteria for morphology to determine below-reference morphology (< 4% normal morphology) (Kruger et al. 1988). Men with values above reference values for all three semen parameters were used as comparison subjects in the logistic regression models. For the CASA motion parameters (VSL, VCL, and LIN), we used multiple linear regression models to assess associations with insecticide metabolites. Nine azoospermic men were excluded from the CASA analyses because motion parameters were not measurable.
Insecticide metabolite concentrations were used both as a continuous measure and categorized into tertiles. For metabolite values below the limit of detection (LOD), corresponding to 0.25 μg/L for TCPY and 0.40 μg/L for 1N, an imputed value equal to one-half the LOD was used. Normality of the metabolite concentrations and semen parameters was assessed, and appropriate transformations were performed before linear regression. Distributions of TCPY, 1N, and sperm concentration were log-transformed in the models. The remaining semen parameters and CASA parameters were normally distributed and not transformed. Semen parameters were stratified by demographic categories to investigate the potential for confounding. Associations between demographic variables and insecticide metabolite levels were also explored. We considered smoking status, race, age, body mass index, a previous exam for infertility, and abstinence time as potential covariates. Inclusion of covariates in the models was based on statistical and biological considerations (Hosmer and Lemeshow 1989). Covariates were entered into the models individually in a forward stepwise manner. Covariates that changed the exposure parameter estimate by greater than 10% were retained in the multivariate model and were considered confounders. There was evidence of confounding by both age and abstinence time in many, but not all, of the models for the various outcome measures. However, because there is evidence that age and abstinence time are associated with semen quality, we included them in all multivariate models (Blackwell and Zaneveld 1992; Kidd et al. 2001). Age was modeled as a continuous independent variable. Abstinence time was modeled as an ordinal variable with five categories: ≤ 2, 3, 4, 5, and ≥ 6 days.
Results
A total of 330 eligible men provided a single semen and urine sample. The distributions of urinary levels of TCPY and 1N for the 330 men are presented in Table 1, as are adjusted metabolite distributions after excluding men with highly concentrated or dilute samples according to creatinine (23 of 330 men; n = 307) or SG (58 of 330 men; n = 272). SG-adjusted TCPY and 1N levels were moderately correlated (Spearman correlation coefficient = 0.3; p < 0.001). Demographic characteristics and semen parameters are described in Table 2. Subjects were primarily white (82%), with a mean (± SD) age of 36.2 ± 5.5 years, and 72% had never smoked. The proportion of men with a previous exam for infertility was higher among all three of the below-reference semen parameter groups (48%, 36%, and 40% for sperm concentration, motility, and morphology groups, respectively) than among the comparison group (25%). The semen parameter categories were not mutually exclusive. A man could contribute data to one, two, or all three of the below-reference groups.
Odds ratios (ORs) for the relationship between dichotomized semen parameters and SG-adjusted metabolite tertiles are presented in Table 3. Compared with men in the lowest 1N tertile, men in both the medium and high SG-adjusted 1N tertiles were more likely to have below-reference sperm concentration {ORs for increasing exposure tertiles = 1.0, 4.2 [95% confidence interval (CI), 1.4–13.0], 4.2 [95% CI, 1.4–12.6]; p-value for trend = 0.01} and sperm motility [1.0, 2.5 (95% CI, 1.3–4.7), 2.4 (95% CI, 1.2–4.5); p-value for trend = 0.01]. Although the ORs for the second and third tertiles were both significantly different from 1.0, the exposure–response trends were not monotonic. There were suggestive associations between SG-adjusted TCPY with sperm concentration (1.0, 2.1, 2.4; p-value for trend = 0.09) and sperm motility (1.0, 1.6, 1.7; p-value for trend = 0.09). However, the estimates for the second and third tertiles suggest that the dose–response relationship was not monotonic. Sperm morphology was weakly associated with both TCPY and 1N.
To further explore potential dose–response relationships, subjects were divided into quintiles based on SG-adjusted 1N and TCPY concentrations (Figures 1 and 2). Although not monotonic, there were relationships between increased 1N and sperm concentration (OR estimates for increasing exposure quintiles were 1.0, 0.7, 2.3, 3.6, 2.4; p-value for trend = 0.02) and decreased sperm motility (1.0, 0.8, 2.8, 2.0, 2.8; p-value for trend = 0.002). A suggestive relationship was found between 1N and abnormal sperm morphology (1.0, 1.1, 1.5, 1.4, 2.3; p-value for trend = 0.09). Point estimates for the associations between TCPY quintiles and below-reference sperm concentration, motility, and morphology were > 1.0, but none of them approached statistical significance.
We conducted sensitivity analyses to test the robustness of the results. Associations between SG-adjusted exposure tertiles and below-reference semen parameters were recalculated after excluding nine azoospermic men. For 1N, ORs were moderately attenuated for sperm concentration (1.0, 3.0, 3.1; p-value for trend = 0.05) but were unchanged for sperm motility. ORs for the highest TCPY tertile with both sperm concentration and motility were slightly larger but remained of borderline statistical significance.
We also reanalyzed the data after retaining the 58 men with SG < 1.01 or > 1.03 (n = 330). Estimates of relationships with 1N tertiles became moderately lower for sperm concentration (1.0, 3.0, 2.6; p-value for trend = 0.05) and motility (1.0, 2.2, 1.9; p-value for trend = 0.03). The suggestive relationship between TCPY tertiles and sperm concentration became slightly stronger (1.0, 1.8, 2.2; p-value for trend = 0.08), whereas relationships of 1N with sperm morphology and TCPY with sperm motility and morphology remained weak.
Results of multivariate linear regression models for continuous semen parameters and continuous urinary metabolites are shown in Table 4. A suggestive association between SG-adjusted 1N concentration and decreased sperm concentration was found (p = 0.06). As in the logistic regression analysis, there was an association between 1N levels and a decreased percentage of motile sperm (p = 0.03). SG-adjusted TCPY did not show associations with decreased concentration or morphology, but there was a suggestive association with motility. Similar results were found in sensitivity analyses that excluded nine azoospermic men (data not shown).
Multivariate linear regression analyses for CASA motion parameters (Table 4) showed significant inverse associations for VSL and LIN with increased SG-adjusted TCPY (p-values < 0.05). SG-adjusted 1N levels were inversely associated with VSL (p = 0.02). CASA motion parameters were also modeled against tertiles of SG-adjusted TCPY and 1N. The association of TCPY with LIN became nonsignificant (linear regression coefficients for increasing exposure tertiles were 0, −1.16, −1.05; p-value for trend = 0.3). An inverse relationship remained for TCPY and VSL (0, −0.13, −2.79; p-value for trend = 0.05) and between 1N and VSL (0, −2.17, −3.50; p-value for trend = 0.01). There was a suggestive inverse relationship between 1N and VCL (0, −0.49, −4.16; p-value for trend = 0.09).
In addition to SG-adjusted values, all statistical analyses were performed with unadjusted and CRE-adjusted TCPY and 1N concentrations (results available from the authors upon request). Results using unadjusted values were similar to those from SG-adjusted values. Creatinine-adjusted results differed from SG-adjusted results. The only relationship in the multivariate logistic models that approached statistical significance was between sperm motility and creatinine-adjusted 1N tertiles (1.0, 1.3, 1.7; p-value for trend = 0.08) and quintiles (1.0, 1.3, 1.6, 1.9, 1.8; p-value for trend = 0.07). No statistically significant associations were found between creatinine-adjusted metabolite levels and outcome measures in the multivariate linear regression analysis.
Discussion
In the present study we found associations between urinary metabolites of contemporary-use insecticides and decreased sperm concentration and motility in humans. Specifically, we found statistically significant inverse dose–response relationships between 1N and sperm concentration and motility, as well as between 1N and VSL. Suggestive associations were found between 1N and sperm morphology, VCL, and LIN and between TCPY and sperm concentration, motility, and VSL.
The present data were generally consistent with laboratory animal studies that have shown an association between exposure to carbaryl and decreased semen quality. A 90-day study of rats found statistically significant dose-related declines in epididymal sperm count and percent motile sperm, as well as increased sperm with abnormal morphology (Pant et al. 1995, 1996). In an earlier study, subacute and chronic reproductive effects of carbaryl were found in male rats (Rybakova 1966; Shtenberg and Rybakova 1968). Subacute exposure induced a decrease in motile sperm by an average of 40% after 50 days, whereas chronic exposure led to a significant decrease in motile sperm among even the lowest of the three exposed groups after 12 months.
Limited animal studies have explored relationships between chlorpyrifos exposure and semen quality. Decreased sperm production and motility was observed in Holstein bulls 6 months after dermal lice treatment with an unknown amount of chlorpyrifos [Agency for Substances and Disease Registry (ATSDR) 1997; Everett 1982]. Other animal studies found no associations between chlorpyrifos exposure and altered male reproductive health (ATSDR 1997; Breslin et al. 1996). However, semen quality was not assessed in these studies, and conclusions were reached in part based on the lack of observed changes in testicular weight. In the carbaryl studies, no change in rat testicular weight was reported for lower doses for which decreased semen quality was observed (Pant et al. 1995, 1996; Rybakova 1966).
Human studies investigating exposure to carbaryl and chlorpyrifos and associations with male reproductive health are limited. Until recently, there were no known human male reproductive health studies that used biological measures of exposure to carbaryl and chlorpyrifos (ATSDR 1997). Swan et al. (2003) found elevated but nonsignificant ORs for low semen quality (sperm concentration, motility, and morphology below the population median) among 24 Missouri men with detectable 1N (OR = 2.7; 95% CI, 0.2–34.2) and TCPY levels (6.4; 95% CI, 0.5–86.3). The numbers of subjects were small, limiting statistical power. In a study among Chinese workers exposed to other organophosphate pesticides (ethylparathion and methamidophos), Padungtod et al. (2000) found significantly lower sperm concentration and sperm motility compared with nonexposed workers but no difference in sperm morphology.
In the present study, the relationship between 1N and sperm concentration below the WHO reference value (WHO 1999) is consistent with two published reports on a cohort of carbaryl production workers (Whorton et al. 1979; Wyrobek et al. 1981). Whorton and co-workers found a higher percentage of exposed workers (15%) had sperm concentrations below the reference value of 20 million sperm/mL compared with non-exposed controls (5.5%, p = 0.07). In contrast to the present study, Wyrobek et al. (1981) reported an association between carbaryl exposure and sperm morphology. The distribution of abnormal sperm morphology was significantly higher for exposed workers (p < 0.005), and the proportion of teratospermic men was larger in the exposed group (29%) compared with controls (12%, p = 0.06). Because of logistical constraints, sperm motility was not measured in the published reports of the carbaryl production worker study.
Functional defects of sperm may be an important factor in male infertility. The role of reactive oxygen species (ROS) in male infertility has been suggested in studies that found higher seminal ROS levels in infertile men compared with fertile controls (Agarwal et al. 1994; Pasqualotto et al. 2000). Sperm cells do not have cytoplasmic defense enzymes (e.g., catalase) that serve as ROS scavengers. Consequently, sperm, which have a high content of polyunsaturated fatty acids, are more susceptible to the oxidative deterioration of polyunsaturated fatty acids known as lipid peroxidation (Sharma and Agarwal 1996). Lipid peroxidation causes the plasma membrane to lose its fluidity and integrity, ultimately leading to loss of sperm function (Aitken 1995). Loss of membrane fluidity also impairs the cell membrane ion exchange that controls sperm movement (Rao et al. 1989). Carbaryl causes lipid peroxidation at low concentrations by either efficiently lowering the intracellular level of glutathione, which is associated with an increase in ROS, or through the inhibition of excision esterases (Soderpalm-Berndes and Onfelt 1988). Thus, it is biologically plausible that exposure to carbaryl may be associated with altered semen quality, particularly sperm motility and sperm motion.
Biomonitoring for insecticide metabolite concentrations in urine is a commonly used indicator of internal dose integrating the various routes through which the contaminant enters the body (Barr et al. 1999). However, nonpersistent insecticides are metabolized and excreted rapidly. For example, TCPY has an estimated half-life of 27 hr in humans (Nolan et al. 1984), and levels of both TCPY and 1N measured in urine reflect insecticide exposure in the previous 24–48 hr (Maroni et al. 2000). Spermatogenesis is a cyclical process that takes approximately 3 months. Although insecticide metabolite levels in urine can vary considerably over time, suggesting that a single urine sample may not be a reliable surrogate for longer-term exposure (MacIntosh et al. 1999), we recently showed that a single urine sample was predictive of the 3-month average urinary insecticide metabolite levels (Meeker et al., in press). A single urine sample correctly classified men in the highest 3-month exposure tertile with a sensitivity (specificity) of 0.6 (0.9) for SG-adjusted 1N and 0.5 (0.8) for SG-adjusted TCPY.
Distributions of unadjusted and creatinine-adjusted TCPY and 1N levels in the present study were compared with those recently reported for males in the National Health and Nutrition Examination Survey (NHANES) 1999–2000 (CDC 2003). Unadjusted TCPY concentrations were slightly higher in the present study, with median and 95th percentile values of 2.69 and 10.6 μg/L, respectively, compared with 1.90 and 9.9 μg/L from NHANES 1999–2000. Median and 95th percentiles for unadjusted 1N concentrations were also higher in the present study (2.86 and 13.3 μg/L, respectively, vs. 1.40 and 11.0 μg/L from NHANES 1999–2000). SG-adjusted TCPY and 1N distributions were not reported by NHANES 1999–2000 (CDC 2003).
In the present study, we obtained similar results using SG-adjusted or unadjusted urine metabolite levels, but our results were different for creatinine-adjusted levels. The inability to detect associations using creatinine-adjusted values may reflect tubular secretion of 1N and thus excretion rates of 1N that are independent of urine flow through the glomerulus and not directly related to the amount of creatinine that is filtered (Boeniger et al. 1993). Adjustment of 1N concentration by urinary dilution using creatinine may introduce additional nondifferential exposure measurement error, further limiting the ability to find associations between exposure and outcome.
Strengths of the present study include its size and high participation rate and the use of biological markers of exposure. To test the robustness of the data analysis, we used several modeling approaches in which exposures and outcomes were used as both continuous and categorical measures. The results were consistent across modeling approaches, suggesting that the data were not sensitive to the statistical analysis methods used. Study weaknesses included collecting only a single urine sample as an estimate of 3-month exposure and collecting only a single semen sample to assess semen quality. However, our earlier work supported the utility of a single urine specimen as predictive of 3-month average exposure (Meeker et al., in press). In conclusion, associations between 1N and sperm concentration and motility were found that are consistent with animal studies of carbaryl exposure. The sperm motion parameter most strongly associated with urinary 1N was VSL, although suggestive associations of 1N with VCL and LIN were also found. There were also suggestive associations between TCPY and sperm concentration and motility, but they are difficult to interpret because there are currently limited human and animal data.
Because most of the U.S. population is exposed to these insecticides (CDC 2003), the public health significance of an association with semen quality is potentially large. For instance, our results suggest that an interquartile range increase in carbaryl metabolite levels in urine is associated with a 4% decrease in sperm motility. Although this may not alter an individual man’s fertility, a 4% decrease in the mean of the distribution of sperm motility among U.S. men may result in a significant increase in the number of men in the lower tail of the sperm motility distribution, increasing the number of subfertile men. Further studies are needed to confirm these preliminary findings and assess the potential public health significance.
Figure 1 Adjusted ORs and 95% CIs for below-reference semen parameters by increasing quintiles of 1N for (A) sperm concentration (p-value for trend = 0.02), (B) motility (p-value for trend = 0.002), and (C) morphology (p-value for trend = 0.09). The quintiles of SG-adjusted 1N (μg/L) are as follows: Q1 (low), < LOD to 1.50; Q2, 1.50–2.67; Q3, 2.67–3.73; Q4, 3.73–5.86; Q5 (high), 5.86–159.7.
Figure 2 Adjusted ORs and 95% CIs for below-reference semen parameters by increasing quintiles of TCPY for (A) sperm concentration (p-value for trend = 0.21), (B) motility (p-value for trend = 0.15), and (C) morphology (p-value for trend = 0.26). The quintiles of SG-adjusted TCPY (μg/L) are as follows: Q1 (low), < LOD to 1.45; Q2, 1.45–2.72; Q3, 2.72–3.85; Q4, 3.85–5.59; Q5 (high), 5.59–40.69.
Table 1 Distribution of insecticide (carbaryl and chlorpyrifos) metabolite levels in urine.
Insecticide metabolite No.a Geometric mean Selected percentiles
10th 25th 50th 75th 90th 95th Maximum
Unadjusted (μg/L)b
1N 330 2.82 0.93 1.61 2.86 4.49 7.61 13.28 139.7
TCPY 330 2.32 0.50 1.49 2.69 4.80 7.60 10.57 32.21
SG-adjustedc
1N 272 3.13 1.02 1.80 3.19 5.03 9.57 13.96 159.7
TCPY 272 2.63 0.58 1.75 3.22 5.03 7.89 9.66 40.69
CR-adjustedd
1N 307 2.32 0.72 1.26 2.21 4.38 7.38 11.04 150.7
TCPY 307 1.97 0.56 1.27 2.29 3.57 5.58 7.08 35.13
a Number of subjects.
b LOD for 1N = 0.40 μg/L; 99.7% of samples > LOD. LOD for TCPY = 0.25 μg/L; 93.9% of samples > LOD.
c Excluded 58 samples with SG > 1.03 or < 1.01.
d Excluded 23 samples with creatinine > 300 or < 30 mg/dL.
Table 2 Demographic categories by semen parametersa (n = 330).
Comparison subjects (n = 157) Sperm concentration < 20 million/mL (n = 44) Sperm motility < 50% motile (n = 147) Sperm morphology < 4% normal (n = 72)
Age (mean ± SD) 35.4 ± 5.2 37.6 ± 6.0 37.0 ± 5.6 36.7 ± 5.6
Abstinence time [n (%)]
≤ 2 days 34 (22) 17 (40) 37 (25) 13 (18)
3 days 52 (33) 9 (20) 44 (30) 23 (32)
4 days 28 (18) 6 (14) 24 (16) 12 (17)
5 days 18 (12) 2 (5) 14 (9) 5 (7)
≥ 6 days 24 (15) 9 (20) 27 (18) 19 (26)
Race [n (%)]
White 134 (85) 32 (73) 113 (76) 59 (82)
Black/African American 7 (4) 4 (9) 11 (7) 5 (7)
Hispanic 5 (3) 2 (5) 11 (7) 3 (4)
Other 11 (7) 6 (14) 13 (9) 5 (7)
Smoking status [n (%)]
Never smoker 117 (75) 25 (59) 102 (70) 48 (67)
Ever smoker
Current smoker 12 (8) 6 (14) 12 (8) 8 (11)
Ex-smoker 27 (17) 11 (25) 30 (20) 15 (21)
Previous exam for infertility [n (%)] 40 (25) 21 (48) 54 (36) 29 (40)
a Information on race missing for one man and on smoking for three men.
Table 3 Adjusted ORsa (95% CIs) for SG-adjusted metabolite tertiles (n = 272).b
Sperm concentration (< 20 million/mL; n = 35)
Sperm motility (< 50% motile; n = 119)
Sperm morphology (< 4% normal; n = 59)
Comparison subjects (n = 130) No.c OR (95% CI) No.c OR (95% CI) No.c OR (95% CI)
1Nd
Low 53 5 1.0 27 1.0 20 1.0
Medium 39 14 4.2 (1.4–13.0)* 45 2.5 (1.3–4.7)* 17 1.4 (0.6–3.0)
High 38 16 4.2 (1.4–12.6)* 47 2.4 (1.2–4.5)* 22 1.6 (0.8–3.5)
p-Value for trend 0.01 0.01 0.20
TCPYe
Low 52 8 1.0 33 1.0 17 1.0
Medium 39 12 2.1 (0.8–5.6) 40 1.6 (0.8–3.0) 16 1.2 (0.5–2.7)
High 39 15 2.4 (0.9–6.3) 46 1.7 (0.9–3.2) 26 1.9 (0.9–4.0)
p-Value for trend 0.09 0.09 0.10
a ORs adjusted for age and abstinence time.
b Excluded 58 subjects with SG > 1.03 or < 1.01.
c Number of subjects in each exposure tertile with below-reference semen parameters. The semen parameter categories were not mutually exclusive; a man could contribute data to one, two, or all three of the below-reference groups.
d SG-adjusted 1N tertiles: low, < LOD to 2.36 μg/L; medium, 2.36–4.02 μg/L; high, 4.02–159.7 μg/L.
e SG-adjusted TCPY tertiles: low, < LOD to 2.30 μg/L; medium, 2.30–4.42 μg/L; high, 4.42–40.7 μg/L.
* p < 0.05.
Table 4 Adjusted regression coefficientsa,b for a change in semen parameters and sperm motion parameters associated with an interquartile range (IQR)c increase in SG-adjusted insecticide metabolite levels (n = 272).
Coefficient (95% CI)
1Nd TCPYd
Semen parameters
Concentratione 0.84 (0.71–1.01) 0.97 (0.83–1.12)
Motility (percent motile) −3.87 (−7.28–−0.45)* −2.16 (−5.05–0.73)
Morphology (percent normal) −0.15 (−0.79–0.49) 0.15 (−0.39–0.68)
Motion parametersf
VSL −1.64 (−2.99–−0.27)* −1.21 (−2.34–−0.08)*
VCL −1.98 (−4.33,–0.35) −0.53 (−2.47–1.42)
LIN −0.79 (−1.79–0.22) −1.07 (−1.90–−0.24)*
a Regression coefficients were adjusted for age and abstinence time.
b Regression coefficients for motility, morphology, and motion parameters represent the change in semen parameter for an IQR change in insecticide metabolite concentration (0, no change in semen parameter for an IQR change in insecticide metabolite concentration; < 0, a decrease in semen parameter for an IQR change in insecticide metabolite concentration; > 0, an increase in semen parameter for an IQR change in insecticide metabolite concentration).
c 1N IQR = 1.80–5.02 μg/L; TCPY IQR = 1.76–5.01 μg/L.
d 1N and TCPY were log transformed for regression analysis.
e Sperm concentration was log transformed. The coefficient represents a multiplicative change in sperm concentration per IQR change in TCPY or 1N (1.0, no change in sperm concentration for an IQR change in insecticide metabolite concentration; < 1.0, a multiplicative decrease in sperm concentration for an IQR change in insecticide metabolite concentration; > 1.0, a multiplicative increase in sperm concentration for an IQR change in insecticide metabolite concentration).
f VSL, VCL, and LIN analyses not performed on 9 azoospermic men; n = 263. TCPY IQR = 1.76–5.08 μg/L; 1N IQR = 1.77–5.02 μg/L.
* p < 0.05.
==== Refs
References
Agarwal A Ikemoto I Loughlin KR 1994 Relationship of sperm parameters with levels of reactive oxygen species in semen specimens J Urol 152 107 110 8201640
Aitken RJ 1995 Free radicals, lipid peroxidation and sperm function Reprod Fertil Dev 7 659 668 8711202
ATSDR 1997. Toxicological Profile for Chlorpyrifos. Atlanta, GA:Agency for Toxic Substances and Disease Registry.
Barr DB Barr JR Driskell WJ Hill RH Jr Ashley DL Needham LL 1999 Strategies for biological monitoring of exposure for contemporary-use pesticides Toxicol Ind Health 15 168 179 10188199
Blackwell JM Zaneveld LJ 1992 Effect of abstinence on sperm acrosin, hypoosmotic swelling, and other semen variables Fertil Steril 58 798 802 1426327
Boeniger MF Lowry LK Rosenberg J 1993 Interpretation of urine results used to assess chemical exposure with emphasis on creatinine adjustments: a review Am Ind Hyg Assoc J 54 615 627 8237794
Breslin WJ Liberacki AB Dittenber DA Quast JF 1996 Evaluation of the developmental and reproductive toxicity of chlorpyrifos in the rat Fundam Appl Toxicol 29 119 130 8838647
CDC 2003. Second National Report on Human Exposure to Environmental Chemicals. Atlanta, GA:Centers for Disease Control and Prevention. Available: http://www.cdc.gov/exposurereport/2nd/report_results.htm [accessed 14 October 2004].
Duty SM Calafat AM Silva MJ Brock JW Ryan L Chen Z 2004 The relationship between human environmental exposure to phthalates and computer aided sperm analysis motion parameters J Androl 25 293 302 14760016
Everett RW 1982 Effect of Dursban 44 on semen output of Holstein bulls J Dairy Sci 65 1781 1794 6183301
Ghosh P Bhattacharya S Bhattacharya S 1990 Impairment of the regulation of gonadal function in Channa punctatus by metacid-50 and carbaryl under laboratory and field conditions Biomed Environ Sci 3 106 112 2109984
Hauser R Chen Z Pothier L Ryan L Altshul L 2003 The relationship between human semen parameters and environmental exposure to polychlorinated biphenyls and p,p ’-DDE Environ Health Perspect 111 1505 1511 12948891
Hill RH Jr Head SL Baker S Gregg M Shealy DB Bailey SL 1995 Pesticide residues in urine of adults living in the United States: reference range concentrations Environ Res 71 99 108 8977618
Hosmer DW JrLemeshow S 1989. Model building strategies and methods for logistic regression. In: Applied Logistic Regression (Hosmer DW Jr, Lemeshow S, eds). New York:John Wiley & Sons, 82–134.
Kidd SA Eskenazi B Wyrobek AJ 2001 Effects of male age on semen quality and fertility: a review of the literature Fertil Steril 75 237 248 11172821
Kruger TF Acosta AA Simmons KF Swanson RJ Matta JF Oehninger S 1988 Predictive value of abnormal sperm morphology in in vitro fertilization Fertil Steril 49 112 117 3335257
Lewis RG 2000. Pesticides. In: Indoor Air Quality Handbook (Samet J, Spenger JD, eds). New York:McGraw-Hill, 35.1–35.21.
Luca D Balan M 1987 Sperm abnormality assay in the evaluation of the genotoxic potential of carbaryl in rats Morphol Embryol (Bucur) 33 19 22 2953961
MacIntosh DL Needham LL Hammerstrom KA Ryan PB 1999 A longitudinal investigation of selected pesticide metabolites in urine J Expo Anal Environ Epidemiol 9 494 501 10554151
Maroni M Colosio C Ferioli A Fait A 2000 Biological monitoring of pesticide exposure: a review. Introduction Toxicology 143 1 118 10675783
Meeker JD Barr DB Bennett DH Ryan L Herrick RF Bravo R In press. Temporal variability of urinary levels of nonpersistent insecticides in adult men. J Expo Anal Environ Epidemiol.
Nolan RJ Rick DL Freshour NL Saunders JH 1984 Chlorpyrifos: pharmacokinetics in human volunteers Toxicol Appl Pharmacol 73 8 15 6200956
Padungtod C Savitz DA Overstreet JW Christiani DC Ryan LM Xu X 2000 Occupational pesticide exposure and semen quality among Chinese workers J Occup Environ Med 42 982 992 11039162
Pant N Shankar R Srivastava SP 1996 Spermatotoxic effects of carbaryl in rats Hum Exp Toxicol 15 736 738 8880208
Pant N Srivastava SC Prasad AK Shankar R Srivastava SP 1995 Effects of carbaryl on the rat’s male reproductive system Vet Hum Toxicol 37 421 425 8592826
Pasqualotto FF Sharma RK Nelson DR Thomas AJ Agarwal A 2000 Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing infertility investigation Fertil Steril 73 459 464 10688996
Rao B Soufir JC Martin M David G 1989 Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility Gamete Res 24 127 134 2793053
Rawlings NC Cook SJ Waldbillig D 1998 Effects of the pesticides carbofuran, chlorpyrifos, dimethoate, lindane, triallate, trifluralin, 2,4-D, and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes J Toxicol Environ Health A 54 21 36 9588346
Rybakova MN 1966 Toxic effect of Sevin on animals Hyg Sanit (USSR) 31 402 407
Sharma RK Agarwal A 1996 Role of reactive oxygen species in male infertility Urology 48 835 850 8973665
Shtenberg AI Rybakova MN 1968 Effect of carbaryl on the neuroendocrine system of rats Food Cosmet Toxicol 6 461 467 5753604
Soderpalm-Berndes C Onfelt A 1988 The action of carbaryl and its metabolite alpha-naphthol on mitosis in V79 Chinese hamster fibroblasts. Indications of the involvement of some cholinester in cell division Mutat Res 201 349 363 3140000
Swan SH Kruse RL Liu F Barr DB Drobnis EZ Redmon JB 2003 Semen quality in relation to biomarkers of pesticide exposure Environ Health Perspect 111 1478 1484 12948887
Teass AW Biagini RE DeBord DG Hull RD 1998. Application of biological monitoring methods. In: NIOSH Manual of Analytical Methods (Eller PM, ed). Cincinnati, OH:National Institute for Occupational Safety and Health, 52–62.
WHO 1999. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed. New York:Cambridge University Press.
Whorton MD Milby TH Stubbs HA Avashia BH Hull EQ 1979 Testicular function among carbaryl-exposed exployees J Toxicol Environ Health 5 929 941 117116
Wyrobek AJ Watchmaker G Gordon L Wong K Moore D II Whorton D 1981 Sperm shape abnormalities in carbaryl-exposed employees Environ Health Perspect 40 255 265 6791917
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7249ehp0112-00167115579411ResearchArticlesEffects of Loud Noise Exposure on DNA Integrity in Rat Adrenal Gland Frenzilli Giada Lenzi Paola Scarcelli Vittoria Fornai Francesco Pellegrini Antonio Soldani Paola Paparelli Antonio Nigro Marco Dipartimento di Morfologia Umana e Biologia Applicata, University of Pisa, Pisa, ItalyAddress correspondence to G. Frenzilli, Dipartimento di Morfologia Umana e Biologia Applicata, Sez. Biologia e Genetica University of Pisa, Via Volta, 4-56126 Pisa, Italy. Telephone: 39-050-2219111. Fax: 39-050-2219101. E-mail:
[email protected] gratefully acknowledge C. Ghezzani for his excellent technical assistance in the image analysis.
This work was supported by the Italian Ministry of Research and by the University of Pisa.
The authors declare they have no competing financial interests.
12 2004 22 9 2004 112 17 1671 1672 12 5 2004 22 9 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Loud noise is generally considered an environmental stressor causing negative effects on acoustic, cardiovascular, nervous, and endocrine systems. In this study, we investigated the effects of noise exposure on DNA integrity in rat adrenal gland evaluated by the comet assay. The exposure to loud noise (100 dBA) for 12 hr caused a significant increase of DNA damage in the adrenal gland. Genetic alterations did not decrease 24 hr after the cessation of the stimulus. We hypothesize that an imbalance of redox cell status is responsible for the induction and persistence of noise-induced cellular damage.
adrenal glandcomet assayDNA damageloud noiserat
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During daily life, people are exposed to potentially hazardous noise levels related to work environment, urban traffic, household appliances, discos, and the like (Kawecka-Jaszcz 1991; Lang et al. 1992). The World Health Organization (Berglund et al. 1999) estimated that approximately 20% of the European population is exposed to noise generated by urban traffic > 65 dBA, a level regarded as a maximum safety threshold, whereas 40% of Europeans are exposed to noise levels between 55 and 65 dBA, which might be responsible for several disorders of both auditory and extra-auditory organs (Berglund et al. 1999).
Extraauditory effects of noise have been related to psychophysiologic stress and the involvement of the pituitary–adrenocortical axis (Axelrod and Reisine 1984; Ising and Braun 2000). Most studies on the effects of noise exposure on the hypothalamus–pituitary–adrenocortical axis have been performed by measuring behavioral, endocrine, and biochemical variables (Alario et al. 1987; Armario et al. 1984; Borrell et al. 1980), whereas few studies have investigated the cellular effects induced by exposure to noise stress. Among these, Pellegrini et al. (1997) and Soldani et al. (1999) demonstrated the occurrence of ultrastructural modifications in the adrenal gland of noise exposed rats. Moreover, recent findings showed that ultra-structural alterations in the rat myocardium detected after loud noise exposure were also accompanied by DNA damage (Lenzi et al. 2003).
The purpose of the present study was to investigate whether levels of loud noise comparable with those present in modern daily life (Baker 1993; Berglund et al. 1999; Brüel 1970; Figure 1) were able to produce DNA damage in rat adrenal gland for the same doses and time intervals previously detected as effective for inducing cellular alterations in the heart (Lenzi et al. 2003).
Materials and Methods
Animals.
Male Wistar rats weighing 200–250 g (Harlan Labs, San Pietro al Natisone, Italy) were used for the experiments. Animals were housed in the animal facility, fed ad libitum, and kept under closely controlled environmental conditions (12 hr light:dark cycle, lights on between 0700 and 1900 hr; room temperature, 21°C). Animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health 1996). All possible efforts were made to reduce animal suffering and minimize the number of animals used.
Experimental procedures.
Noise level was set at 100 dBA by the use of two loud speakers (15 W) (Lenzi et al. 2003) and lasted for 12 hr. Control rats were placed in the same kind of cage without being exposed to noise. Animals were randomly assigned to experimental and control groups, each consisting of four specimens. Experimental rats were sacrificed either soon after cessation of the noise stimulus or 24 hr later by decapitation, to avoid the interference of deep anesthesia with DNA integrity, and the adrenal gland was immediately dissected.
Light microscopy.
To check for potential occurrence of cell death, we processed tissue samples using routine histologic procedures. Briefly, 8-μm-thick sections were cut with a microtome and stained with hematoxylin-eosin and toluidine blue. No cell death was observed.
Evaluation of DNA damage.
We evaluated DNA integrity in rat adrenal gland by the use of alkaline single-cell gel electrophoresis or comet assay, according to Singh et al. (1988), with minor modifications (Lenzi et al. 2003). Electrophoretic DNA migration is proportional to the level of DNA damage producing cometlike images under a fluorescence microscope (magnification 200×) (Figure 2). We used an image analyzer (Komet, version 4; Kinetic Imaging Ltd., Bromborough, UK) to quantify the percentage of DNA migrated in the tail of at least 100 cells per animal. We used multifactor analysis of variance to assess the significance of factor effects such as animals, slides, and doses. For statistical analysis we used the software Statgraphics Plus for Windows (version 2.1; Microsoft Corp., Redmond, WA, USA).
Results
We evaluated the effect of loud noise on the presence of DNA damage in single cells dissociated from adrenal gland as the percentage of migrated DNA after electrophoresis in exposed and control rats. We observed a significant increase of DNA migration (p < 0.001), compared with controls, in the adrenal gland soon after the cessation of acoustic stress, as shown in Figure 3. This pattern of DNA migration persisted 24 hr after noise exposure, suggesting the absence of recovery (Figure 3). Light microscopy did not reveal the occurrence of cell death. This finding excludes the possibility that the number of strand breaks observed in the present study is due to nonspecific loss of DNA integrity related to cell death processes, providing supporting evidence of a genotoxic effect induced by loud noise.
Discussion
This study demonstrates that loud noise exposure produces a significant loss of DNA integrity in the rat adrenal gland. This effect persisted almost unchanged 24 hr after the cessation of the stimulus. We can exclude the possibility that the elevation of DNA strand breaks was due to cell-death–associated fragmentation; indeed, light microscopy revealed a negligible occurrence of necrotic events. The same level and duration of the acoustic stress (100 dBA for 12 hr) were previously demonstrated to be effective in inducing ultrastructural alterations in rat adrenal cells, mainly involving the mitochondria and endoplasmic reticulum (Pellegrini et al. 1997). The adrenal gland is known to react to stressful stimuli, including noise. According to Ising and Braun (2000), habitual noise produces sympathetic activation and chronic increases in noradrenaline; nonhabitual noise produces an acute increase of noradrenaline and adrenaline; and extremely intense noise produces a defeat reaction with an increase of cortisol and adrenal stress hormone. The intense functional stimulation has been reported as potential cause for morphologic changes in subcellular structure, involving those organelles where steroids are synthesized, such as smooth endoplasmic reticulum and mitochondria (Simpson and Waterman 1988; Soldani et al. 1999).
Concerning the persistence of genetic damage, it is noteworthy that DNA single-strand breaks are usually repaired within 15 min and that DNA double-strand breaks are repaired within 2 hr (Plappert et al. 1997; Vijayalaxmi et al. 1993). Thus, such a maintenance of genotoxic effects 24 hr after noise exposure might be the consequence of a long-lasting clastogenic agent.
Our results on DNA damage might be interpreted as the output of two main events, namely, the clastogenic effect of oxyradicals and/or the DNA repair of oxidized bases, which implies the expression of alkali-labile sites, detected by the alkaline comet assay.
The negative effects of noise on cell structure and function were supposed to be, at least in part, mediated by the increase of reactive oxygen species (ROS) (Lenzi et al. 2003). ROS levels in the cochlea were found the be significantly higher 1 hr after exposure to 110 dB noise (Ohlemiller et al. 1999a), persisting after the cessation of the exposure (Ohlemiller et al. 1999b). In this respect, it is worthy to note that DNA is a main target of ROS toxicity (Cross et al. 1987; Lemasters et al. 1992). Oxidative damage of DNA is known to induce single-strand breaks and inter-/intrastrand cross-links (Caraceni et al. 1997). The involvement of ROS might play a causal role in the induction and persistence of genetic damage related to loud noise exposure also in extra-auditory organs. Indeed, Van Campen et al. (2002) reported an elevation of 8-hydroxy-2′-deoxyguanosine in brain and liver (besides the higher cochlear involvement) of rats exposed to loud noise (120 dB). According to these findings, the association between noise exposure, oxidative processes, and persisting DNA damage deserves further attention due to the long-lasting consequences in term of mutagenic and carcinogenic risk (Emerit 1994; Preston-Martin et al. 1989).
Figure 1 Sources and levels of noise exposure. Data represent a synthesis of data from different sources (see “Materials and Methods”).
*WHO safeness threshold limit (Berglund et al. 1999).
Figure 2 Images of ethidium-bromide–stained nuclei exhibiting different degrees of DNA damage after electrophoresis. The amount of DNA damage increases from A to E, as shown by the percentage of “tail” DNA: (A) 0.5%, (B) 10%, (C) 45%, (D) 93%, and (E) 99%. Bar = 20 μm.
Figure 3 DNA damage induced by loud noise exposure in rat adrenal gland cells soon after 12 hr of noise exposure (t = 0) and 24 hr after the cessation of the stimulus (t = 24). Data are expressed as mean ± SD of DNA migrations.
*p < 0.01. **p < 0.001.
==== Refs
References
Alario P Beato MJ Trancho G 1987 Body weight gain, food intake and adrenal development in chronic noise stressed rats Physiol Behav 40 29 32 3039551
Armario A Castellanos JM Balash J 1984 Adaptation of anterior pituitary hormones to chronic noise stress in rats Behav Neural Biol 41 71 76 6431962
Axelrod J Reisine TD 1984 Stress hormones: their interaction and regulation Science 224 452 459 6143403
Baker DE 1993. Noise: The Invisible Hazard. Columbia, MO:University of Missouri Extension.
Berglund B Lindvall T Schwela DH eds. 1999. Guidelines for Community Noise. London:World Health Organization.
Borrell J Torrellas A Guaza C Borrell S 1980 Sound stimulation and its effects on pituitary-adrenocortical function and brain catecholamines in rats Neuroendocrinology 31 53 59 7393405
Brüel PV 1970 Do we measure damaging noise correctly? B & K Technical Review 1 3 32
Caraceni P De Maria N Ryu HS Colantoni A Roberts L Maidt ML 1997 Proteins but not nucleic acids are molecular targets for the free radical attack during reoxygenation of rat hepatocytes Free Radic Biol Med 23 339 344 9199897
Cross DE Halliwell B Borish ET Pryor WA Ames BA Saul RS 1987 Oxygen radicals and human disease Ann Intern Med 107 526 545 3307585
Emerit I 1994 Reactive oxygen species, chromosome mutation, and cancer Free Radical Biol Med 16 99 109 8300000
Kawecka-Jaszcz K 1991 Effect of professional work and environmental factors on arterial blood pressure Med Pract 42 291 296
Ising H Braun C 2000 Acute and chronic endocrine effects of noise: review of the research conducted at the Institute for Water, Soil and Air Hygiene Noise Health 7 7 24 12689468
Lang T Fouriaud C Jacquinet-Salord MC 1992 Length of occupational noise exposure and blood pressure Int Arch Occup Environ Health 63 369 372 1544682
Lemasters JJ Caldwell-Kenkel JC Gao W Nieminen AL Herman B Thurman RG 1992. Hypoxic, ischemic and reperfusion injury in the liver. In: Pathophysiology of Reperfusion Injury (Das DK, ed). Boca Raton, FL:CRC, 101–135.
Lenzi P Frenzilli G Gesi M Ferrucci M Lazzeri G Fornai F 2003 DNA damage associated with ultrastructural alterations in rat myocardium after loud noise exposure Environ Health Perspect 111 467 471 12676600
National Institutes of Health 1996. Guidelines for Care and Use of Laboratory Animals. Washington, DC:National Academies Press.
Ohlemiller KK McFadden SL Ding DL Flood DG Reaume AG Hoffman EK 1999a Targeted deletion of the cytosolic Cu/Zn-superoxide dismutase gene (Sod1) increases susceptibility to noise-induced hearing loss Audiol Neurootol 5 237 246 10436316
Ohlemiller KK Wright JS Dugan LL 1999b Early elevation of cochlear reactive oxygen species following noise exposure Audiol Neurootol 4 229 236 10436315
Pellegrini A Soldani P Gesi M Lenzi P Natale G Paparelli A 1997 Effect of varying noise stress duration on rat adrenal gland: an ultrastructural study Tissue Cell 29 597 602 9364808
Plappert UG Stocker B Fender H Fliedner TM 1997 Changes in the repair of blood cells as a biomarker for chronic low-dose exposure to ionizing radiation Environ Mol Mutagen 30 153 160 9329640
Preston-Martin S Thomas DC Wright WE Henderson BE 1989 Noise trauma in the aetiology of acoustic neuromas in men in Los Angeles County. 1978–1985 Br J Cancer 59 783 786 2736213
Simpson ER Waterman MR 1988 Regulation of the synthesis of steroidogenic enzymes in adrenal cortical cells by ACTH Annu Rev Physiol 50 427 440 2837136
Singh NP McCoy MT Tice RR Schneider EL 1988 A simple technique for quantitation of low levels of DNA damage in individual cells Exp Cell Res 175 184 191 3345800
Soldani P Gesi M Lenzi P Natale G Fornai F Pellegrini A 1999 Long-term exposure to noise modifies rat adrenal cortex ultrastructure and corticosterone plasma levels J Submicrosc Cytol Pathos 31 441 448
Van Campen LE Murphy WJ Franks JR Mathias PI Toraason MA 2002 Oxidative DNA damage is associated with intense noise exposure in the rat Hear Res 164 29 38 11950522
Vijayalaxmi Strauss GHS Tice RR 1993 An analysis of γ -ray-induced DNA damage in human blood leukocytes, lymphocytes and granulocytes Mutat Res 292 123 128 7692248
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7005ehp0112-00167315579412ResearchArticlesProspective Study of Blood and Tibia Lead in Women Undergoing Surgical Menopause Berkowitz Gertrud S. Wolff Mary S. Lapinski Robert H. Todd Andrew C. Department of Community and Preventive Medicine, Mount Sinai School of Medicine, New York, New York, USAAddress correspondence to G.S. Berkowitz, Department of Community and Preventive Medicine, Mount Sinai School of Medicine, Box 1172, One Gustave L. Levy Place, New York, NY 10029-6574 USA. Telephone: (212) 241-8954. Fax: (212) 241-3475. E-mail:
[email protected] thank K. Paulate, J. Hutagalung, N. Ginde, and J. Tolman for their analysis of blood and bone lead levels for this study.
This research was supported by grant P42 ES07384 from the National Institute of Environmental Health Sciences.
The authors declare they have no competing financial interests.
12 2004 7 9 2004 112 17 1673 1678 6 2 2004 7 9 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Despite the dramatic decline in environmental lead exposure in the United States during the past couple of decades, concern has been expressed regarding mobilization during menopause of existing lead stored in bone. To investigate whether bone lead concentrations decrease and blood lead levels increase, we conducted a prospective study of 91 women who were scheduled to undergo a bilateral oophorectomy for a benign condition at Mount Sinai Hospital in New York City during October 1994 through April 1999. We excluded women who were younger than 30 years of age or who were postmenopausal at the time of the surgery. We observed a small but significant increase in median blood lead levels between the baseline visit and the 6-month visit (0.4 μg/dL, p < 0.0001), particularly for women who were not on estrogen replacement therapy (0.7 μg/dL, p = 0.008). No significant change was observed in blood lead values between 6 and 18 months postsurgery, nor was there evidence of significant changes in tibia lead concentrations during the follow-up period. These findings do not point to substantial mobilization of lead from cortical bone during menopause.
blood leadbone turnoverestrogen replacement therapylead mobilizationtibia lead
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Although there has been a substantial decline in lead exposure in the United States during the past couple of decades (Pirkle et al. 1994), mobilization of existing lead stored in bone potentially represents an important endogenous source of exposure. Specifically, it has been hypothesized that lead may be mobilized from skeletal stores during conditions of high bone turnover, such as during menopause (Silbergeld et al. 1988). Approximately 90–95% of the total body burden of lead is retained in bone (Barry 1975; Barry and Mossman 1970), where the half-life can be several decades (Börjesson et al. 1997; Gerhardsson et al. 1993; Nilsson et al. 1991; Price et al. 1992; Rabinowitz et al. 1976). During menopause, calcium and other minerals are mobilized from bone (Pounds 1984; Bronner 1992; O’Flaherty 1992; Simons 1993). Lead is covalently bound in the mineral matrix, apparently in close chemical association with calcium and phosphate (Wittmers et al. 1988). Furthermore, lead is concentrated selectively according to the type of bone, with higher accumulations in trabecular as opposed to cortical bone (Wittmers et al. 1988; Inskip et al. 1992; Lindquist et al. 1981). It has been estimated that up to 50% of trabecular bone and 30% of cortical bone is lost during a woman’s lifetime, particularly during the early menopausal years (Lindquist et al. 1981; Heaney et al. 1978; Riggs and Melton 1986). Lead is mobilized from the bone into the blood compartment. Lead in blood can then be transferred to soft tissues, including the central nervous system, where it could affect cognitive and motor functions (Landrigan et al. 1982; Ryan et al. 1987).
Age-adjusted data from the second National Health and Nutrition Examination Survey (NHANES II, 1976–1980; Silbergeld et al. 1988), the Hispanics HANES (HHANES, 1982–1984; Symanski and Hertz-Picciotto 1995), and NHANES III (1988–1994; Nash et al. 1998) showed higher blood lead levels among postmenopausal women compared with premenopausal women. Similarly, blood lead levels were higher in postmenopausal women compared with premenopausal women in a subsample of the Nurses Health Study (Korrick et al. 2002) and two studies in Mexico City (Garrido Latorre et al. 2003; Hernandez-Avila et al. 1998). However, these studies were all based on cross-sectional data, and only two investigations (Garrido Latorre et al. 2003; Korrick et al. 2002) had any information on bone lead concentrations.
This investigation represents a longitudinal study with repeated measures of blood and tibia lead and bone mineral density (BMD) measurements among women undergoing surgically induced menopause. In addition to assessing whether there was any evidence of increased endogenous lead exposure as a result of the surgical menopause, we aimed to evaluate the effects of BMD, estrogen replacement therapy (ERT), serum ferritin, and endogenous estrogen levels on changes in blood and bone lead measurements.
Materials and Methods
The study population was recruited from women ≥ 30 years of age who had a surgical admission or discharge diagnosis of a bilateral oophorectomy for a benign condition at Mount Sinai Hospital during October 1994 through April 1999. Excluded were women with preexisting neurologic or psychiatric diseases and any medical condition that could affect bone homeostasis. Also excluded were women who were taking corticosteroids, thyroid hormone replacement, or antiseizure medications. Women who had had no menses within the previous 6 months or fewer than nine menstrual periods within the past year were considered to be postmenopausal and were therefore not included. The final study population comprised 91 premenopausal or perimenopausal women.
The study protocol included a baseline visit before or shortly after surgery and follow-up assessments at 6 and 18 months after surgery. At the baseline visit, a structured questionnaire was administered; 25 cc blood was obtained for blood lead, serum ferritin, and hormone analysis; tibia lead concentration was determined via 109Cd-based K shell X-ray fluorescence (XRF) analyses; and BMD was measured by dual energy X-ray absorptiometry (DXA). The 6-month visit included all of the preceding measures except for the BMD assessment. The 18-month assessment was identical to the baseline evaluation. The research protocol was approved by the institutional review board of Mount Sinai Hospital, and written informed consent was obtained from all patients.
An attempt was made to obtain the baseline assessment of each patient before surgery. This was often not feasible because of the short lead time for the surgical admission and the fact that the decision to perform a bilateral as opposed to a unilateral oophorectomy was frequently not made until during the procedure. As a result, 58.2% had a baseline assessment before surgery, and 41.8% had the baseline assessment within 2–29 days after the procedure.
Information on covariates, such as socio-demographic characteristics; height and weight; occupational and environmental exposures; medical, gynecologic, and obstetrical history, and use of medications including ERT; physical activity, cigarette smoking, and alcohol consumption, was obtained from the questionnaire. Serum ferritin levels, which are indicative of iron stores, were determined at each visit because there is some evidence that high ferritin levels are associated with lower blood lead concentrations (Baghurst et al. 1987). Estradiol levels were assessed as an indicator of endogenous estrogen levels. Levels of follicle-stimulating hormone, which is a marker of reproductive senescence, were also assessed to verify that the patients were not postmenopausal at the time of the surgery.
Blood lead was determined using graphite furnace atomic absorption spectrophotometry with Zeeman background correction (model 4100ZL; Perkin Elmer, Norwalk, CT) using the method of Parsons (1992) at the Mount Sinai Lead Laboratory. The lead laboratory was certified by the Occupational Safety and Health Administration (OSHA) and participated in two proficiency testing programs for blood lead (Centers for Disease Control and Prevention and the Wisconsin State Laboratory of Hygiene and College of American Pathologists). OSHA certification requires that proficiency tests come within 6 μg/dL of the target value (or all-method mean) if that value is < 40 μg/dL or within 15% of the target (or all-method mean) if the value is > 40 μg/dL. During a 1-year period while these samples were being analyzed, the accuracy was within 5% or on average < 0.2 μg/dL deviation from target values for 48 proficiency test samples (analyzed in masked fashion) across a wide range of values (0–100 μg/dL). A subsample of triplet samples (baseline, 6-month, and 18-month specimens) was run on the same day in the same laboratory batch for 37 women.
The bone lead measurements were performed on the anterior, middiaphysis of the left tibia, which consists primarily of cortical bone. BMD was measured for left radius/ulna, left hip femoral neck, left hip trochanter, whole left leg (which included both the tibia and the femur), lumbar spine, and whole body. The measurements were obtained with a Hologic QDR 2000 DXA densitometer (Hologic, Bedford, MA) at the Bone Densitometry Laboratory at Mount Sinai Hospital. The scans were analyzed according to computer software protocols for each site provided by the manufacturer.
The XRF method sometimes produces negative results for low bone lead concentrations. This is because the method produces an unbiased (Todd et al. 2002) point estimate of the true concentration that oscillates, because of measurement uncertainty, around the true bone lead concentration. Other researchers (Hu et al. 1998; Kim et al. 1995) have examined the retention of the negative values in the analyses of data from epidemiologic studies and have recommended the retention of all data because alternative procedures (e.g., setting the negative values to zero or to half the value of the detection limit) introduce bias.
It is not possible to assign a specific detection limit to the XRF measurements. Each lead X ray (and the coherent scatter) peak of each in vivo bone lead measurement spectrum has a detection limit (defined in any one of a number of ways). There is therefore no single spectrum-based detection limit value for an individual bone lead measurement. Furthermore, there is an “instrumental detection limit,” which is usually superior to the more realistic method detection limit. In addition, there is a “system performance level” (Todd et al. 1993) and other detection limit definitions described by the International Union on Pure and Applied Chemistry (Todd et al. 2001, 2002). The XRF measurement uncertainty could be used to establish a degree of confidence in the lead concentration, but those uncertainties have been shown to underestimate the standard deviation of repeated measurements (Todd et al. 2001). Nevertheless, most of the tibia lead levels in this study could be described as at or near the method detection limit.
Because tests of normality showed that the blood lead and tibia lead values were not normally distributed (Shapiro-Wilk’s test, p < 0.0001 and < 0.03, respectively), medians are presented. The distributions of blood and tibia lead levels were evaluated by Wilcoxon rank sum test or, if there were more than two categories, the Kruskal-Wallis test. Covariates that were either categorical or continuous were assessed by chi-square or Student’s t-test, respectively. Relationships between BMD and blood or tibia lead concentrations were evaluated by Spearman’s correlation coefficient. Changes in blood and bone lead levels from baseline to 6 months, baseline to 18 months, and from 6 to 18 months were evaluated by the Wilcoxon signed rank test. Changes in blood and tibia lead levels adjusted for covariates were evaluated with multiple linear regressions.
Results
The study population consisted of 91 premenopausal and perimenopausal women ≥ 30 years of age who were scheduled to undergo a bilateral oophorectomy for a benign condition at Mount Sinai Hospital during October 1994 through April 1999.
Among the 91 women who enrolled in the study, 71 completed the 6-month visit and 63 completed the 18-month visit. The age distribution of the 91 women was as follows: 15.4% 30–44 years of age, 53.9% 45–49 years of age, and 30.8% 50–54 years of age. With respect to race/ethnicity, 52.8% were white, 16.5% were African American, 9.9% were Hispanic, and 2.3% were Asian. The participants were generally well educated: almost 70% had received college or higher education. Regarding reproductive characteristics, 67.4% had previously been pregnant and 56.2% had previously delivered a live birth.
The proportion of women who reported ERT use was 78.9% at 6 months postsurgery and 77.8% at 18 months. The proportion of ERT users who were taking a dose of 0.625 mg was 83.9% at the 6-month follow-up and 74.5% at the 18-month assessment. Among the users, 80.8% had stayed on ERT for the period between the surgery and the 6-month visit, and 53.0% had remained on ERT during the period between surgery and the 18-month visit. Current smokers comprised 18.7% of the women, and 50.5% reported consuming one or more alcoholic drinks per week. Those who were lost to follow-up were less well educated (p = 0.02) and had a marginally higher body mass index (BMI; p = 0.06) than did those who completed the follow-up visits. Other characteristics did not differ between the two groups. Furthermore, there was no significant difference in blood lead levels at baseline for those who were lost to follow-up compared with those who remained in the study, although the former group had a somewhat higher blood lead level (3.1 μg/dL vs. 2.4 μg/dL; p = 0.23).
The median blood lead (2.5 μg/dL; range, 0.3–11.7 μg/dL) and tibia lead (6.0 μg/g bone mineral; range, −22.2 to 36.4 μg/g) levels were low at baseline. The median blood lead levels were not significantly different for those who had the blood drawn before (2.2 μg/dL) as opposed to after the surgery (2.6 μg/dL, p = 0.65). There were no significant differences in median blood lead levels or changes in the blood lead levels over time when the triplicate samples that were analyzed in the same batch were compared with the samples analyzed in separate batches. Table 1 presents the median blood lead and tibia lead levels according to selected sociodemographic and lifestyle characteristics. A significant positive association was observed between number of alcoholic drinks per week and median blood lead level. There was some suggestion that blood lead levels increased with age, decreased with increasing BMI, and were lower for women who had never smoked and those who were on ERT at 6 months, but none of these results was statistically significant. The blood lead levels for the four racial/ethnic groups were similar. No association was seen for parity (data not shown).
With respect to tibia lead, significant positive associations were observed both for current cigarette smoking and the number of alcoholic drinks per week. Tibia lead levels tended to increase with age, as expected, and tended to be lower for those on ERT at 6 months. The tibia lead levels were slightly higher for African Americans and Hispanics compared with whites or Asians.
Assessment of other potential lead-related variables such as occupations, hobbies, and residential characteristics (e.g., peeling paint) revealed no significant findings, although women who reported a hobby involving potential lead exposure, such as making jewelry or stained glass, had slightly higher blood lead levels than did those who had no such hobby (Table 2). The increased blood lead levels for those who exercised on a regular basis (> 1 hr/week) is difficult to understand, because bone turnover is generally less in women who exercise (Wolff et al. 1999). Women who had ever used herbal medicines had a borderline significant elevated blood lead level. Apart from the higher tibia lead levels among women who reported a history of hyperthyroidism, no other significant findings were observed with respect to characteristics potentially related to tibia lead levels.
As expected, significant negative declines from the baseline to the 18-month BMD assessments were seen for the lumbar spine (paired t-test, p < 0.0001), the left hip femoral bone (p = 0.004), and the left hip trochanter (p = 0.005). The decline was particularly marked for the lumbar spine for those who had not been taking ERT, but a significant decrement at this site did occur even for those who had used ERT (p = 0.003). There was only a slight and nonsignificant (p > 0.05) drop in the left whole-leg BMD between the baseline and 18-month follow-up assessment, which was limited to those who were not on ERT. However, because bone lead was measured in the left tibia, adjustment for BMD was only based on the left leg. No significant correlations (based on the Spearman correlation coefficient) were seen between left-leg BMD and blood lead values either at baseline or the 18-month follow-up or the change in blood lead levels. With respect to the correlation with tibia lead concentrations, there was a significant positive relationship between the left-leg BMD at baseline and the change in tibia lead between 0 baseline and 6 months (rs = 0.31, p = 0.02) but no correlation at the 18-month follow-up.
Table 3 shows the median blood and tibia lead levels at baseline and at the 6-month and 18-month follow-up visits. Two women had no blood lead determinations at baseline, and seven women did not have a tibia lead assessment at baseline. The sample sizes for the 6-month and 18-month assessments are also given in Table 3. Because not all women participated in the 6-month and 18-month follow-up assessments, changes in individual blood and tibia lead levels are of greater relevance. Table 4 summarizes the median changes over the follow-up period. It may be seen that median blood lead levels increased significantly during the first 6 months but did not change significantly between 6 and 18 months postsurgery. Although the increase during the first 6 months was greater for women who were not on ERT, both women with and without ERT experienced significant increases. Similar changes were observed for the tibia lead concentrations for all women, but none of the changes was statistically significant. The tibia lead changes according to ERT status are more difficult to interpret. There was a marginal decline in tibia lead concentrations between 6 and 18 months for women who took ERT, but not for those who did not take ERT. It should be noted that some of these results may reflect the different sample sizes.
Assessment of Spearman correlation coefficients between the change in blood lead and tibia lead showed a borderline significant result for the change in blood lead and tibia lead between baseline and 18 months (rs = −0.26, p = 0.05). The rs for the change in blood and tibia lead between 6 and 18 months was 0.22 (p = 0.10). Because the correlations went in the opposite directions, these findings are not easily interpretable.
Multiple regression analysis was used to further explore the significant increase in blood lead levels between baseline and 6 months postsurgery. Variables that were considered included blood lead at baseline, alcohol consumption, estradiol and serum ferritin levels at 6 months, tibia lead adjusted for BMD of the left leg at baseline, and change in tibia lead between baseline and 6 months adjusted for BMD. The results are summarized in Table 5. The r
2 for this model was 0.22. It may be seen that the endogenous level of estradiol at 6 months, the BMD-adjusted tibia lead level at baseline, and the change in BMD-adjusted tibia lead level between baseline and 6 months were significant predictors of the change in blood lead between baseline and 6 months. Blood lead at baseline was not significant but was included in the model because exclusion of this variable resulted in a borderline significance for estradiol (p = 0.06). No significant interaction was observed between ERT use and baseline tibia lead level adjusted for BMD in this model (p = 0.38).
Discussion
Despite the dramatic decline in environmental lead exposure that has occurred in the United States since the 1980s, certain subgroups, such as poor innercity residents and minorities, remain more likely to have elevated levels of blood lead. Pregnant and lactating women (Gulson et al. 2003; Tellez-Rojo et al. 2002) and those undergoing menopause (Nash et al. 1998) have been identified as additional groups who may be at risk for increased blood lead levels because of potential lead mobilization during conditions of high bone turnover. To date, however, there are no published prospective studies that have assessed blood lead, bone lead, and BMD changes during these conditions. Possible increases in levels of blood lead during menopause are of concern because studies of adults have shown neuro-cognitive deficits (Muldoon et al. 1994; Payton et al. 1998) and increased blood pressure (Nash et al. 2003; Symanski and Hertz-Picciotto 1995) even at relatively low blood lead levels.
Our data suggest a slight but significant increase in blood lead between baseline and 6 months after a bilateral oophorectomy. This increase was evident both for those on ERT and those who were not on ERT, although the increase was greater for the latter group. No significant changes in blood lead levels were seen between 6 and 18 months postsurgery. With respect to tibia lead concentrations, there was some suggestion of an increase between baseline and the 6-month follow-up for those who were not on ERT therapy and a decline between 6 and 18 months for those who were on ERT therapy. Thus, these findings do not point to any substantial lead mobilization during menopause. The fact that close to 80% of the women were on ERT postsurgery may explain the findings because ERT reduces bone resorption (Prestwood et al. 2000). Alternatively, current bone lead concentrations may be sufficiently low to result in the release of only small amounts of lead into the bloodstream.
Previous studies on the effects of ERT on blood and tibia lead levels are not entirely consistent. A small cross-sectional study of blood lead concentrations among postmenopausal women either on ERT or calcium supplementation found that ERT may reduce the release of lead from bone (Webber et al. 1995). However, this was evident only for cortical (tibia) and not trabecular (calcaneus) bone, even though trabecular bone is thought to be more sensitive to estrogen declines than is cortical bone. Furthermore, ERT had no effect on blood lead concentrations in the latter study. Analysis of a subgroup from the Nurses’ Health Study (Korrick et al. 2002) found higher blood lead levels in postmenopausal women who were not taking estrogens than either premenopausal women or postmenopausal women who were using ERT. Bone lead was positively associated with blood lead only among postmenopausal women who were not using ERT, and this was true both for trabecular (patella) and cortical (tibia) bone lead. A Mexican cross-sectional osteoporosis-screening study reported that trabecular bone lead (patella) was an important predictor of blood lead in postmenopausal women both for those with a natural or surgical menopause (Garrido Latorre et al. 2003). Users of ERT also had lower blood lead levels than did nonusers in this study. In contrast, another Mexican study found significantly higher blood lead values in women with a natural compared with a surgical menopause but no difference according to ERT use (Hernandez-Avila et al. 2000). Analysis of NHANES III data for 1988–1994 showed lower blood lead levels among postmenopausal women who were current ERT users compared with past or never users (Nash et al. 1998). Two studies that also assessed BMD found no association between BMD and blood lead values (Garrido Latorre et al. 2003; Muldoon et al. 1994).
With respect to other correlates of blood lead levels, positive associations have been reported with increasing age (Hernandez-Avila et al. 2000; Korrick et al. 2002; Muldoon et al. 1994; Weyermann and Brenner 1997), cigarette smoking (Muldoon et al. 1994; Weyermann and Brenner 1997), and alcohol consumption (Korrick et al. 2002; Muldoon et al. 1994; Weyermann and Brenner 1997). Alcohol consumption was significantly associated with increased blood lead levels in our data, and nonsignificant positive trends were evident for age and cigarette smoking. Use of herbal remedies has been previously linked to lead poisoning (Centers for Disease Control and Prevention 1993; Markowitz et al. 1994). In the study by Muldoon et al. (1994) of women 65–74 years of age, moderate physical activity was related to decreased blood lead values, but more strenuous activity was associated with increased lead levels. We observed higher blood lead values among women who exercised > 1 hr/week, but our numbers were too small to detect a dose–response relationship.
Only limited data are available on characteristics influencing bone lead concentrations. In a study of tibia lead concentrations, Kosnett et al. (1994) reported positive associations with age and cigarette smoking and a negative relationship with a history of lactation. Korrick et al. (2002) found that older age and lower parity were associated with higher tibia lead but only age was related to patella lead levels. We similarly found a significant positive association between tibia lead and cigarette smoking and a positive trend with age. In addition, alcohol consumption significantly increased bone lead concentration. A history of hyperthyroidism was also a significant predictor in our study. Hyperthyroidism, which can cause bone turnover (Goldman et al. 1994), would, however, be expected to be related to higher blood but not bone lead levels.
Because there were significant declines in BMD for the lumbar spine, left hip femoral neck, and left hip trochanter (but not in the left leg or the radius/ulna), there is evidence of bone turnover in this study population. However, the possibility that a release of lead from bone with subsequent redeposition cannot be discounted because tibia lead concentrations did not change significantly over the follow-up period. Nevertheless, tibia bone lead concentrations were adjusted for left-leg BMD in the final model. Another limitation of this study is the fact that the whole-leg BMD rather than just the tibial shaft BMD was measured. Because both the tibia and the femur primarily consist of cortical bone, measurement of the whole leg should not have had any major effect on our results.
Conclusion
We observed a small but significant increase in blood lead between the baseline assessment and the 6-month postsurgical visit, particularly for women who were not on ERT after the surgical menopause. However, no significant changes were observed for the period between 6 and 18 months, nor were there any significant changes in tibia lead concentrations postsurgery. Thus, these data do not support the hypothesis of substantial lead mobilization from cortical bone during menopause.
Table 1 Baseline median blood and tibia lead levels according to sociodemographic and lifestyle characteristics among 91 women with a surgical menopause, Mount Sinai Hospital, 1994–1999.
Blood lead
Tibia lead
Characteristic Median (μg/dL) No. p-Valuea Median (μg/g) No. p-Valuea
Age (years)
30–44 2.1 14 2.4 12
45–49 2.5 47 5.7 46
50–54 2.7 28 0.39 7.6 26 0.32
Race/ethnicity
White 2.6 55 5.7 50
African American 2.1 19 7.5 18
Hispanic 2.4 12 7.2 13
Asian 2.6 3 0.61 6.0 3 0.44
Education
Less than high school 3.4 9 4.4 9
High school graduate 2.5 12 8.6 12
Some college 2.0 19 6.1 18
College graduate 2.6 47 0.39 5.6 45 0.33
BMI (kg/m2)
< 25 2.6 43 6.3 40
25–29.9 2.2 24 6.7 24
> 30.0 2.1 20 0.59 4.5 20 0.49
Cigarette smoking
Never 2.2 40 4.4 39
Ex-smoker 2.5 31 7.1 28
Current 3.4 15 0.14 11.4 16 0.02
Alcohol consumption (drinks/week)
0 1.9 42 3.4 42
1–6 2.6 35 7.6 35
> 7 3.5 9 0.001 9.5 9 0.03
Coffee consumption
0 2.1 22 3.3 23
1–2 2.6 40 7.5 38
> 3 2.7 15 0.63 7.1 14 0.28
ERT at 6 months
No 3.8b 15 10.4b 15
Yes 3.0 56 0.15c 5.8 55 0.11c
a p-Value is based on the Kruskal-Wallis test, unless otherwise indicated.
b Median blood and tibia lead levels at 6 months after oophorectomy.
c p-Value is based on the Wilcoxon rank sum test.
Table 2 Baseline median blood and bone lead levels according to other potential lead-related characteristics among 91 women with a surgical menopause, Mount Sinai Hospital, 1994–1999.
Blood lead
Bone lead
Characteristic Median (μg/dL) No. p-Valuea Median (μg/g) No. p-Valuea
Ever had lead-related hobby
No 2.1 40 5.2 38
Yes 2.6 44 0.12 7.6 43 0.53
History of hyperthyroidism
No 2.5 84 5.7 80
Yes 3.9 3 0.32 13.1 4 0.02
Physical exercise (> 1 hr/week)
No 2.1 28 4.7 27
Yes 2.6 56 0.04 6.9 54 0.21
Ever used herbal medicines
No 2.3 51 6.6 50
Yes 2.6 31 0.05 4.8 29 0.95
a p-Value based on Wilcoxon rank sum test.
Table 3 Median blood and tibia lead levels at baseline, 6 months, and 18 months after oophorectomy by ERT status among 91 women with a surgical menopause, Mount Sinai Hospital, 1994–1999.
Blood lead (μg/dL)
Tibia lead (μg/g)
Follow-up period Median (range) No. Median (range) No.
All women
Baseline 2.5 (0.3–11.7) 89 6.1 (−22.2–36.4) 84
6 months postsurgery 3.2 (0.4–12.0) 71 6.8 (−14.2–29.0) 70
18 months postsurgery 3.1 (0.5–9.1) 63 5.8 (−15.4–24.2) 62
Women on ERT
6 months postsurgery 3.0 (0.4–12.0) 56 5.8 (−14.2–24.3) 55
18 months postsurgery 3.1 (0.5–9.1) 49 4.2 (−15.4–24.2) 46
Women not on ERT
6 months postsurgery 3.8 (1.3–11.6) 15 10.4 (−6.9–29.0) 15
18 months postsurgery 3.2 (1.6–6.7) 14 6.9 (−4.0–19.9) 16
Table 4 Median changes in blood and tibia lead levels during the follow-up period by ERT status among 91 women with a surgical menopause, Mount Sinai Hospital, 1994–1999.
Blood lead (μg/dL)
Tibia lead (μg/g)
Follow-up period Median change (range) p-Valuea No. Median change (range) p-Valuea No.
All women
0–6 months 0.4 (−2.0–9.6) < 0.0001 71 1.8 (−33.3–26.3) 0.22 69
6–18 months −0.1 (−7.6–3.9) 0.36 60 −2.2 (−18.6–22.2) 0.16 58
0–18 months 0.3 (−6.2–4.7) 0.06 63 −0.4 (–34.0–31.3) 0.86 62
Women on ERT postsurgery
0–6 months 0.3 (−2.0–4.4) 0.003 56 0.8 (−33.3–26.3) 0.59 55
6–18 months −0.1 (−7.6–3.9) 0.62 46 −2.8 (−18.6–22.2) 0.06 42
0–18 months 0.2 (−6.2–4.7) 0.16 49 0.1 (−34.0–31.3) 0.86 46
Women not on ERT
0–6 months 0.7 (−0.6–9.6) 0.008 15 5.0 (−10.5–19.7) 0.08 14
6–18 months −0.2 (−2.6–2.1) 0.31 14 2.2 (−14.0–14.9) 0.72 16
0–18 months 0.4 (−1.8–2.2) 0.21 14 −0.8 (−22.4–7.4) 0.49 16
a p-Value is based on the Kruskal-Wallis test.
Table 5 Multivariate model of blood lead changes between baseline and 6 months postsurgery among 91 women with a surgical menopause, Mount Sinai Hospital, 1994–1999.
Variable Parameter estimate SE p-Valuea Partial r2
Intercept 1.36 0.58 0.02 —
Endogenous estradiol level at 6 months −0.01 0.01 0.03 0.09
Blood lead at baseline −0.17 0.12 0.16 0.04
Tibia lead at baseline adjusted for BMDb 0.07 0.03 0.008 0.13
Change in tibia lead at 0–6 months adjusted for BMDb 0.06 0.02 0.01 0.12
a Based on Student’s t-test.
b Tibia lead concentration multiplied by mean left-leg BMD.
==== Refs
References
Baghurst PA McMichael AJ Vimpani GV Robertson EF Clark PD Wigg NR 1987 Determinants of blood lead concentrations of pregnant women living in Port Pirie and surrounding areas Med J Aust 146 69 73 3796424
Barry PS 1975 A comparison of concentrations of lead in human tissues Br J Ind Med 32 119 139 1131339
Barry PS Mossman DB 1970 Lead concentrations in human tissues Br J Ind Med 27 339 351 5488693
Börjesson J Mattsson S Stromberg U Gerhardsson L Schutz A Skerfving S 1997 Lead in fingerbone: a tool for retrospective exposure assessment Arch Environ Health 52 104 112 9124869
Bronner F 1992 Bone and calcium homeostasis Neurotoxicology 13 775 782 1302303
Centers for Disease Control and Prevention 1993 Lead poisoning associated with use of traditional ethnic remedies MMWR Morbid Mortal Wkly Rep 42 521 524
Garrido Latorre F Hernandez-Avila M Tamayo Orozco J Albores Medina CA Aro A Palazuelos E 2003 Relationship of blood and bone lead to menopause and bone mineral density among middle-age women in Mexico City Environ Health Perspect 111 631 636 12676627
Gerhardsson L Attewell R Chettle DR Englyst V Lundstrom NG Nordberg GF 1993 In vivo measurements of lead in bone in long-term exposed lead smelter workers Arch Environ Health 48 147 156 8333784
Goldman RH White R Kales SN Hu H 1994 Lead poisoning from mobilization of bone stores during thyrotoxicosis Am J Ind Med 25 417 424 8160659
Gulson BL Mizon KJ Korsch MJ Palmer JM Donnelly JB 2003 Mobilization of lead from human bone tissue during pregnancy and lactation—a summary of long-term research Sci Total Environ 303 79 104 12568766
Heaney RP Recker RR Saville PD 1978 Menopausal changes in bone remodeling J Lab Clin Med 92 964 970 739174
Hernandez-Avila M Smith D Meneses F Sanin LH Hu H 1998 The influence of bone and blood lead on plasma lead levels in environmentally exposed adults Environ Health Perspect 106 473 477 9681974
Hernandez-Avila M Villalpando CG Palazuelos E Hu H Villalpando ME Martinez DR 2000 Determinants of blood lead levels across the menopausal transition Arch Environ Health 55 355 360 11063411
Hu H Rabinowitz M Smith D 1998 Bone lead as a biological marker in epidemiologic studies of chronic toxicity: conceptual paradigms Environ Health Perspect 106 1 8 9417769
Inskip MJ Franklin CA Subramanian KS Blenkinsop J Wandelmaier F 1992 Sampling of cortical and trabecular bone for lead analysis: method development in a study of lead mobilization during pregnancy Neurotoxicology 13 825 834 1302308
Kim R Aro A Rotnitzky A Amarasiriwardena C Hu H 1995 K X-ray fluorescence measurements of bone lead concentration: the analysis of low-level data Phys Med Biol 40 1475 1485 8532760
Korrick SA Schwartz J Tsaih SW Hunter DJ Aro A Rosner B 2002 Correlates of bone and blood lead levels among middle-aged and elderly women Am J Epidemiol 156 335 343 12181103
Kosnett MJ Becker CE Osterloh JD Kelly TJ Pasta DJ 1994 Factors influencing bone lead concentration in a suburban community assessed by noninvasive K X-ray fluorescence JAMA 271 197 203 8277545
Landrigan PJ Baker EL Jr Himmelstein JS Stein GF Weddig JP Straub WE 1982 Exposure to lead from the Mystic River Bridge: the dilemma of deleading N Engl J Med 306 673 676 7057827
Lindquist O Bengtsson C Hansson T Roos B 1981 Bone mineral content in relation to age and menopause in middle-aged women. A study of bone density in lumbar vertebrae by dual photon absorptiometry in a population sample of women Scand J Clin Lab Invest 41 215 223 7313505
Markowitz SB Nunez CM Klitzman S Munshi AA Kim WS Eisinger J 1994 Lead poisoning due to hai ge fen. The porphyrin content of individual erythrocytes JAMA 271 932 934 8120963
Muldoon SB Cauley JA Kuller LH Scott J Rohay J 1994 Lifestyle and sociodemographic factors as determinants of blood lead levels in elderly women Am J Epidemiol 139 599 608 8172171
Nash D Magder L Lustberg M Sherwin RW Rubin RJ Kaufmann RB 2003 Blood lead, blood pressure, and hypertension in perimenopausal and postmenopausal women JAMA 289 1523 1532 12672769
Nash D Silbergeld E Mager L Stolley P 1998 Menopause, hormone replacement therapy (HRT), and blood lead levels among adult women from NHANES III, 1988–1994 Am J Epidemiol 147 S93
Nilsson U Attewell R Christoffersson JO Schutz A Ahlgren L Skerfving S 1991 Kinetics of lead in bone and blood after end of occupational exposure Pharmacol Toxicol 68 477 484 1891443
O’Flaherty EJ 1992 Modeling bone mineral metabolism, with special reference to calcium and lead Neurotoxicology 13 789 797 1302305
Parsons PJ 2002 Monitoring human exposure to lead: an assessment of current laboratory performance for the determination of blood lead Environ Res 57 149 162 1568437
Payton M Riggs KM Spiro A III Weiss ST Hu H 1998 Relations of bone and blood lead to cognitive function: the VA Normative Aging Study Neurotoxicol Teratol 20 19 27 9511166
Pirkle JL Brody DJ Gunter EW Kramer RA Paschal DC Flegal KM 1994 The decline in blood lead levels in the United States. The National Health and Nutrition Examination Surveys (NHANES) JAMA 272 284 291 8028141
Pounds JG 1984 Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: a review Neurotoxicology 5 295 331 6151637
Prestwood KM Gunness M Muchmore DB Lu Y Wong M Raisz LG 2000 A comparison of the effects of raloxifene and estrogen on bone in postmenopausal women J Clin Endocrinol Metab 85 2197 2202 10852452
Price J Grudzinski AW Craswell PW Thomas BJ 1992 Repeated bone lead levels in Queensland, Australia—previously a high lead environment Arch Environ Health 47 256 262 1497378
Rabinowitz MB Wetherill GW Kopple JD 1976 Kinetic analysis of lead metabolism in healthy humans J Clin Invest 58 260 270 783195
Riggs BL Melton LJ III 1986 Involutional osteoporosis N Engl J Med 314 1676 1686 3520321
Ryan CM Morrow L Parkinson D Bromet E 1987 Low level lead exposure and neuropsychological functioning in blue collar males Int J Neurosci 36 29 39 3654090
Silbergeld EK Schwartz J Mahaffey K 1988 Lead and osteoporosis: mobilization of lead from bone in postmenopausal women Environ Res 47 79 94 3168967
Simons TJ 1993 Lead-calcium interactions in cellular lead toxicity Neurotoxicology 14 77 85 8247414
Symanski E Hertz-Picciotto I 1995 Blood lead levels in relation to menopause, smoking, and pregnancy history Am J Epidemiol 141 1047 1058 7771441
Tellez-Rojo MM Hernandez-Avila M Gonzalez-Cossio T Romieu I Aro A Palazuelos E 2002 Impact of breast-feeding on the mobilization of lead from bone Am J Epidemiol 155 420 428 11867353
Todd AC Landrigan PJ Bloch P 1993 Workshop on the X-ray fluorescence of lead in bone: conclusions, recommendations and summary Neurotoxicology 14 145 154 8361673
Todd AC Parsons PJ Carroll S Geraghty C Khan FA Tang S 2002 Measurements of lead in human tibiae. A comparison between K-shell X-ray fluorescence and electrothermal atomic absorption spectrometry Phys Med Biol 47 673 687 11900198
Todd AC Parsons PJ Tang S Moshier EL 2001 Individual variability in human tibia lead concentration Environ Health Perspect 109 1139 1143 11712999
Webber CE Chettle DR Bowins RJ Beaumont LF Gordon CL Song X 1995 Hormone replacement therapy may reduce the return of endogenous lead from bone to the circulation Environ Health Perspect 103 1150 1153 8747022
Weyermann M Brenner H 1997 Alcohol consumption and smoking habits as determinants of blood lead levels in a national population sample from Germany Arch Environ Health 52 233 239 9169635
Wittmers LE Jr Aufderheide AC Wallgren J Rapp G Jr Alich A 1988 Lead in bone. IV. Distribution of lead in the human skeleton Arch Environ Health 43 381 391 3196073
Wolff I van Croonenborg JJ Kemper HC Kostense PJ Twisk JW 1999 The effect of exercise training programs on bone mass: a meta-analysis of published controlled trials in pre- and postmenopausal women Osteoporos Int 9 1 12 10367023
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7295ehp0112-00167915579413ResearchArticlesThe Sources of Inflammatory Mediators in the Lung after Silica Exposure Rao K. Murali Krishna Porter Dale W. Meighan Terence Castranova Vince Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USAAddress correspondence to K.M.K. Rao, Box 2015, PPRB/HELD/NIOSH, 1095 Willowdale Road, Morgantown, WV 26505 USA. Telephone: (304) 285-6169. Fax: (304) 285-5938. E-mail:
[email protected] authors declare they have no competing financial interests.
12 2004 16 8 2004 112 17 1679 1685 28 5 2004 16 8 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. The expression of 10 genes implicated in regulation of the inflammatory processes in the lung was studied after exposure of alveolar macrophages (AMs) to silica in vitro or in vivo. Exposure of AMs to silica in vitro up-regulated the messenger RNA (mRNA) levels of three genes [interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2)] without a concomitant increase in the protein levels. AMs isolated after intratracheal instillation of silica up-regulated mRNA levels of four additional genes [granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-1β, IL-10, and inducible nitric oxide synthase]. IL-6, MCP-1, and MIP-2 protein levels were elevated in bronchoalveolar lavage fluid. Fibroblasts under basal culture conditions express much higher levels of IL-6 and GM-CSF compared with AMs. Coculture of AMs and alveolar type II cells, or coculture of AMs and lung fibroblasts, in contact cultures or Transwell chambers, revealed no synergistic effect. Therefore, such interaction does not explain the effects seen in vivo. Identification of the intercellular communication in vivo is still unresolved. However, fibroblasts appear to be an important source of inflammatory mediators in the lung.
alveolar macrophagesalveolar type II cellscytokinesfibroblastsgene expressionlungsilica
==== Body
Occupational exposure to crystalline silica is associated with the development of pulmonary silicosis (Hnizdo and Vallyathan 2003; Reiser and Last 1979) and an increased risk for lung cancer (McDonald 1996). Silica can cause direct DNA damage and mammalian cell transformation (Daniel et al. 1995; Shi et al. 1994). The initial event, however, is an inflammatory response, including oxidant production and recruitment of inflammatory cells into the lung.
Numerous inflammatory mediators have been implicated in silica-induced pathology. Among them are cytokines, such as interleukin-(IL) 1β(Goodman et al. 1982), IL-6 (Gosset et al. 1991), IL-10 (Huaux et al. 1998), tumor necrosis factor-α(TNF-α) (Dubois et al. 1989), and transforming growth factor (TGF) (Williams et al. 1993; Williams and Saffiotti 1995); chemokines, such as monocyte chemoattractant protein-1 (MCP-1) (Barett et al. 1999) and macrophage inflammatory protein-2 (MIP-2) (Driscoll et al. 1993); the nonprotein inflammatory mediator nitric oxide, generated mainly through inducible nitric oxide synthase (iNOS) (Castranova et al. 1998); and adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) (Hubbard and Giardina 2000; Nario and Hubbard 1996). However, the changes in the inflammatory mediators have been studied in different contexts. Some were studied in vitro, some in vivo, some in cell lines, and some in primary cells, making it difficult to conclude which of these mediators are involved in the initial phase of the lung inflammatory response and which become important in later stages of the response. A second aspect that has not received much attention is the source of these inflammatory mediators and the importance of cell–cell interactions in the production of these mediators.
In our studies of silica-induced production of inflammatory mediators in alveolar macrophages (AMs), we found the responses of AMs after in vitro stimulation were quite different compared with AMs isolated after in vivo exposure to silica. This suggests that cell–cell interactions may a play an important role in silica-induced production of inflammatory mediators in the lung. Previous studies have indicated a role for interaction between alveolar epithelial type II cells and AMs in the production of iNOS (Pechkovsky et al. 2002) and a role for interaction between fibroblasts and AMs in the production of granulocyte/macrophage-colony stimulating factor (GM-CSF) (Fitzgerald et al. 2003).
The recent technical advances in polymerase chain reaction (PCR) methodology make it possible to study the expression of several genes simultaneously even with small amounts of RNA. Therefore, to understand the role of inflammatory mediators in silica-induced pathology, we studied the expression of several inflammatory mediators in AMs after in vitro exposure to silica or after in vivo exposure by intratracheal instillation. The expression was studied at the message level by real-time reverse transcription (RT) PCR and, where appropriate, at the protein level by enzyme-linked immunosorbent assays (ELISAs). In addition, we studied the interactions between AMs and type II cells or fibroblasts in in vitro culture systems. Our studies indicate that the lung fibroblasts are an important source of inflammatory mediators after silica exposure.
Materials and Methods
Animals.
The animals used in these experiments were specific pathogen-free Sprague-Dawley rats [HLA:(SD)CVF; Hilltop Laboratories, Scottdale, PA] weighing 250–300 g (~ 8 weeks old at arrival). The animals were housed in an environmentally controlled facility that was accredited by the Association for Assessment and Accreditation of Laborary Animal Care. The rats were monitored to be free of endogenous viral pathogens, parasites, mycoplasmas, Helicobacter, and cilia-associated respiratory bacillus. Rats were acclimated for at least 5 days before use and were housed in ventilated cages, which were provided with HEPA-filtered air and used Alpha-Dri virgin cellulose chips (Shepherd Specialty Papers, Watertown, TN) and hardwood Beta chips (NEPCO, Warrensburg, NY) as bedding. The rats were maintained on 2018S Teklad Global 18% rodent diet (Harlan Teklad, Madison, WI) and tap water, both of which were provided ad libitum.
Reagents.
Rat cytokine kits for IL-6, MCP-1, MIP-2, and TNF-αwere obtained from Biosource (Camarillo, CA). Lactate dehydrogenase (LDH) was measured within 24 hr on refrigerated samples with a COBAS MIRA Plus analyzer (Roche Diagnostics, Indianapolis, IN) using kits from Roche. Lipopolysaccharide B (LPS; from E. coli 026:B6) was obtained from Difco Laboratories (Detroit, MI). The culture medium consisted of Dulbecco’s modified Eagle medium (BioWhittaker, Walkersville, MD), 1 mM glutamine (Sigma, St. Louis, MO), 10 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethane-sulfonic acid] (HEPES; Sigma), 100 U/mL penicillin–streptomycin (GIBCO Life Technologies, Grand Island, NY), 100 μg/mL kanamycin (GIBCO), and 10% (vol/vol) heat-inactivated fetal bovine serum (GIBCO).
Source of silica.
We obtained MIN-U-SIL 5 from U.S. Silica (Berkeley Springs, WV). It was examined by proton-induced X-ray emission spectrometry for inorganic contaminants and for desorbable organic compounds by gas chromatography mass spectroscopy. The results of these analyses have been reported elsewhere (Porter et al. 2001). Silica samples were found to be > 99% pure quartz. Mean particle count diameter, determined by scanning electron microscopy, was 2.14 μm, with 99% of the particles < 5 μm. Silica was weighed and dry heated at 170°C for 24 hr to sterilize. Sterile medium was then added to the silica, which was vortexed into suspension before being added to the cell culture.
Isolation of AMs.
The animals were anesthetized with pentobarbital sodium (150 mg/kg body weight) and exsanguinated by cutting the abdominal aorta. AMs were obtained by bronchoalveolar lavage (BAL) according to the method of Myrvik et al. (1961). The lungs from each animal were lavaged eight times with 5 mL phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 9.4 mM Na2HPO4, and 1.9 mM NaH2PO4, pH 7.4) per gram lung weight. The cells were separated from the lavage fluid by centrifugation at 300 × g for 5 min and then washed three times by alternate centrifugation and resuspension in phosphate-buffered medium. The cells were then resuspended in the culture medium for use in all experiments. Cell number was determined by an electronic cell counter (model ZB; Coulter Electronics, Hialeah, FL).
Isolation of type II cells.
We isolated type II cells as described previously (Miles et al. 1997). Briefly, the procedure involves perfusing the lung to remove blood, removing free AMs by BAL, digestion of lung tissue with elastase, and purification of type II cells by centrifugal elutriation. Cells isolated and purified by this method were > 85% pure type II cells as determined microscopically after staining with phosphine 3R (Jones et al. 1982).
The cells were cultured on collagen gels similar to those described by Lee et al. (1984) for growing hamster tracheal epithelial cells. Collagen gels were prepared from stock solution of collagen type I from rat tail (Sigma-Aldrich, St. Louis, MO) dissolved in 1:1,000 dilution of acetic acid in sterile distilled water overnight at 4°C. A six-well plate was layered with 0.775 mL (each well) of ice-cold collagen gel mixture consisting of 0.5 mL collagen stock, 0.15 mL 10× modified Eagle medium, and 0.125 mL 0.5 N NaOH. The mixture was allowed to polymerize for 4 hr at a humidified atmosphere of 5% CO2 at 37°C. The polymerized collagen gels were washed with 1 mL epithelial cell growth medium before cells were plated and grown overnight.
Isolation of lung fibroblasts.
We isolated lung fibroblasts as described by Reist et al. (Reist et al. 1991). Briefly, the lungs were perfused with normal saline, lavaged with phosphate-buffered saline (PBS) containing 0.1% glucose, and sectioned four times at 0.5-mm intervals with a McIlwain tissue chopper. The chopped lung tissue from a single rat was digested in 20 mL of HEPES-buffered solution (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 505 mM glucose, and 10 mM HEPES, pH 7.4), containing collagenase (0.1%), elastase (40 U/mL), bovine serum albumin (0.5%), and DNAse (0.018%) in a shaker water bath for 30 min at 37°C. The digested mixture was filtered through two layers of sterile gauze that had been washed with culture medium. The cells were sedimented by centrifugation and plated in six-well culture plates. The medium was changed 24 hr later, and the cells were allowed to grow to confluence.
Coculture of type II cells and AMs.
Type II cells cultured overnight on collagen gels in six-well plates (catalog no. 353046; tissue culture treated by vacuum gas plasma, polystyrene, nonpyrogenic; Becton Dickinson, Franklin Lakes, NJ), as described above, were incubated for an additional 4 hr at 37°C in a CO2 incubator with freshly isolated AMs (1 million cells) with or without silica. Controls were type II cells alone with or without silica. The collagen gels were dissolved in a solution containing 1 mg of Sigma blend collagenase type F made up in 1 mL of type II cell growth medium for each well to be dissolved. The cells were then spun down and used for isolation of total RNA.
Coculture of lung fibroblasts and AMs.
Lung fibroblasts were cultured until they became confluent. The cells were trypsinized and 2 × 106 cells were plated in six-well plates. After overnight culture, freshly isolated AMs (2 × 106 cells) were added to the wells and cultured for an additional 4 hr with or without silica. Controls were fibroblasts alone with or without silica. The culture medium was aspirated and spun down, and the supernatant was stored at –80°C. The cells were scraped and combined with the cell pellet from the above step and used for isolation of total RNA.
Transwell experiments with fibroblasts and AMs.
To measure messenger RNA (mRNA) expression in separated cell populations and to study the interaction of soluble mediators released by cell populations on each other, we conducted experiments in Transwell chambers (CoStar, Corning, NY). For these experiments, cultured lung fibroblasts were trypsinized, and 1 million cells were plated in the outer well of a Transwell plate and cultured for an additional 24 hr. At the end of the 24-hr period, freshly isolated AMs (1 million cells) were placed in the inserts. Silica was added either to the macrophages in the inner wells or to the fibroblasts in the outer well and incubated for 4 hr. Total RNA was isolated from each population separately.
Preparation of AM- and polymorpho-nuclear neutrophil–enriched fractions.
We obtained AM- and polymorphonuclear neutrophil (PMN)–enriched fractions from BAL fluid obtained from rats treated with silica in vivo, as described by Huffman et al. (2003). Briefly, the method consisted of layering BAL cell populations obtained by lavage onto a Histopaque double-density gradient composed of equal amounts of Histopaque 1083 and Histopaque 1119 (Sigma). The gradients were then centrifuged (400 × g, 30 min, room temperature). The AM-enriched fraction localized at the interface between PBS diluent and Histopaque 1083, and the PMN-enriched fraction was located at the bottom as a pellet. This method yields about 60% AMs in the AM-enriched fraction and 90% PMN in the PMN-enriched fraction (Huffman et al. 2003).
Measurement of cytokines.
We measured the cytokines in culture supernatants after a 24-hr incubation with either 200 μg/mL silica or 1 μg/mL LPS. IL-6, MCP-1, MIP-2, and TNF-α were measured by ELISA kits according to manufacturer instructions (Biosource International, Camarillo, CA). The values were expressed as nanograms or picograms per million cells. For measurement of cytokines in the BAL fluid, lavage fluid from the first wash was collected and spun down to sediment the cellular elements. The supernatant was stored at –80°C for later measurement of cytokine levels by ELISA.
Quantitation of mRNAs by RT-PCR.
We measured cytokine mRNA levels using a SYBR Green PCR kit with the ABI 5700 Sequence Detector (PE Applied Biosystems, Foster City, CA). Total RNA was isolated using RNAqueous 4PCR kits (Ambion, Austin, TX) from AMs (≈2 million cells) or lung tissue after alveolar lavage (≈50 mg wet tissue). One to two micrograms of the DNAse I–treated RNA was reverse transcribed, using Superscript II (Life Technologies, Gaithersburg, MD). The complementary DNA generated was diluted 1:100, and 15 μL was used to conduct the PCR reaction according to the SYBR Green PCR kit instructions. The comparative CT (threshold cycle) method was used to calculate the relative concentrations (User Bulletin no. 2; ABI PRISM 7700 Sequence Detector, PE Applied Biosystems). Briefly, the method involves obtaining the CT values for the cytokine of interest, normalizing to a housekeeping gene (18S in the present case), and deriving the fold increase compared with the control, unstimulated cells. Table 1 lists the primer sets used for these experiments. In preliminary experiments, the products were analyzed by gel electrophoresis, and a single product was obtained with each primer set. In addition, dissociation curves yielded single peaks.
In vitro experiments.
All experiments were performed on pooled AMs from several animals. AMs were placed in six-well plates, incubated for 2 hr at 37°C, and washed to remove nonadherent cells. Then the cells were incubated with silica (200 μg/mL) or LPS (1 μg/mL) for 4 hr for mRNA measurements, or 24 hr for the measurement of inflammatory cytokines.
In vivo experiments.
Rats were anesthetized with an intraperitoneal injection of 30–40 mg/kg body weight sodium methohexital (Brevital; Eli Lilly and Company, Indianapolis, IN) and were intratracheally instilled using a 20-gauge 4-inch ball-tipped animal feeding needle. Silica (MIN-U-SIL 5) was suspended in endotoxin-free, Ca2+/Mg2+-free PBS (BioWhittaker, Walkersville, MD), and rats received either 2 mg silica/100 g body weight or an equivalent volume of PBS. The animals were sacrificed 4 hr postexposure, and AMs were isolated as described above. The lavaged lung tissue was used for isolation of total RNA.
Statistical methods.
A paired t-test was used for in vitro experiments. A t-test assuming unequal variance or a Z-test for means was used to evaluate the in vivo data. The significance was set at < 0.05.
Results
Effects of silica treatment on cell viability.
In initial experiments, the effect of silica treatment (200 μg/mL) on cell viability was assessed by measuring the release of LDH into the medium at the end of the 4-hr incubation time. The means ± SEs (U/L) for control versus silica-treated cells (n = 3) were, for fibroblasts, 49 ± 14 versus 49 ± 15; for type II cells, 87 ± 9 versus 91 ± 7; and for macrophages, 101 ± 13 versus 100 ± 7.
Effects of silica or LPS on mRNA expression in AMs in vitro.
AMs were stimulated with either 200 μg/mL silica or 1 μg/mL LPS for 4 hr. The expression of 10 genes, implicated in the induction of an inflammatory response, was measured by real-time RT-PCR. The message levels of only three cytokines (MCP-1, MIP-2, and IL-6) showed a significant increase at 4 hr after in vitro exposure to silica (Figure 1). In contrast, mRNA levels for GM-CSF, ICAM-1, IL-1β, IL-10, iNOS, TGF-β1, and TNF-αwere not significantly elevated after this treatment.
To compare the effect of silica with that of bacterial endotoxin, LPS, we also measured mRNA levels of these inflammatory mediators in AMs stimulated with LPS for 4 hr in vitro (Figure 2). LPS stimulation increased message levels of IL-1β, IL-6, GM-CSF, iNOS, MCP-1, MIP-2, and TNF-αbut not those of ICAM-1, IL-10, and TGF-β1.
Effects of in vivo silica treatment on mRNA expression in cells obtained by BAL.
Figure 3 shows the mRNA expression in cells isolated from rats 4 hr after intratracheal instillation of silica (2 mg/100 g body weight). The three cytokines that showed an increase at 4 hr in vitro (IL-6, MCP-1, and MIP-2) also showed an increase in vivo. In addition, the expression of four other genes (GM-CSF, IL-1β, IL-10, and iNOS) was increased. Three genes (TGF-β1, TNF-α, and ICAM-1) showed no change either in vitro or in vivo.
Effects of silica on mRNA expression in the lung tissue after intratracheal instillation.
We also measured cytokine expression in the lavaged lung tissue 4 hr after the intratracheal instillation of silica (Figure 4). The results were mostly similar to those seen in AMs (Figure 3). There was a significant increase in the message levels of GM-CSF, IL-1β, IL-6, iNOS, MCP-1, MIP-2, and TNF-α. No increase was seen for ICAM-1, IL-10, and TGF-β1.
mRNA expression of cytokines in AM-enriched and PMN-enriched fractions.
One major difference between cells obtained by BAL from control rats versus silica-treated rats is the presence of a large number of PMNs in the silica-treated animals. One explanation for the differences seen in gene expression after in vitro and in vivo exposure may be that the neutrophils produce additional cytokines not seen with AMs alone. To determine the role of PMN in mRNA expression after silica treatment, we obtained AM-enriched and PMN-enriched fractions from BAL fluid of animals treated with silica in vivo. The mRNA levels were expressed in relation to the expression levels in relation to AMs. These AMs have been exposed to silica in vivo and express high levels of mRNA, as shown in Figure 3. The mRNA expression in AMs was assigned an arbitrary value of 1 for Figure 5. Figure 5 shows that mRNA expression of the seven cytokines studied was essentially the same in the two fractions, indicating that PMN enrichment is not the cause for differences between in vitro and in vivo treatments.
Cytokine/chemokine expression at the protein level.
We measured the levels of four cytokines/chemokines (IL-6, MCP-1, MIP-2, and TNF-α) in the supernatants of AM cultures after 4 hr incubation with either silica or LPS. There was no increase in the protein levels of these mediators with silica, but LPS produced very high levels of these cytokines/ chemokines (Figure 6A–D). There was no increase in these mediators even after 24 hr incubation with silica. In contrast, the cytokine levels of IL-6, MCP-1, and MIP-2 were increased in the alveolar lavage fluid when the animals were exposed to silica for 4 hr in vivo (Figure 7). There was no increase in TNF-α levels 4 hr after exposure to silica either in vitro or in vivo (data not shown).
Coculture of type II cells and AMs on gene expression.
To determine whether the differences seen in mRNA expression in AMs exposed to silica in vitro and in vivo may be related interaction between AMs and type II cells, we performed coculture experiments. We focused on four genes that were up-regulated only after in vivo exposure. Table 2 shows the expression of these four genes in cocultures of AMs and type II cells. Essentially, there was no difference in the expression of these genes when the cells were cocultured with or without silica. These results indicate that the expression of these inflammatory mediators is not mediated by interaction between AMs and type II cells.
Gene expression in lung fibroblasts.
Figure 8 shows the expression of five genes in fibroblasts cultured alone or in the presence of silica. We included IL-6 because lung tissue showed very high levels of IL-6 mRNA levels (Figure 4). The mRNA levels were expressed relative to mRNA levels of AMs alone. It is clear that the mRNA levels for IL-6 and GM-CSF are very high in resting lung fibroblasts. IL-6 protein levels, as determined by ELISA, were 100-fold higher in culture supernatants of lung fibroblasts compared with culture supernatants of AMs (210 ± 75 vs. 2.4 ± 1.1, n = 7), indicating that the message is being translated into protein. In addition, in vitro exposure to silica caused a significant increase in mRNA levels, but this increase in mRNA levels was not reflected in an increase in protein synthesis (210 ± 75 vs. 231 ± 62, control vs. silica, n = 7). This is similar to that seen in AMs. We did not measure the GM-CSF protein levels.
Gene expression in AMs and fibroblast cocultures.
Table 3 shows the relative expression of the five genes in coculture experiments. Although coculture of AMs and lung fibroblasts seems to enhance iNOS and IL-10 mRNA levels seen over and above that seen with each cell type alone, the results were too variable to draw a definitive conclusion concerning a synergistic effect. With regard to IL-6 and GM-CSF, the main source seems to be fibroblasts, but results were too variable to conclude whether cocultures with or without silica enhance the mRNA levels.
mRNA expression in AMs and lung fibro-blasts in Transwell experiments.
The coculture experiments do not allow determination of mRNA expression in individual cell types. Therefore, we conducted Transwell experiments to isolate RNA from each cell type and to study the roles of cell–cell contact versus soluble mediators in these interactions (Table 4). Two conclusions can be drawn from these experiments: First, there is no difference in the mRNA levels of IL-1, IL-10, and iNOS under the different conditions tested; and second, the main sources of IL-6 and GM-CSF are lung fibroblasts. Although AMs seem to enhance IL-6 and GM-CSF mRNA levels in fibroblasts, the extreme variation in the results does not permit a definitive conclusion.
Combining the observations from coculture experiments and Transwell experiments, it appears that factors in addition to cell–cell contact and soluble mediators secreted by these two cell types are involved in regulating the inflammatory mediators in in vivo situations.
Discussion
Exposure to silica causes inflammatory and fibrotic lung disease (Hnizdo and Vallyathan 2003). Silica-induced inflammatory response has been implicated in the pathogenesis of fibrosis. In this study, we measured expression of 10 genes that are involved in regulating the inflammatory processes in the lung, at the message level. The studies were conducted in BAL cells and lung tissue after in vivo exposure, and in AMs, type II cells, and lung fibroblasts after in vitro exposure.
Exposure of AMs to silica in vitro increased message levels of only three genes: IL-6, MCP-1, and MIP-2. MCP-1 plays an important role in accumulation of monocytes (Leonard and Yoshimura 1990). MIP-2 is a potent chemotactic factor for neutrophils (Driscoll 1994). Up-regulation of these two genes very early, after silica exposure, may account for the rapid accumulation of these cells in the lung. IL-6 is a pleiotropic cytokine with multiple biologic activities (Van Snick 1990) that has been shown to be up-regulated after silica (Hetland et al. 2001) and asbestos (Simeonova et al. 1997) exposure of human lung epithelial cells. Here, we show that it is one of the early genes expressed in AMs after silica exposure. Up-regulation of these genes requires only the interaction between the silica particles and the macrophages.
When mRNA levels were measured in BAL cells harvested from rats instilled with silica, the mRNA levels of four other genes (GM-CSF, IL-1, IL-10, and iNOS) went up in addition to the three genes mentioned above.
The production of NO in isolated AMs from in vivo silica-treated animals and lack of NO production after in vitro treatment has been reported previously (Huffman et al. 1998). We confirm that observation. To determine whether cell–cell interactions may be involved in the production of NO and the expression of three other genes belonging to this group, AMs were cocultured with either type II cells or lung fibroblasts. The data (Table 2) clearly indicate that coculture with type II cells does not up-regulate these genes under any of the conditions studied.
Coculture of AMs with fibroblasts showed that iNOS and IL-10 mRNA levels may go up, but the response was extremely variable, and no definitive conclusions could be drawn. In addition, the Transwell experiments show that the coculture of AMs and fibroblasts does not significantly increase the message levels of GM-CSF, IL-1, IL-10, and iNOS levels in AMs. Therefore, the factors responsible for up-regulation of these genes after intratracheal instillation of silica remains elusive.
Three other genes (ICAM-1, TGF-1β, and TNF-α) did not show any change in BAL cells and lung tissue after in vivo treatment or in AMs after in vitro treatment. The observation that the release of TNF-αis increased in the blood monocytes of miners with coal workers’ pneumoconiosis (Borm et al. 1988) has led to several studies showing an increase in TNF-α levels from AMs stimulated with silica (Baer et al. 1998; Dubois et al. 1989; Gossart et al. 1996). However, others have shown that in vitro treatment with silica does not induce TNF-αlevels in human AMs (Gosset et al. 1991). In some cases, where an increase in TNF-αproduction was shown, the levels were minimally increased and the levels were at least a couple of orders of magnitude less than what is seen with LPS stimulation (Kanj et al. 2002; Rojanasakul et al. 1999; Shi et al. 1999), raising the question of their biologic relevance. In one in vivo study in rats with silica, an increase in mRNA levels of TNF-αin AMs was not seen until 3 days after intratracheal instillation (Snadrin et al. 1996), and even later in a silica inhalation study (Porter et al. 2002). These data make the role of TNF-αin the initial stages of silica-induced inflammation questionable, even though TNF-αhas been reported to be a key mediator in the eventual development of fibrosis (Piguet et al. 1990). In our studies, we did not observe any increase in mRNA in AMs for TNF-αat 4 hr in vitro or at 4 hr in vivo. However, there was an increase in the mRNA expression in TNF-αin the lung tissue 4 hr after intratracheal instillation of silica.
The cytokine TGF-β1 and the adhesion molecule ICAM-1 have also been implicated in the pathogenesis associated with silica exposure (Matrat et al. 1998; Nario and Hubbard 1996). We have not detected any increase in the message levels of these two genes after either in vitro or in vivo silica exposure. TGF-β1 is shown to be critical in acute lung injury (Pittet et al. 2001) but may not play a role in particle-induced lung disease, at least in the initial stages. ICAM-1 has been shown to be up-regulated in LPS-induced lung inflammation (Madjdpour et al. 2000; Nathms et al. 1998). LPS stimulation in vivo has been shown to increase ICAM-1 expression both in AMs (Grigg et al. 1994) and in the lung tissue (Nathms et al. 1998). There was no increase in ICAM-1 message in AMs after in vitro exposure to silica or LPS in the present study. A third gene that did not show any change with LPS was IL-10. Our findings are consistent with previous findings that there is no up-regulation of TGF-β1 in AMs (Xing et al. 1994) or IL-10 in lung tissue (Johnston et al. 1998) after LPS stimulation. With regard to ICAM-1, an increase was demonstrated in AMs after in vivo exposure to LPS (Grigg et al. 1994). We evaluated ICAM-1 in AMs after only in vitro exposure.
We observed significant increases in the mRNA levels of IL-6 and genes for two chemokines (MCP-1 and MIP-2) at 4 hr after in vitro treatment with silica. Although the message levels showed an increase, there was no increase in the protein levels measured in the supernatants of the cultures at 4 hr. However, when silica was administered intra-tracheally, there was considerable increase in both message levels and protein levels at 4 hr. Our findings with regard to the production of MCP-1 and MIP-2 are consistent with previous observations demonstrating an increase in these two chemokines after silica exposure (Driscoll 2000; Driscoll et al. 1998; Hubbard et al. 2002). The observation that the in vitro treatment up-regulates the message levels without increasing the protein levels, but in vivo both message levels and protein levels go up, indicates that cell–cell interactions and/or other influences might play an important role in the expression of these cytokines at the protein level.
The Transwell experiments revealed that a major source of IL-6 and GM-CSF in the lung could be lung fibroblasts. When mRNA levels were expressed relative to AMs (Table 4), the IL-6 levels in lung fibroblasts were several hundred-fold higher than those in AMs. Similarly, mRNA levels of GM-CSF were much higher in fibroblasts compared with AMs. Further, the number of fibroblasts (interstitial cells) is 10-fold higher than AMs in the lung tissue (Stone et al. 1992). These observations indicate that the fibroblasts are a major of source of these inflammatory mediators in the lung.
MCP-1 has significant involvement in the inflammatory disorders of the lung (Rose et al. 2003). It has been shown to regulate alveolar epithelial cell inhibition of fibroblast proliferation (Moore et al. 2002). In addition to monocytes, fibroblasts are an important source of MCP-1 (Galindo et al. 2001; Hao et al. 2003). We found MCP-1 mRNA levels were severalfold higher in lung fibroblasts compared with AMs (data not shown). Therefore, the main source of both IL-6 and MCP-1 in the BAL fluid after silica exposure could be lung fibroblasts. This is consistent with the observation that silica can directly stimulate lung fibroblasts (Arcangeli et al. 2001; Baroni et al. 2001). We have not evaluated the sources of MIP-2 in this study.
GM-CSF is purported to play an important role in numerous respiratory illnesses, including asthma (Xing et al. 1996). It is generated by a variety of lung cell types (Bergman et al. 2000; Blau et al. 1994; Christensen et al. 2001; Churchill et al. 1992; Fitzgerald et al. 2003; O’Brien et al. 1998; Smith et al. 1990; Soloperto et al. 1991; Trapnell and Whitsett 2002). GM-CSF was not produced by AMs when stimulated with silica in vitro, but an increase in message levels were seen in both BAL cells and lung tissue after intratracheal instillation. This confirms the reported need for cell–cell interactions in the up-regulation of GM-CSF (Fitzgerald et al. 2003).
The importance of cell–cell interactions in the production of inflammatory mediators has been emphasized in several studies. Direct contact between human peripheral blood mononuclear cells and renal fibroblasts facilitates the expression of MCP-1 (Hao et al. 2003). Similarly, macrophage/fibroblast interactions are important for the production of GM-CSF (Fitzgerald et al. 2003). Both soluble mediators and adhesion molecules have been implicated in these interactions (Hao et al. 2003; Zickus et al. 2004). The lack of effect on the expression of several genes in coculture experiments with contact or without contact (Transwell experiments) indicates that some additional factors may be involved in the regulation of cytokine production in the lung after silica exposure. During inflammation a variety of cells are recruited into the lung and a number products are generated. Any one of these factors may influence the expression of inflammatory mediators. In this regard, it is important to keep in mind the role of lung surfactant. Lung surfactant is known to modulate immune functions in the lung (Wright 1997); we mention its role in particular because we have some preliminary data to suggest that lung surfactant may enhance cytokine production in the lung fibroblasts.
In summary, we found that exposure of AMs to silica in vitro up-regulates only three genes (IL-6, MCP-1, and MIP-2). However, in BAL cells harvested after intratracheal instillation of silica, four additional genes (IL-1, IL-10, iNOS, and GM-CSF) were up-regulated. Cocultures of AMs with alveolar epithelial type II cells or lung fibroblasts did not enhance mRNA level of the four additional genes that were expressed after in vivo exposure. There is need to evaluate the role of other mediators in regulating the production of inflammatory mediators in the lung, perhaps the role of lung surfactant. Most of the studies concerning silica-induced inflammatory processes in the lung have been focused on the role of AMs; our Transwell studies show that lung fibroblasts are an important source of IL-6 and GM-CSF. These observations indicate that the fibroblast-derived inflammatory mediators may also play an important role after silica exposure.
Figure 1 mRNA expression in AMs stimulated in vitro with silica (200 μg/mL) at 4 hr postexposure. Error bars represent fold increase above control (mean ± SE of at least four experiments for each cytokine).
*Significantly greater than control,
p < 0.05.
Figure 2 mRNA expression in AMs stimulated in vitro with LPS (1 μg/mL) at 4 hr postexposure. Error bars represent fold increase above control (mean ± SE) in the message levels from a minimum of four different experiments with LPS.
*Significantly different from control except ICAM-1, IL-10, and TGF-β1 (TGF).
Figure 3 mRNA expression in cells obtained by BAL from animals at 4 hr after intratracheal instillation of silica (2 mg/100 g body weight). Error bars represent fold increase above control (mean ± SE) in the message levels from a minimum of five different animals (in the control and treated groups).
*Significantly greater than control,
p < 0.05.
Figure 4 mRNA expression in the lavaged lung tissue isolated from animals at 4 hr after intratracheal instillation of silica (2 mg/100 g body weight). Error bars represent fold increase above control (mean ± SE) in the message levels from a minimum of five different animals (in the control and treated groups).
*Significantly greater than control,
p < 0.05.
Figure 5 mRNA expression in separated AMs and PMNs isolated from animals at 4 hr after intratracheal instillation of silica (2 mg/100 g body weight). Error bars represent fold increase above control (mean ± SE) in the message levels from three different animals.
Figure 6 Cytokine/chemokine protein levels as determined by ELISA in culture supernatants of AMs treated in vitro with silica (200 μg/mL) or LPS (1 μg/mL) for 4 hr, or silica for 24 hr. There were no differences in (A) IL-6, (B) MCP-1, (C) MIP-2, or (D) TNF-αlevels between control and silica-treated cells at either exposure time. In contrast, LPS produced a large increase in all four cytokine/chemokines as early as 4 hr. Error bars represent mean ± SE from four separate experiments.
*Significantly greater than control,
p < 0.05.
Figure 7 Cytokine/chemokine protein levels as determined by ELISA in alveolar lavage fluid from animals at 4 hr after the intratracheal instillation of silica (2 mg/100 g body weight). Error bars represent mean ± SE from five different animals (in the control and silica-treated groups). Silica treatment increased protein levels of the three mediators, (A) IL-6 and MCP-1 and (B) MIP-2.
*Significantly greater than control,
p < 0.05.
Figure 8 mRNA expression in lung fibroblasts (Fibro) stimulated in vitro with silica (Si; 200 μg/mL) at 4 hr postexposure. The mRNA values were measured relative to that found in freshly isolated AMs incubated for 4 hr. Error bars represent fold increase above control (mean ± SE of at least four experiments for each inflammatory mediator).
*Significantly greater than control,
p < 0.05.
Table 1 Primer sets used.
Gene Primers Product (bp)
GM-CSF Sense: GAC ATG CGT GCT CTG GAG AAC G 144
Antisense: GCC ATT GAG TTT GGT GAG GTT GC
ICAM-1 Sense: AAT CTG ACC TGC AGC CGG AAA G 108
Antisense: GGA GCT AAA GGC ACG GCA CTT G
IL-1β Sense: AGC TCC ACG GGC AAG ACA TAG G 155
Antisense: GGA TGG CTT CCA AGC CCT TGA C
IL-6 Sense: CCC AAC TTC CAA TGC TCT CCT AAT G 141
Antisense: GCA CAC TAG GTT TGC CGA GTA GAC C
IL-10 Sense: GGC TCA GCA CTG CTA TGT TGC C 116
Antisense: AGC ATG TGG GTC TGG CTG ACT G
iNOS Sense: GTC ACC TAT CGC ACC CGA GAT G 117
Antisense: GCC ACT GAC ACT CCG CAC AAA G
MCP-1 Sense: TCA CGC TTC TGG GCC TGT TG 131
Antisense: CAG CCG ACT CAT TGG GAT CAT C
MIP-2 Sense: GGC AAG GCT AAC TGA CCT GGA AAG 113
Antisense: CAC ATC AGG TAC GAT CCA GGC TTC
TGF-β 1 Sense: GCT AAT GGT GGA CCG CAA CAA C 103
Antisense: TGG CAC TGC TTC CCG AAT GTC
TNF-α Sense: CGT CAG CCG ATT TGC CAT TTC 116
Antisense: TGG GCT CAT ACC AGG GCT TGA G
18S rRNA Sense: GGA CCA GAG CGA AAG CAT TTG C 115
Antisense: CGC CAG TCG GCA TCG TTT ATG
Table 2 Relative mRNA expression in AMs and type II alveolar epithelial cell cocultures stimulated with silica (Si; 200 μg/mL) for 4 hr.
mRNA AM AM + Si Type II Type II + Si AM + type II AM + type II + Si
IL-1β 1.47 ± 0.53 0.87 ± 0.34 0.61 ± 0.41 0.41 ± 0.27 1.10 ± 0.60 1.03 ± 0.49
IL-10 1.04 ± 0.13 0.30 ± 0.11 2.58 ± 1.01 5.74 ± 2.62 1.41 ± 1.46 2.21 ± 2.32
iNOS 1.04 ± 0.12 0.67 ± 0.24 0.49 ± 0.23 0.44 ± 0.16 1.75 ± 1.63 1.93 ± 1.47
GM-CSF 1.13 ± 0.27 1.00 ± 0.34 2.02 ± 1.19 3.88 ± 2.38 3.49 ± 0.71 5.01 ± 3.20
Values are mean ± SE from at least three different experiments relative to mRNA levels in AMs.
Table 3 Relative mRNA expression in AMs and lung fibroblast (fibro) cocultures stimulated with silica (Si; 200 μg/mL) for 4 hr.
mRNA AM AM + Si Fibro Fibro + Si AM + Fibro AM + Fibro + Si
IL-1β 1.03 ± 0.08 0.71 ± 0.12 0.08 ± 0.07 0.31 ± 0.29 1.49 ± 0.46 2.41 ± 0.91
IL-10 1.16 ± 0.11 1.04 ± 0.16 0.33 ± 0.17 0.78 ± 0.40 25.9 ± 15.8 26.8 ± 13.4
iNOS 1.14 ± 0.11 0.83 ± 0.19 0.58 ± 0.33 1.54 ± 0.93 8.22 ± 3.54 16.2 ± 9.95
GM-CSF 1.23 ± 0.21 1.06 ± 0.17 18.4 ± 7.0 58.9 ± 22.4 6.44 ± 2.4 58.0 ± 43.7
Values are mean ± SE from at least three different experiments relative to mRNA levels in AMs.
Table 4 Relative mRNA expression in AMs and lung fibroblasts (fibro) stimulated with silica (Si; 200 μg/mL) for 4 hr in Transwell experiments.
Insert/well
mRNA AMa/none None/fibroa AMa/fibro AM/fibroa AMa + Si/fibro AM + Si/fibroa AMa/fibro + Si AMa/fibroa + Si
IL-1β 1.0 ± 0.1 0.3 ± 0.2 4.7 ± 2.5 3.1 ± 2.5 1.1 ± 0.5 0.6 ± 0.2 2.8 ± 2.1 1.0 ± 0.5
IL-6 0.7 ± 0.1 912 ± 565 1.1 ± 0.8 14,487 ± 11,702 16 ± 9.6 3,009 ± 1,344 4.8 ± 4.3 14,573 ± 16,524
IL-10 1.4 ± 0.4 1.6 ± 0.6 1.2 ± 0.6 3.5 ± 2.0 0.8 ± 0.5 1.3 ± 1.1 0.1 ± 0.1 0.7 ± 0.4
iNOS 0.8 ± 0.1 1.0 ± 0.1 2.6 ± 1.4 3.4 ± 1.6 3.9 ± 2.8 1.9 ± 1.5 1.5 ± 1.1 1.0 ± 0.4
GM-CSF 1.0 ± 0.3 4.2 ± 1.9 2.1 ± 0.7 41.6 ± 24 1.6 ± 0.9 42 ± 39 0.9 ± 1.0 19.3 ± 7.2
Values are mean ± SE from three different experiments relative to mRNA levels in AMs.
a Source of the cells in which the mRNA levels were measured.
==== Refs
References
Arcangeli G Cupelli V Giuliano G 2001 Effects of silica on human lung fibroblasts in culture Sci Total Environ 270 135 139 11327386
Baer M Dillner A Schwartz RC Seldon C Nedospasov S Johnson PF 1998 Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-κB p50 Mol Cell Biol 18 5678 5689 9742085
Baroni T Bodo M D’Alessandro A Conte C Calvitti M Muzi G 2001 Silica and its antagonistic effects on transforming growth factor-βin lung fibroblast extracellular matrix production J Invest Med 49 146 156
Barrett EG Johnston C Oberdorster G Finkelstein JN 1999 Antioxidant treatment attenuates cytokine and chemokine levels in murine macrophages following silica exposure Toxicol Appl Pharmacol 158 211 220 10438654
Bergmann M Barnes PJ Newton R 2000 Molecular regulation of granulocyte macrophage colony-stimulating factor in human lung epithelial cells by interleukin (IL)-1β, IL-4, and IL-13 involves both transcriptional and post-transcriptional mechanisms Am J Respir Cell Mol Biol 22 582 589 10783130
Blau H Riklis S Kravtsov V Kalina M 1994 Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A Am J Physiol 266 L148 L155 8141310
Borm PJ Palmen N Engelen JJ Buurman WA 1988 Spontaneous and stimulated release of tumor necrosis factor-α(TNF-α) from blood monocytes of miners with coal workers’ pneumoconiosis Am Rev Respir Dis 138 1589 1594 2849351
Castranova V Huffman L Judy DJ Bylander JE Lapp LN Weber SL 1998 Enhancement of nitric oxide production by pulmonary cells following silica exposure Environ Health Perspect 106 suppl 5 1165 1169 9788892
Christensen PJ Bailie MB Goodman RE O’Brien AD Toews GB Paine R III 2000 Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis Am J Physiol 279 L487 L495
Churchill L Friedman B Schleimer RP Proud D 1992 Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells Immunology 75 189 195 1537596
Daniel LN Mao Y Williams AO Saffiotti U 1995 Direct interaction between crystalline silica and DNA—a proposed model for silica carcinogenesis Scand J Work Environ Health 21 suppl 2 22 26 8929683
Driscoll KE 1994 Macrophage inflammatory proteins: biology and role in pulmonary inflammation Exp Lung Res 20 473 490 7882902
Driscoll KE 2000 TNF-αand MIP-2: role in particle-induced inflammation and regulation by oxidative stress Toxicol Lett 112–113 177 183
Driscoll KE Carter JM Howard BW Hassenbein D Janssen YM Mossman BT 1998 Crocidolite activates NF-κB and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants Environ Health Perspect 106 suppl 5 1171 1174 9788893
Driscoll KE Hassenbein DG Carter JM Poynter J Asquith TN Grant RA 1993 Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibro-blasts, and epithelial cells and in rat lung after mineral dust exposure Am J Respir Cell Mol Biol 8 311 318 8383510
Dubois CM Bissonnette E Rola-Pleszczynski M 1989 Asbestos fibers and silica particles stimulate rat alveolar macrophages to release tumor necrosis factor: autoregulatory role of leukotriene B4 Am Rev Respir Dis 139 1257 1264 2540688
Fitzgerald SM Chi DS Hall HK Reynolds SA Aramide O Lee SA 2003 GM-CSF induction in human lung fibroblasts by IL-1β, TNF-α, and macrophage contact J Interferon Cytokine Res 23 57 65 12744771
Galindo M Santiago B Rivero M Rullas J Alcami J Pablos JL 2001 Chemokine expression by systemic sclerosis fibroblasts: abnormal regulation of monocyte chemoattractant protein 1 expression Arthritis Rheum 44 1382 1386 11407698
Goodman MG Chenoweth DE Weigle WO 1982 Induction of interleukin 1 secretion and enhancement of humoral immunity by binding of human C5a to macrophage surface C5a receptors J Exp Med 156 912 917 6809882
Gossart S Cambon C Orfila C Seguelas M-H Lepert J-C Rami J 1996 Reactive oxygen intermediates as regulators of TNF-αproduction in rat lung inflammation induced by silica J Immunol 156 1540 1548 8568258
Gosset P Lassalle P Vanhee D Wallaert B Aerts C Voisin C 1991 Production of tumor necrosis factor-αand interleukin-6 by human alveolar macrophages exposed in vitro to coal mine dust Am J Respir Cell Mol Biol 5 431 436 1657062
Grigg J Kukielka GL Berens KL Dreyer WJ Entman ML Smith CW 1994 Induction of intercelluar adhesion molecule-1 by lipopolysaccharide in canine alveolar macrophages Am J Respir Cell Mol Biol 11 304 311 7522015
Hao L Okada H Kanno Y Inoue T Kobayashi T Watanabe Y 2003 Direct contact between human peripheral blood mononuclear cells and renal fibroblasts facilitates the expression of monocyte chemoattractant protein-1 Am J Nephrol 23 208 213 12771503
Hetland RB Schwarze PE Johansen BV Myran T Uthus N Refsnes M 2001 Silica-induced cytokine release from A549 cells: importance of surface area versus size Hum Exp Toxicol 20 46 55 11339625
Hnizdo E Vallyathan V 2003 Chronic obstructive pulmonary disease due to occupational exposure to silica dust: a review of epidemiological and pathological evidence Occup Environ Med 60 237 243 12660371
Huaux F Louahed J Hudspith B Meredith C Delos M Renauld JC 1998 Role of interleukin-10 in the lung response to silica in mice Am J Respir Cell Mol Biol 18 51 59 9448045
Hubbard AK Giardina C 2000 Regulation of ICAM-1 expression in mouse macrophages Inflammation 24 115 125 10718114
Hubbard AK Timbin CR Shukla A Rincon M Mossman BT 2002 Activation of NF-κB-dependent gene expression by silica in lungs of luciferase reporter mice Am J Physiol 282 L968 L975
Huffman LJ Judy DJ Castranova V 1998 Regulation of nitric oxide production by rat alveolar macrophages in response to silica exposure J Toxicol Environ Health A 53 29 46 9447227
Huffman LJ Prugh DJ Millechia L Schuller KC Cantrell S Porter DW 2003 Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica J Biosci 28 29 37 12682422
Johnston CJ Finkelstein JN Gelein N Oberdorster G 1998 Pulmonary cytokine and chemokine mRNA levels after inhalation of lipopolysaccharide in C57BL/6 mice Toxicol Sci 46 300 307 10048133
Jones GS Miles PR Lanz RC Hinton DE Castranova V 1982 Ionic content and regulation of cellular volume in rat alveolar type II cells J Appl Physiol 53 258 268 7118639
Kanj R Kang J Castranova V 2002 Comparison of the responses of primary alveolar macrophages (AM’s), primary alveolar type II cells (TII), and a rat alveolar type II cell line (RLE-6TN) to lipopolysaccharide (LPS) and silica exposure [Abstract] FASEB J 16 A1146
Lee TC Wu R Brody AR Barrett JC Nettesheim P 1984 Growth and differentiation of hamster tracheal epithelial cells in culture Exp Lung Res 6 27 45 6734541
Leonard EJ Yoshimura T 1990 Human monocyte chemoattractant protein-1 (MCP-1) Immunol Today 11 97 101 2186747
Madjdpour C Oertli B Ziegler U Bonvini JM Pasch T Beck-Schimmer B 2000 Lipopolysaccharide induces functional ICAM-1 expression in rat alveolar epithelial cells in vitro Am J Physiol Lung Cell Mol Physiol 278 L572 L579 10710530
Matrat M Lardot C Huaux F Broeckaert F Lison D 1998 Role of urokinase in the activation of macrophage-associated TGF-βin silica-induced fibrosis J Toxicol Environ Health A 55 359 371 9829559
McDonald JC McDonald AD 1995 Chrysotile, tremolite, and mesothelioma Science 267 776 777 7710525
Miles PR Bowman L Rengasamy A Huffman L 1997 Alveolar type II cells cNOS activity and ATP levels are increased by lung surfactant or DPPC vesicles Am J Physiol Lung Cell Mol Physiol 273 L339 346
Moore BB Peters-Golden M Christensen PJ Lama V Kuziel WA Paine R III 2003 Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2 Am J Physiol Lung Cell Mol Physiol 284 L342 L349 12388376
Myrvik QN Leake ES Fariss B 1961 Lysozyme content of alveolar and peritoneal macrophages from the rabbit J Immunol 86 133 136 13727274
Nario RC Hubbard AK 1996 Silica exposure increases expression of pulmonary intercellular adhesion molecule-1 (ICAM-1) in C57Bl/6 mice J Toxicol Environ Health 49 599 617 8977627
Nathens AB Bitar R Watson RW Issekutz TB Marshall JC Dackiw AP 1998 Thiol-mediated regulation of ICAM-1 expression in endotoxin-induced acute lung injury J Immunol 160 2959 2966 9510200
O’Brien AD Standiford TJ Christensen PJ Wilcoxen SE Paine R III 1998 Chemotaxis of alveolar macrophages in response to signals derived from alveolar epithelial cells J Lab Clin Med 131 417 424 9605106
Pechkovsky DV Zissel G Stamme C Goldmann T Ari Jaffe H Einhaus M 2002 Human alveolar epithelial cells induce nitric oxide synthase-2 expression in alveolar macrophages Eur Respir J 19 672 683 11998997
Piguet PF Collart MA Grau GE Sappino AP Vassalli P 1990 Requirement of tumor necrosis factor for development of silica-induced pulmonary fibrosis Nature 344 245 247 2156165
Pittet JF Griffiths MJ Geiser T Kaminski N Dalton SL Huang X 2001 TGF-βis a critical mediator of acute lung injury J Clin Invest 107 1537 1544 11413161
Porter DW Ramsey D Hubbs AF Battelli L Ma J Barger M 2001 Time course of pulmonary response of rats to inhalation of silica: histological results and biochemical indices of damage, lipidosis and fibrosis J Environ Pathol Toxicol Oncol 20 suppl 1 1 14 11570667
Porter DW Ye J Ma J Barger M Robinson VA Ramsey D 2002 Time course of pulmonary response of rats to inhalation of crystalline silica: NF-κB activation, inflammation, cytokine production, and damage Inhal Toxicol 14 349 367 12028809
Reiser KM Last JA 1979 Silicosis and fibrogenesis: fact and artifact Toxicology 13 51 72 229587
Reist R Bryner K Wearden P Blackford J Vrana K Castranova V 1991 Development of a bioassay for pulmonary cell production of fibrogenic factors Toxicol Methods 1 53 65
Rojanasakul Y Ye J Chen F Wang L Cheng N Castranova V 1999 Dependence of NF-κB activation and free radical generation on silica-induced TNF-βproduction in macrophages Mol Cell Biochem 200 119 125 10569191
Rose CE Jr Sung SS Fu SM 2003 Significant involvement of CCL2 (MCP-1) in inflammatory disorders of the lung Microcirculation 10 273 288 12851645
Shi X Ding M Dong Z Chen F Ye Y Wang S 1999 Antioxidant properties of aspirin: characterization of the ability of aspirin to inhibit silica-induced lipid peroxidation, DNA damage, NF-κB activation, and TNF-αproduction Mol Cell Biochem 199 93 102 10544957
Shi X Mao Y Daniel LN Saffiotti U Dalal NS Vallyathan V 1994 Silica radical-induced DNA damage and lipid peroxidation Environ Health Perspect 102 suppl 10 149 154 7705289
Simeonova PP Toruimi W Kommineni C Ekran M Munson AE Rom WN 1997 Molecular regulation of IL-6 activation by asbestos in lung epithelial cells: role of reactive oxygen species J Immunol 159 3921 3928 9378980
Smith SM Lee DKP Lacy J Coleman DL 1990 Rat tracheal epithelial cells produce granulocyte/macrophage colony-stimulating factor Am J Respir Cell Mol Biol 2 59 68 2407273
Soloperto M Mattoso VL Fasoli A Mattoli S 1991 A bronchial epithelial cell-derived factor in asthma that promotes eosinophil activation and survival as GM-CSF Am J Physiol 260 L530 L538 1647680
Stone KC Mercer RR Gehr P Stockstill P Crapo JD 1992 Allometric relationships of cell numbers and size in the mammalian lung Am J Respir Cell Mol Biol 6 235 243 1540387
Trapnell BC Whitsett JA 2002 GM-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense Annu Rev Physiol 64 775 802 11826288
Van Snick J 1990 Interleukin-6: an overview Annu Rev Immunol 8 253 278 2188664
Williams AO Flanders KC Saffiotti U 1993 Immunohistochemical localization of transforming growth factor-β1 in rats with experimental silicosis, alveolar type II hyperplasia, and lung cancer Am J Pathol 142 1831 1840 8389528
Williams AO Saffiotti U 1995 Transforming growth factor beta1, ras and p53 in silica-induced fibrogenesis and carcinogenesis Scand J Work Environ Health 21 suppl 2 30 34 8929685
Wright JR 1997 Immunomodulatory functions of surfactant Physiol Rev 77 931 962 9354809
Xing Z Braciak TA Ohkawara Y Sallenave GM Foley R Sime PJ 1996 Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation J Leukoc Biol 59 481 488 8613693
Xing Z Joradana M Kirpalani H Driscoll KE Schall TJ Gauldie J 1994 Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor, macrophage inflammatory protein-1, interleukin-1 and inter-leukin-6, but not RANTES or transforming growth factor β mRNAs expression in acute lung inflammation Am J Respir Cell Mol Biol 10 148 153 8110470
Zickus C Kunkel SL Simpson K Glass M Streiter RM Lukacs NW 1998 Differential regulation of C-C chemokines during fibroblast-monocyte interactions: adhesion vs. inflammatory cytokine pathways Mediators Inflamm 7 269 274 9792337
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7286ehp0112-00168615579414ResearchArticlesEstimating the Independent Effects of Multiple Pollutants in the Presence of Measurement Error: An Application of a Measurement-Error–Resistant Technique Zeka Ariana Schwartz Joel Exposure, Epidemiology, and Risk Program, Environmental Health Department, Harvard School of Public Health, Boston, Massachusetts, USAAddress correspondence to A. Zeka, Exposure, Epidemiology, and Risk Program, Environmental Health Department, Harvard School of Public Health, 401 Park Dr., Suite 415 W, P.O. Box 15698, Boston, MA 02215 USA. Telephone: (617) 998-1001. Fax: (617) 384-8745. E-mail:
[email protected] thank B. Coull, M.S. O’Neill, and D.Q. Rich for their useful suggestions in the revision of the manuscript.
This work was supported by the Harvard Environmental Protection Agency Center, grant R827353. The views expressed are those of the authors and not the U.S. Environmental Protection Agency.
The authors declare they have no competing financial interests.
12 2004 7 9 2004 112 17 1686 1690 25 5 2004 7 9 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Misclassification of exposure usually leads to biased estimates of exposure–response associations. This is particularly an issue in cases with multiple correlated exposures, where the direction of bias is uncertain. It is necessary to address this problem when considering associations with important public health implications such as the one between mortality and air pollution, because biased exposure effects can result in biased risk assessments. The National Morbidity and Mortality Air Pollution Study (NMMAPS) recently reported results from an assessment of multiple pollutants and daily mortality in 90 U.S. cities. That study assessed the independent associations of the selected pollutants with daily mortality in two-pollutant models. Excess mortality was associated with particulate matter of aerodynamic diameter ≤10 μm/m3 (PM10), but not with other pollutants, in these two pollutant models. The extent of bias due to measurement error in these reported results is unclear. Schwartz and Coull recently proposed a method that deals with multiple exposures and, under certain conditions, is resistant to measurement error. We applied this method to reanalyze the data from NMMAPS. For PM10, we found results similar to those reported previously from NMMAPS (0.24% increase in deaths per 10-μg/m3 increase in PM10). In addition, we report an important effect of carbon monoxide that had not been observed previously.
air pollutioncarbon monoxidedaily mortalitymeasurement errorparticulate matter
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Growing evidence from published studies has shown increased all-cause and specific-cause mortality from short-term exposures to air pollution (Fairley 1999; Katsouyanni et al. 1997; Pope at al. 1995; Schwartz 1993; Schwartz and Dockery 1992). An important piece of that evidence comes from the National Mortality and Morbidity Air Pollution Study (NMMAPS) conducted across 90 U.S. cities (Dominici et al. 2000a; Samet et al. 2000a, 2000b, 2000c). Recent updates of this study reported excess mortality in association with exposures to particulate matter of aerodynamic diameter ≤10 μm (PM10), whereas no independent associations with gaseous pollutants were observed (Dominici et al. 2002, 2003). In these previous studies, effects of pollutants were examined using two- and multiple-pollutant models.
From the public health perspective, when considering the evidence of a positive association between air pollution and mortality, it is important to determine whether such an effect is biased due to exposure misclassification and, if so, to correct for that bias.
The magnitude and direction of uncertainty in the observed effects of air pollution due to exposure measurement error have been argued by several investigators to be limitations in making causal inference for the link between air pollution and health outcomes (Lipfert and Wyzga 1997, 1999). In a single-pollutant model, exposure measurement error, due to the nondifferential misclassification, will underestimate the “true” effects of exposure–response associations (bias toward the null). Because of this, risk assessments based on the findings of observational epidemiologic studies may underestimate the benefits of reducing exposures. This is particularly true for air pollution studies, which, unlike cancer risk assessment, rely on maximum likelihood estimates of risk coefficients and not on upper confidence estimates.
The situation is more complex in the case of multiple correlated pollutants. Here, the measurement error in one pollutant will tend to bias the risk coefficient of that pollutant toward the null. However, measurement error in the second pollutant will contribute some bias to the coefficient of the first pollutant. The direction of the bias will depend on the sign of the correlation between the pollutants. In rare cases, when the correlation is high between the two pollutants and the measurement error in the second is large, this can produce an upward bias in the risk coefficient of the first pollutant. This may lead to an overestimation of exposure effects of the better measured pollutant (bias away from the null) (Schwartz 2000; Zeger et al. 2000). In the context of studies of air pollution, the upward bias has been cited as one reason for the positive associations between air pollutants and health outcomes (Lipfert and Wyuzga 1997, 1999). In recent analysis of this issue, Zeger et al. (2000) demonstrated that in the case of two pollutants measured with error, the correlation between the two pollutants, the variances of measurement errors of these two pollutants, and the correlation between the two errors would predict the magnitude and direction of bias. The study showed that even with hypothetically large differences in the four parameters, upward bias was unlikely (Zeger et al. 2000). Schwartz and Coull (2003) reported a similar finding in a simulation study, where under certain assumptions such as high correlation between the pollutants and their errors (> 0.95) and/or large difference in the error variances, upward bias was not likely to occur.
Why is there measurement error in air pollution studies? A recent development in air pollution epidemiology, called time-series design, is based on series of air pollution concentrations and health outcomes (events) over a certain period of observation (which may be months or years). Through this, one can estimate the average number of events that occur with changes in air pollution concentrations. The unit of analysis is the day, and the outcome data are the counts of events (mortality, or other health outcomes). Exposure data are usually ambient concentrations of different air pollutants measured continuously (hourly or daily) from fixed-site monitoring stations. However, monitored ambient concentrations of air pollutants are not representative of personal exposures, which are important when evaluating the relation of exposure and health outcome at the individual level. Unfortunately, in time-series studies of air pollution, there are no available calibration data (routinely measured series of personal exposure data) on which to base a measurement error correction. Dominici et al. (2000b) described a method to estimate a correction factor, using information on ambient and personal exposures in several cities in the United States, addressing only one pollutant (PM10). However, the applicability of that approach to the population-based time series is difficult because a more complex scenario of multiple air pollutants is generally present and usually information on only ambient exposures is available.
In recent reports, Schwartz and Coull (Schwartz 2000; Schwartz and Coull 2003) have developed an approach that uses hierarchical modeling to assess exposure–health outcome associations, which is resistant to exposure measurement error. The method is useful in studies with multiple exposures, such as air pollution time-series studies, and can provide bias-corrected estimates for the multiple exposures in the presence of measurement error. In the present study, we had two goals. First, to validate recent findings in air pollution epidemiology, we applied this approach to examine the independent effects of PM10 and several gaseous air pollutants on daily deaths, using recent data and results from NMMAPS. Second, we demonstrated the method with the intention to make it compelling to other researchers as a useful tool in assessing causal relationships.
Materials and Methods
The NMMAPS study analyzed the association between air pollution and daily deaths in 90 U.S. cities. The cities included essentially the entire urban U.S. population living in counties with regular air pollution monitoring. Data on daily deaths in this study were obtained from records of the National Center for Health Statistics (Hyattsville, MD), and air monitoring data were obtained from the U.S. Environmental Protection Agency (Washington, DC), for the period from 1987 to 1994. This study conducted Poisson regressions, relating the daily death counts as a function of each day to air pollution concentrations on the same day, the previous day, and 2 days before the event, controlling for weather and season. Under such model, a Poisson process is assumed to describe the number of deaths per day, with events following a binomial process with low probability of occurrence. The model had the form log[E(Y )] = α+ ∑βjXj + ∑βkZk, where E (Y ) was the expected daily death count, βj represented the coefficients measuring the effects of j pollutants, and βk represented the coefficients measuring the effects of k predictors (weather, season). The study looked at the effects on daily mortality from ambient concentrations of PM10, sulfur dioxide, nitrogen dioxide, carbon monoxide, and ozone. Further details have been published elsewhere (Dominici et al. 2003; Samet et al. 2000b, 2000c).
The hierarchical model.
The approach from Schwartz and Coull (2003) applied to this study was as follows: If an outcome were linearly associated with two exposures—in our case two pollutants (X1 and X2)—then we would have a model such as
where E (Y ) is the expected daily mortality and β1 and β2 are the unbiased effects of, respectively, X1 and X2. If X1 and X2 were correlated with each other, then we could also fit a model like the following:
If we now substitute X2 in Equation 1 with Equation 2, then we would obtain
If we were to regress Y against X1 alone, then
And by comparing Equations 3 and 4, we would obtain
Hence, as Equation 5 shows, by regressing the coefficient relating X1 to mortality (δ1) against the coefficient relating X2 to X1 (γ1), we can recover β2, the coefficient relating X2 to mortality. If instead of substituting for X2 we had substituted for X1, then we would similarly have obtained an estimate of β1.
The advantage of this method is seen when we consider the impact of measurement error. If X1 and X2 are both measured with error, then the coefficients γ1 and δ1 in Equations 2 and 4 are both biased; however, that bias depends only on the variance of X1 and its measurement error, which are the same in both equations and cancel out in Equation 5. This results in the estimate of β 2 in Equation 5 being unbiased. The extension of the approach to models with additional predictors, and Poisson regressions, is provided in Schwartz and Coull (2003).
We applied the hierarchical model to the NMMAPS study to estimate the unbiased independent effects of each of the five pollutants (PM10, SO2, NO2, CO, and O3) on daily mortality. One can think of the application of the hierarchical approach of Schwartz and Coull (2003) as a three-step analysis. As an example, we are presenting each step using two pollutants (e.g., SO2 and PM10). If we were to estimate the “true” effects of each of the two pollutants on daily mortality, then β1 (in Equation 1) would represent the unbiased effect of SO2, and β2 the unbiased effect of PM10. However, we are unable to estimate β1 and β2 directly, due to measurement error in each pollutant. The three-stage method then comes into play.
In the first stage, daily values of the various air pollutants, in each city, were regressed against each other to obtain regression slopes for each pollutant pair, using least-squares linear regression. In this step, we applied Equation 2, which in our example would take the form PM10 = γ0 + γ1SO2 + e. To enhance comparability with the original NMMAPS results, we obtained the daily air pollution data used by the NMMAPS researchers, so that their cleaning and averaging procedures would be reflected in our analysis. These data have been made publicly available as part of the Internet-based Health and Air Pollution Surveillance System (IHAPSS 2003).
The second stage involved fitting single-pollutant models to the mortality data, in each city, for each of the pollutants being examined (Equation 4). In such case, if estimating the effects of SO2 on daily mortality, then E (Y ) = δ0 + δ1SO2. The NMMAPS study already provided results for the single-pollutant models in each city, relating daily death counts to daily concentration of each pollutant (PM10, SO2, NO2, CO, and O3) on the day before (lag 1) the event, using Poisson regression modeling (Dominici et al. 2003; Samet et al. 2000b). Therefore, the slopes relating mortality and each of the air pollutants the day before death, by city, as provided by the NMMAPS, were the coefficients (δ1) of the second stage of the hierarchical approach.
Finally, the slopes (δ1) obtained from the second stage were regressed against those (γ1) obtained from the first stage, across the 90 U.S. cities (Equation 5), using least-squares linear regression. The slope of this final regression is, ideally, an unbiased estimate of β2, the effect of PM10 on daily mortality, controlling for the effect of SO2 and of measurement error.
The method allows one to recover the independent effect of each pollutant, controlling for any other pollutant, using an approach that is, in principle, unbiased by measurement error and that, in simulations, appears to be relatively unbiased under moderate violations of the model assumptions (Schwartz and Coull 2003). Obviously, for this approach to work, the independent variable (γ1) in Equation 5 must vary across the cities.
In the application of the three-stage analyses, the five pollutants were paired with each other. That is, for each pollutant, four unbiased independent estimates relating that pollutant to daily mortality were obtained, each estimate controlling for the effects of the other four pollutants. One limitation of the hierarchical modeling is loss in precision in these estimates, because the last regression (Equation 5) has only 90 observations. This is reflected in the confidence limits of the estimates. We could improve power in the effect of each pollutant, by averaging its four estimates, using the following formula:
where β̄j is the weighted average slope (meta-slope) for pollutant j (i.e., PM10) defined over k number slopes (obtained from third-stage regressions), and βji is the unbiased slope of pollutant j (i.e., PM10) controlling for pollutant i (i.e., SO2), where i = 1 to k. Weights for the slopes were defined as follows:
where vji is the variance of βji. The variance of β̄j is calculated as
Results
The relationship between pollutant pairs differed across the 90 cities (Table 1). The heterogeneity in the pollutant–pollutant regression slopes (γ1) among all the cities, as shown in Table 1, assured sufficient variability in the independent variable of the third-stage regression to proceed with the analysis. Power in a linear regression is increased by increasing the variability in the independent variable (γ1 in this case) and by reducing variability in the residuals. Table 1 indicates that at least the first condition is met. The range of variability in slopes relating PM10 to other pollutants was lower for traffic-related pollutants (CO and NO2) than for SO2 or O3. This likely reflects traffic particles always being a substantial component of PM10, whereas the correlation with SO2 is more varied because it depends on the sulfur content of fuel. SO2 is poorly correlated with O3, resulting in a very small mean slope and large range of variation. The range of variation in the slope relating NO2 to SO2 was smaller than for the other pollutants. It is possible that this reflects the importance of diesel emissions for NO2 concentrations in urban areas. Diesel fuel has much higher sulfur content than does gasoline, and this may contribute to a tighter spread of the association between the two pollutants across cities.
The bias-corrected estimates from the third-stage analysis are presented in Table 2. The results presented are percent increase in daily deaths for a 10-μg/m3 increase in PM10, or a 10-ppb increase in each of the gaseous air pollutants, except CO (100 ppb). For PM10, the percent increase in daily mortality ranged from 0.14 to 0.35% (controlling for other pollutants), with an overall estimate of 0.24% [95% confidence interval (CI), 0.05–0.42%]. In contrast, we found small and nonsignificant associations of daily deaths with SO2, NO2, and O3.
We found increased daily mortality in association with CO in the present analyses, with an overall relative excess daily mortality of 0.06% per increments of 100 ppb of CO, estimated with fair precision (95% CI, 0.02–0.10%).
Discussion
The validity of exposure–response associations in epidemiologic studies depends on the precision of exposure measurements (Dominici et al. 2000b; Schwartz and Coull 2003; Zeger et al. 2000). Environmental studies of air pollution often lack precisely measured exposures, which can lead to exposure misclassification and biased estimates of exposure–response associations (Dominici et al. 2000b; Schwartz and Coull 2003; Zeger et al. 2000). Usually, an exposure measured with error will bias the association toward the null. A more complicated problem occurs when two or more exposures are measured with error and are correlated with each other. Zeger et al. (2000) reported that, in such a case, the better measured exposure may, rarely, capture some of the effect of the other exposure, which could bias away from the null. In any event, the extent of downward bias in each pollutant effect still depends on the measurement error in the other exposure. Hence, environmental studies of multiple air pollutants may inherit bias in both directions and with varying degrees. Underestimating the public health consequences of air pollution exposure can result in suboptimal measures to reduce these health consequences, particularly when cost–benefit or cost-effectiveness analysis is used as part of the decision process. Uncertainties about upward bias in the effect estimates can undermine the credibility of observed associations, raising questions about the appropriateness of proposed air quality standards. Hence, reducing both potential errors can improve environmental health.
Schwartz and Coull (2003) recently described a method that uses hierarchical modeling to deal with confounding and measurement error bias in epidemiologic studies. Their approach yielded an exposure–response estimate that was unbiased under certain assumptions and showed small downward biases when those assumptions were not met—for example, when the measurement errors among the multiple exposures were correlated. Although not entirely eliminated, the bias effect in this case was much less than the one produced by the two-pollutant model under the same circumstances (Schwartz and Coull 2003).
In the present study we applied the method of Schwartz and Coull (2003) to data and results from NMMAPS to estimate the independent effects of air pollutants on daily mortality. The application of this approach to the NMMAPS results was used in the case of two concurrent pollutants, both assumed to be measured with error. The method provided minimally biased independent effect estimates for each pollutant–daily mortality association. The price of this reduced bias was a reduction in precision. However, when we averaged over the results for each different pollutant, important associations appeared for PM10 and CO.
Recent results of NMMAPS had shown positive associations between PM10 and daily mortality for 90 U.S. cities (Dominici et al. 2003; Samet et al. 2000b, 2000c). The relative increase ± SE in daily mortality was 0.21 ± 0.06% per 10-μg/m3 increase of PM10 concentration 1 day before the event. The presence of other pollutants in the model did not change this effect (Dominici et al. 2003). Our estimate for this pollutant was slightly greater after reducing measurement error bias (0.24 ± 0.09% increase in mortality).
The NMMAPS had reported no independent associations between daily mortality and concentration of other air pollutants 1 day before the event, including SO2, NO2, CO, and O3 (Dominici et al. 2003). The findings for gaseous pollutants from that study were based on two- and multiple-pollutant models. For comparison with our results, we report the estimates from this previous study for increments in concentration of 10 ppb. The estimates for SO2 from the two- and multiple-pollutant models from the NMMAPS ranged between 0.4 and 0.5% increase in daily mortality. NO2 showed relative increases in daily mortality from 0.3 to about 0.4%. Relative increase in daily mortality for O3 varied between 0.08 and 0.2%. Percent increases of 0.02–0.06 in daily mortality were associated with increments of 100 ppb in CO concentrations. None of these reported effects was estimated precisely, which resulted in the summary of the evidence from this previous study of no association between any of the gaseous pollutants and daily mortality (Dominici et al. 2003).
We found the effects for SO2, controlling for other pollutants, to vary greatly, with an overall estimate of 0.1% increase in daily mortality. For NO2, we found essentially no effect on daily mortality (estimate = –0.004%). O3 effects in our study were found to be between two and three orders of magnitude smaller than the observed effects for the same pollutant from the NMMAPS. However, none of these estimates was precise, which made our summary finding for these pollutants qualitatively similar to the one reported from the NMMAPS study.
Unlike in the NMMAPS finding, we observed an association between CO and daily mortality. The estimates, controlling for other pollutants, ranged from −0.02 to 0.09%, with the greater effects being estimated fairly precise. The pooled effect of CO showed a 0.06% increase in mortality with a tight confidence interval (95% CI, 0.02–0.10% per 100 ppb).
One explanation for the different finding for CO in our study, compared with that of NMMAPS, could be related to the high degree of measurement error in this pollutant. There is a possibility that the spatial heterogeneity in ambient concentrations of CO is greater than that of any other air pollutant. This would produce a greater amount of measurement error when monitoring ambient concentrations of CO from a central monitoring site. Whether the greater effect seen using the hierarchical modeling approach reflects a true association with CO per se, or whether CO is a surrogate for traffic particles or some other component of vehicular exhaust (Sarnat et al. 2001), is not clear. Nevertheless, the results for CO indicate the potential use of the approach and suggest that attention should be focused on CO or on traffic pollution. The similar results of this study with those of NMMAPS for PM10 were reassuring.
Several limitations in the application of the Schwartz and Coull (2003) approach must be acknowledged. First, because the third-stage regression had only 90 observations, the power of the method was reduced. The power depends in part on the R
2 values of the third-stage models. In our case, these were low (Table 2), forcing us to apply meta-analysis as an ad hoc approach to improve power. In other applications, R2 may be larger, and this approach may be unnecessary. We are currently developing a multivariate version of the approach that is less ad hoc in improving power, by using multiple predictors at the last-stage regression.
Second, we did not have season-specific regression coefficients relating air pollution to mortality in the NMMAPS. The use of this approach with season-specific relationships offers increased power because of the increased number of risk coefficients by pollutant, and also because the relationship between many pollutants varies seasonally. We expect these to be available from NMMAPS in the future.
Third, in the present analyses we assumed that the concentration–response relation between air pollution and daily deaths is linear. This question has been subject to a number of investigations using splines and smoothing in single-city studies and combining splines or smooth curves in multicity studies (Schwartz and Zanobetti 2000; Schwartz et al. 2002). At least for PM10, linear associations were seen in concentration ranges similar to those of the present study.
Finally, our model implies that each pollutant, as measured at a central site in each city, is a surrogate for exposure to the same pollutant. However, Sarnat et al. (2001) have proposed recently that ambient concentrations of gaseous air pollutants may be serving as surrogates not for exposure to those gases but for exposure to particles or particles from particular sources. For example, in Baltimore, CO was a good surrogate for exposure to particles from traffic (Sarnat et al. 2001). This suggests that there must be caution in interpreting the present results for CO. We think that personal exposure studies of intermediate outcomes may be necessary to resolve the question.
Despite these limitations, the approach of Schwartz and Coull (2003) is useful to studies of air pollution and mortality. In our study, it provided evidence of a slightly greater effect of PM10 than that reported previously by NMMAPS, when controlling for measurement error in other pollutants, and suggested that greater attention be paid to CO and possibly traffic pollution.
Table 1 Mean ± SD of the distribution of pollutant–pollutant regression slopes (γ1),a and their coefficients of variation (CV), across 90 U.S. cities, for each pollutant pair.
Pollutant–pollutant regression variables
Pollutant–pollutant regression slopes
Dependent pollutant Independent pollutant Mean ± SD CV (%)
PM10 SO2 1.31 ± 1.45 110.3
NO2 0.83 ± 0.64 77.2
CO 0.01 ± 0.01 62.7
O3 0.27 ± 0.38 143.6
SO2 PM10 0.09 ± 0.08 87.9
NO2 0.22 ± 0.20 91.6
CO 0.003 ± 0.003 92.0
O3 −0.03 ± 0.10 377.6
NO2 SO2 2.64 ± 5.09 192.8
PM10 −0.22 ± 1.83 831.1
CO −0.01 ± 0.08 1030.7
O3 0.19 ± 2.03 1064.6
CO SO2 61.58 ± 74.64 121.2
NO2 34.19 ± 19.39 56.7
PM10 13.73 ± 9.26 67.4
O3 −9.58 ± 9.47 98.8
O3 SO2 136.66 ± 535.82 392.1
NO2 24.76 ± 95.82 387.0
CO −0.59 ± 3.26 550.6
PM10 −63.99 ± 292.61 457.2
a The slopes were obtained from first-stage regressions (Equation 2), pairing each of the five pollutants with the other four (pollutant pairs), for each U.S. city.
Table 2 Percent increase in daily deaths associated with each pollutant, controlling for measurement error in other pollutants, based on data and results from the NMMAPS study, including the period between 1987 and 1994.
Independent effects of pollutants on daily mortalitya
PM10 SO2 NO2 CO
O3
Paired pollutantb R2c Sloped Te R2 Slope T R2 Slope T R2 Slope T R2 Slope T
PM10 NA NA NA 0.048 1.14 1.77 0.008 0.033 0.69 0.029 0.078 1.56 0.008 0.0003 0.77
SO2 0.047 0.28 1.73 NA NA NA < 0.001 −0.004 0.06 0.154 0.086 3.25 0.012 −0.0004 0.83
NO2 0.012 0.16 0.83 0.007 −0.29 0.54 NA NA NA 0.007 0.032 0.64 0.024 −0.0019 1.15
CO 0.006 0.14 0.69 0.030 0.65 1.33 0.001 −0.004 0.27 NA NA NA 0.045 0.0011 1.87
O3 0.036 0.35 1.70 0.039 −0.76 1.52 0.007 −0.025 0.63 0.002 −0.018 0.34 NA NA NA
Meta-slopef NA 0.24 2.80 NA 0.10 0.34 NA −0.004 0.27 NA 0.062 3.16 NA 0.0002 0.74
NA, nonapplicable.
a Corrected slopes (effects) removing the effect of each pollutant shown in the first column.
b The independent pollutant of first-stage regression.
c Adjusted R2 from regressions of the third stage (see text for details).
d Slope for PM10 presented as percent increase per 10 μg/m3. Slopes for other pollutants are presented as percent increase per 10 ppb (100 ppb for CO).
e t-Statistic from regressions of the third stage.
f Meta-slope of the four alternative estimates.
==== Refs
References
Dominici F McDermott A Daniels M Zeger SL Samet JM 2003. Mortality among Residents of 90 Cities. Revised Analyses of Time-Series Studies of Air Pollution and Health. Special Report. Montpelier, VT:Capital City Press, 9–24.
Dominici F McDermott A Zeger SL Samet JM 2002 On the use of generalized additive models in time-series studies of air pollution and health Am J Epidemiol 156 193 203 12142253
Dominici F Samet J Zeger S 2000a Combining evidence on air pollution and daily mortality from the twenty largest US cities: a hierarchical modeling strategy J R Stat Soc Ser A 163 263 302
Dominici F Zeger SL Samet JM 2000b A measurement error model for time-series studies of air pollution and mortality Biostatistics 1 157 175 12933517
Fairley D 1999 Daily mortality and air pollution in Santa Clara County, California: 1989–1996 Environ Health Perspect 107 637 641 10417361
IHAPSS 2003. Internet-based Health and Air Pollution Surveillance Systems. Baltimore, MD:Johns Hopkins Bloomberg School of Public Health. Available: http://www.ihapss.jhsph.edu/data/data.htm [accessed 15 September 2003].
Katsouyanni K Touloumi G Spix C Schwartz J Balducci F Medina S 1997 Short-term effects of ambient sulphur dioxide and particulate matter on mortality in 12 European cities: results from time series data from the APHEA project Br Med J 314 1658 1663 9180068
Lipfert FW Wyzga RE 1997 Air pollution and mortality: the implications of uncertainties in regression modeling and exposure measurement J Air Waste Manag Assoc 47 517 523 9130440
Lipfert FW Wyzga RE 1999 Statistical considerations in determining the health significance of constituents of airborne particulate matter J Air Waste Manag Assoc 49 182 191 11002835
NMMAPS 2003. National Morbidity and Mortality Air Pollution Study. Baltimore, MD:Johns Hopkins University. Available: http://www.biostat.jhsph.edu/biostat/research/update.main.htm [accessed 15 September 2003].
Pope CA III Bates DV Raizenne ME 1995 Health effects of particulate air pollution: time for reassessment? Environ Health Perspect 103 472 480 7656877
Samet JM Dominici F Curriero FC Coursac I Zeger SL 2000a Fine particulate air pollution and mortality in 20 U.S. cities, 1987–1994 N Engl J Med 343 1742 1749 11114312
Samet JM Dominici F Zeger SL Schwartz J Dockery DW 2000b The National Morbidity, Mortality, and Air Pollution Study. Part I: Methods and methodologic issues Res Rep Health Eff Inst 94 pt 1 5 14
Samet JM Zeger SL Dominici F Curriero F Coursac I Dockery DW 2000c The National Morbidity, Mortality, and Air Pollution Study. Part II: Morbidity and mortality from air pollution in the United States Res Rep Health Eff Inst 94 5 7
Sarnat JA Schwartz J Catalano PJ Suh HH 2001 Gaseous pollutants in particulate matter epidemiology: confounders or surrogates? Environ Health Perspect 109 1053 1061 11675271
Schwartz J 1993 Air pollution and daily mortality in Birmingham, Alabama Am J Epidemiol 137 1136 1147 8317443
Schwartz J 2000 Daily deaths are associated with combustion particles rather than SO2 in Philadelphia Occup Environ Med 57 692 697 10984342
Schwartz J Coull B 2003 Control for confounding in the presence of measurement error in hierarchical models Biostatistics 4 569 582 14557112
Schwartz J Dockery DW 1992 Increased mortality in Philadelphia associated with daily air pollution concentrations Am Rev Respir Dis 145 600 604 1546841
Schwartz J Laden F Zanobetti A 2002 The concentration-response relation between PM(2.5) and daily deaths Environ Health Perspect 110 1025 1029 12361928
Schwartz J Zanobetti A 2000 Using meta-smoothing to estimate dose-response trends across multiple studies, with application to air pollution and daily death Epidemiology 11 666 672 11055627
Zeger SL Thomas D Dominici F Samet JM Schwartz J Dockery D 2000 Exposure measurement error in time-series studies of air pollution: concepts and consequences Environ Health Perspect 108 419 426 10811568
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Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Science 10.1289/ehp.7199ehp0112-00169115579415ResearchArticlesEpidemiologic Evaluation of Measurement Data in the Presence of Detection Limits Lubin Jay H. 1Colt Joanne S. 1Camann David 2Davis Scott 3Cerhan James R. 4Severson Richard K. 5Bernstein Leslie 6Hartge Patricia 11Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA2Southwest Research Institute, San Antonio, Texas, USA3Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington, USA4Mayo Clinic, College of Medicine, Rochester, Minnesota, USA5Karmanos Cancer Institute and Department of Family Medicine, Wayne State University, Detroit, Michigan, USA6Department of Preventive Medicine, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, USAAddress correspondence to J. Lubin, National Cancer Institute, Biostatistics Branch, 6120 Executive Boulevard, Room 8042, Rockville, MD 20852 USA. Telephone number: (301) 496-3357. Fax: (301) 402-0081. E-mail:
[email protected] for this study included contracts with the National Cancer Institute: N01-PC-67010, N01-PC-67008, N02-PC-71105, N01-PC-67009, and N01-PC-65064.
The authors declare they have no competing financial interests.
12 2004 13 9 2004 112 17 1691 1696 21 4 2004 13 9 2004 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Quantitative measurements of environmental factors greatly improve the quality of epidemiologic studies but can pose challenges because of the presence of upper or lower detection limits or interfering compounds, which do not allow for precise measured values. We consider the regression of an environmental measurement (dependent variable) on several covariates (independent variables). Various strategies are commonly employed to impute values for interval-measured data, including assignment of one-half the detection limit to nondetected values or of “fill-in” values randomly selected from an appropriate distribution. On the basis of a limited simulation study, we found that the former approach can be biased unless the percentage of measurements below detection limits is small (5–10%). The fill-in approach generally produces unbiased parameter estimates but may produce biased variance estimates and thereby distort inference when 30% or more of the data are below detection limits. Truncated data methods (e.g., Tobit regression) and multiple imputation offer two unbiased approaches for analyzing measurement data with detection limits. If interest resides solely on regression parameters, then Tobit regression can be used. If individualized values for measurements below detection limits are needed for additional analysis, such as relative risk regression or graphical display, then multiple imputation produces unbiased estimates and nominal confidence intervals unless the proportion of missing data is extreme. We illustrate various approaches using measurements of pesticide residues in carpet dust in control subjects from a case–control study of non-Hodgkin lymphoma.
dustenvironmental exposureimputationmissing datanon-Hodgkin lymphomapesticides
==== Body
Epidemiologic studies often collect quantitative measurement data to improve precision and reduce bias in exposure assessment and in the estimation of the effect of exposure on risk of disease, as measured by odds ratios (Hatch and Thomas 1993; Sim 2002). Some measurements serve as biomarkers for “dose”—for example, residual radiation in tooth enamel as a marker of exposure to ionizing radiation (Desrosiers and Schauer 2001)—whereas other measures are more indirect—for example, urinary cotinine level as an indicator of exposure to environmental tobacco smoke (Woodward and Al Delaimy 1999). Problems in the analysis of measurement data commonly arise because measurement procedures often have detection limits (DLs). A DL may represent a floor value, a ceiling value, or an interval where precise quantitative levels cannot be determined. For example, exposure assessment for nuclear workers relied on radiation film badges that record radiation levels only above a fixed minimum, because of limits in film photosensitivity (Gilbert et al. 1996; Kerr 1994). Investigators encountered ceiling levels of particle-bound polycyclic aromatic hydrocarbons in rural Chinese dwellings when values exceeded 60,000 ng/m3, the upper limit of the measurement protocol (Ligman et al. 2004).
Although values below or above a DL are “missing,” data are not missing at random in the usual sense, because their absence reflects levels of exposure. This type of missing data is called “nonignorable missing,” and the simple exclusion of such “interval-measured” data can bias results (Little and Rubin 1987; Schafer 1997).
Analytic procedures for environmental measurement data with DLs are often presented in the context of environmental monitoring where the primary goal is estimation of distributional parameters when numbers of measurements are limited (Gleit 1985; Haas and Scheff 1990; Helsel 1990; Persson and Rootzen 197; Singh and Nocerino 2002; Travis and Land 1990). In epidemiologic studies, measurement data are used to characterize exposures of study subjects and are typically employed in two ways: a) to develop regression models to examine the relationship between a measured value (dependent variable) and covariates (independent variables); and b) as covariates in a risk analysis to estimate the relationship between a binary disease outcome and exposure measures and other factors. In this article, we focus on the first application, namely, the regression of an exposure measurement on covariate factors. The use of measurements with DLs in risk regression will be considered in another article.
Investigators apply various strategies for measurement data with DLs, including replacement of measurements below a DL with a single value, for example, DL, DL/2, or DL/√2 (Helsel 1990; Hornung and Reed 1990). Less frequently, measurements below a DL are assigned a value of zero. Unless such measurements indicate a true zero exposure, this latter strategy clearly distorts results and is not considered further in this article. If the distribution of the measurement data is known—for example, measurements are log-normally distributed—then an alternative strategy replaces values below the DL with expected values of the missing measurements, conditional on being less than the DL (Garland et al. 1993; Gleit 1985). For measurement Z and detection limit DL, we denote this value E[Z |Z < DL]. Calculation of the conditional expected value requires the investigator to either know or estimate parameters of the measurement distribution.
Substitution schemes like those described above are simple, because one value replaces all measurements below the DL, and, except for E[Z|Z < DL], distributional assumptions are not considered. However, because a single value represents all measurements below the DL, parameter estimates and their variances are likely biased, unless the proportion is small, which potentially distorts inference. This limitation led to a single-impute “fill-in” method (Helsel 1990; Moschandreas et al. 2001a, 2001b). An investigator first characterizes the form of the distribution and estimates its parameters and then assigns randomly sampled values below the DL from the estimated distribution. Fill-in values along with measured values above the DL are then used in analyses. With appropriate estimation techniques, this approach accommodates multiple DLs.
As described by Helsel (1990) and applied by Moschandreas et al. (2001b), the fill-in method did not include complex modeling of regression factors. In addition, although the fill-in approach assigned random values from an appropriate distribution, it did not account for the variability of the imputation process, because the inserted values are not real data. In this article, we illustrate methods for epidemiologic data that account for measurements with DLs, using data from a case–control study of non-Hodgkin lymphoma (NHL) (Colt et al. 2004). The example evaluates the relationship between concentrations of pesticide analytes in carpet dust and use of pesticide products in and around the home. We restrict analysis to control subjects, with adjustment for study design factors.
Example Data from a Case–Control Study of NHL and Pesticides
The principal exposure of the general population to pesticides occurs in the home (Nigg et al. 1990) as the result of indoor use, track-in or drift from outdoors, intrusion of vapors from foundation treatments, or take-home contamination from occupational use (Bradman et al. 1997; Lewis et al. 1999, 2001). Pesticide residues are retained in carpets, migrating into the underlying foam pad, and may persist for months or years.
Data source.
We consider data from controls from a multicenter, population-based case–control study of NHL, conducted in the United States: the Detroit, Michigan, metropolitan area; the state of Iowa; Los Angeles County, California; and the Seattle, Washington, metropolitan area (Colt et al. 2004). Controls include 1,057 residents 20–74 years of age, frequency matched to cases on age, sex, race, and study area, with an oversampling of African-American subjects in Los Angeles and Detroit.
Interviewers collected information from respondents on lifetime residential history and the frequency and form of pesticides used to treat various types of pests (e.g., flying insects, crawling insects, lawn weeds). Interviewers obtained vacuum cleaner bags from 95% of subjects who had used their vacuum cleaners within the past year and had owned at least half of their carpets or rugs for 5 years or more. Bags were shipped in insulated boxes by overnight mail to Southwest Research Institute and placed in freezers. Samples were collected and analyzed for 513 control subjects.
Measurement of carpet dust.
The protocol for the collection and measurement of dust samples has been described previously (Colt et al. 2004). Briefly, before extraction and analysis, dust samples were sieved through a 100-mesh sieve to obtain the fine (< 150 μm) dust. Neutral extractions were carried out for 25 pesticides (18 insecticides, six herbicides, and ortho-phenylphenol), seven polycyclic aromatic hydrocarbons, and five polychlorinated biphenyl congeners. Acid extractions were carried out for four herbicides and pentachlorophenol. Extracts were analyzed using gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode. Analyte amounts were quantified using the internal standard method. In the full study, GC/MS analysts were blinded to disease status.
After analyzing about half of the samples, investigators began monitoring additional ions for some neutral analytes to clarify identification at low levels, resulting in raised DLs for 14 pesticides. DLs were also raised when < 2 g dust were available. An additional problem with some dust samples involved the presence of interfering compounds (i.e., compounds that coeluted with the target analyte), creating uncertainty and prohibiting assignment of specific concentrations.
For three scenarios analysts could provide concentrations only within an interval, which we accommodated by defining a lower bound (LB) and an upper bound (UB) of possible values. If the analyte was not detected and no interferences were present (type I), the LB was set to zero and the UB was set to the specified DL. If there was an interfering compound but insufficient evidence for the presence of the target analyte (type II), the GC/MS analyst reported the result as a nondetect with a DL equal to the entire peak of the coeluting compounds. We set the LB to zero and the UB to 20% of the raised peak or to the DL, whichever was larger. If the target analyte and the interference were both present (type III), the analyst reported an “elevated detect” with a concentration equal to the entire peak of the coeluting compounds. We set the LB bound to the maximum of 20% of the recorded peak, or the DL, and the UB to the maximum of 90% of the reported peak, or the DL, resulting in an interval bounded away from zero.
For ease of presentation, we allow the replacement of measurements below the DL with DL/2 (which applies to missing data types I and II) to refer more generally to the replacement with (LB + UB)/2 (which applies to missing data types I, II, and III).
Methods and Analysis
Preliminary analysis indicates that measurement data are consistent with a log-normal distribution. If Z denotes the measured value of an analyte and is log-normally distributed, denoted Z ~ LN(μ,σ2), then by definition log(Z) is a normal random variable with mean μ and variance σ2, denoted log(Z) ~ N(μ,σ2) (Singh et al. 1997). Suppose X = (X0, … , XK)t is a column vector of covariates, with X0 = 1, and β= (β0, … , βK)t is a column vector of regression parameters, where t denotes vector transpose. If data are complete, then a linear regression equation has the form log(Z) = βtX + ɛ, where ɛ~ N(0, σ2). For each X, the model implies that Z is log-normally distributed with mean βtX; that is, Z ~ LN(βtX, σ2).
Regression analysis in control data.
We evaluate the association between analyte concentration and pesticide use by fitting a linear regression model of the logarithm of the analyte level on subject characteristics. Regression (independent) covariates include indicator variables for season of sample collection, presence of oriental rugs, study center, sex, age (< 45, 45–64, ≥ 65 years), race (African American, Caucasian, other), type of home (single family, townhouse/duplex/apartment, other), year of home construction (< 1940< 1940–1959–1960–1979, ≥ 1980), and educational level (< 12, 12–15, ≥ 16 years). As in Colt et al. (2004), covariates vary slightly with analyte. Models also include five variables describing the use of insect treatment products: ever/never used products to treat for crawling insects, flying insects, fleas/ticks, termites, and lawn/garden insects. We use data from current homes only.
Regression analysis is hampered by the presence of measurements known only within bounds. We assume that the probability distributions of measurements below the DL (more precisely, within the LB and UB interval) depend only on observed data; that is, the interval-measured concentrations arise from the same distributions that generate the measured values. Let F(•) be the cumulative distribution function and f(•) the probability density function for a log-normal random variable. Suppose Xi = (Xi0, … ,XiK)t is the covariate vector for the ith of i = 1, … , n subjects. LBi and UBi are recorded for i = 1, … , n0 individuals, whereas a specific Zi measurement is recorded for i = n0 + 1, … , n0 + n1 individuals. LB and UB are subscripted to allow different DLs. Using a Tobit regression approach (Gilbert 1987; Persson and Rootzen 1977; Tobin 1958), the log-likelihood function has the form
The first summand derives from the n0 interval measured values and involves the difference of the cumulative distribution function F evaluated at UB and at LB; that is, the probability the measurement lies between the LB and UB. The second summand derives from the n1 detected values. Maximum likelihood estimates (MLEs) for β and their covariance matrix are obtained by maximizing Equation 1 and computing the inverse information matrix using standard methods.
Imputation of missing concentrations.
If the goal is to evaluate pesticide use and analyte levels in carpet dust, represented by the β parameters, then the Tobit regression of Equation 1 is sufficient and no imputation is required. For further analysis or for graphical display, it is useful to generate values for measurements below DLs. We consider several different approaches, including inserting DL/2, inserting E[Z|Z < DL], or using a single or multiple imputation (Little and Rubin 1987).
A multiple imputation procedure is carried out as follows. Using all data (measured concentrations, missing data types I–III, and covariates), we create the log-likelihood function 1, solve for the MLEs of β and σ2 (denoted β̂ and ς̂2), and impute a value by randomly sampling from a log-normal distribution with the estimated parameters. However, in selecting fill-in values we cannot ignore that β̂ and ς̂2 are themselves estimates with uncertainties. We therefore do not use β̂ and ς̂2 for the imputation, but rather β̃ and σ̃2, which are estimated from a bootstrap sample of the data (Efron 1979). Bootstrap data are generated as described below by sampling with replacement, and represent a sample from the same universe as the original data. We repeat the process to create multiple data sets, which are then independently analyzed and combined in a way that accounts for the imputation. Differences in regression results in the multiple data sets reflect variability due to the imputation process.
This procedure, however, omits a source of variability. We have tacitly assumed that the LB and UB are fixed and known in advance. When there are no interfering compounds (missing type I), the assumption is justified because the DL is determined before the GC/MS dust analysis. When there are interfering compounds (missing types II and III), the assumption cannot be fully justified because the bounds depend on the amount of interference and therefore are random. In the NHL data, we assume this uncertainty is small relative to other uncertainties. The imputation proceeds as follows:
Step 1: Create a bootstrap sample and obtain estimates β̃ and σ̃2 based on Equation 2. Bootstrap data are generated by sampling with replacement n times from the n subjects. Sampling “with replacement” selects one record at random and then “puts it back” and selects a second record. After n repetitions, some subjects are selected multiple times, whereas other subjects are not selected at all. If wi is the number of times the ith subject is sampled, then the log-likelihood function for the bootstrap data is
Step 2: Impute analyte values based on sampling from LN (β̃tX, σ̃2). For the ith subject, assign the value
This quantity consists of various elements. F(LBi; β̃tX, σ̃2) and F(UBi; β̃tX, σ̃2) are the cumulative probabilities at ULi and UBi, respectively, based on parameters β̃, σ̃2. Both values lie between zero and one. Select randomly from a uniform distribution on the interval [a, b], denoted Unif[a, b], in particular the interval [F(LBi; β̃tXi, σ̃2), F(UBi; β̃tXi, σ̃2)]. The inverse cumulative distribution function, F−1(•), is the required imputed value in original units between LBi and UBi. Repeat using the same β̃, σ̃2 for each missing value. Detected values are not altered.
Step 3: Repeat steps 1 and 2 to create M plausible (or “fill-in”) data sets. Remarkably, M need not be large, and a recommended value is between 3 and 5, with larger values if greater proportions of data are missing (Little and Rubin 1987; Rubin 1987). We select M = 10 to fully account for the variance from the imputation.
Step 4: Fit a regression model to each of the M data sets and obtain M sets of parameter estimates and covariance matrices. Combine the M sets of estimates to account for the imputation (Little and Rubin 1987; Schafer 1997). The imputation procedure results in confidence intervals (CIs) that are wider than the single-imputation, fill-in approach.
Simulation study.
We conducted a simulation study, using a simple regression model with zero intercept and no covariates, to evaluate the imputation approaches, the effects of the proportion of data below the DL, and sample size. We generated data sets of size n by sampling from a log-normal distribution with parameters (μ,σ2), and defined the DL such that in expectation p percent of the samples falls below the DL; that is, DL = F−1(p; μ, σ2). The simulation involves 5,000 independent data sets for each set of parameters. We compared five approaches: a) direct estimation (Tobit regression) of MLEs (μ̂ and ς̂2) using Equation 1; b) multiple imputation with allowance for uncertainty in model parameters; c) single imputation based on a random fill-in value for each datum below the DL, using MLEs (μ̂ and ς̂2) from Equation 1; d) insertion of DL/2 for all data below the DL; and e) insertion of E[Z|Z < DL] for data below the DL with the expected value based on the MLEs (μ̂ and ς̂2) from Equation 1. For approaches b) through e), estimators are the mean and variance of the logarithm of the observed and imputed data, with adjustment for multiple imputation in b). We compare results with estimates based on complete data.
For the NHL example, we use SAS (SAS System for Windows, version 8.2; SAS Institute Inc., Cary, NC) to generate bootstrap samples, fit linear regressions (PROC REG), solve log-likelihood Equations 1 and 2 (PROC LIFEREG), and combine results from multiple data sets (PROC MIANALYZE). The simulation was conducted using MATLAB (version 7.0; MathWorks Inc., Natick, MA).
Results
We limited results to four insecticides, which exhibited various types and proportions of missing data: propoxur and carbaryl, both carbamate insecticides; chlorpyrifos, an organophosphate; and α-chlordane, an organochlorine.
Regression analysis in control subjects.
After omitting subjects’ missing questionnaire data, there are 478 control subjects with carpet dust measurements and all regression variables. The percentages of measurements below DLs or known only within bounds vary from 25.7% for propoxur to 67.0% for carbaryl (Table 1). The arithmetic mean (AM), geometric mean (GM), and geometric standard deviation (GSD), with fill-in imputations for interval-measured values, indicate that concentrations for the individual analytes varied substantially. For carbaryl and α-chlordane, the GM falls within the range of missing data. Figure 1A and B show quantile plots for measurements of propoxur and carbaryl and reveals good concordance with a log-normal distribution; Figure 1A and B show values used for imputation based on DL/2, denoted by stars, and the conditional expected value, denoted by triangles. For carbaryl, DL/2 values are nearly twice the conditional expected values. By construction, the fill-in values conform to the estimated distribution.
In several instances, estimates for the various types of pests treated differ substantially, particularly for analytes with a high percentage of missing data. The multiplicative standard errors for the replacement approaches (i.e., inserting DL/2, E[Z|LB < Z < UB], or a fill-in value) are smaller than standard errors from the multiple imputation approach and direct estimation. The smaller standard errors result from an inadequate account of missing data and result in CIs that are too narrow and inflated type I error rates. Table 2 shows several variables that do not achieve traditional significance levels when imputation is taken into account. In some instances, there are marked differences in estimates. Estimated increases in carpet dust levels of α-chlordane among subjects treating for termites are 2.6- and 3.1-fold based on DL/2 insertion and fill-in methods, respectively, and 3.7-fold based on multiple imputation and direct estimation approaches.
Comparing the two fill-in approaches, standard errors are smaller when the covariate information is included than when covariate information is omitted. Fill-in values are obtained from regression models by sampling from LN(β̂tXi, ς̂2). Figure 1C and D show quantile plots of residuals, that is, from exp[log(Z) – β̂tX]for each subject. Although GMs of the residuals are close to the expected value of 1.0 for the error distributions, plots suggest a slight underprediction at extreme values for propoxur and carbaryl.
Simulation study.
For the simulation study, we set μ = 0 and σ2 = 1 without loss of generality and present results for n = 50, 100, 200, and 400 and with DLs such that the expected proportions of values below the DL are p = 10, 30, 50, and 70%. With 5,000 repetitions, the standard error for coverage of 95% CIs is 0.003. Table 3 shows that estimates of μ based on Tobit regression, multiple imputation, and single impute fill-in approaches are generally unbiased. Insertion of DL/2 or E[Z|Z < DL] results in substantial bias unless the proportion of missing data is small, ≤ 10%. Table 3 also shows coverage of the 95% CI for the estimate of μ. In comparison with complete data, Tobit regression and the multiple imputation approaches are the only methods that achieve nominal coverage over a broad range of simulation parameters, although the multiple imputation begins to degrade when more than about 50% of the measurements are below DLs. The single imputation approach results in anomalous CIs when about ≥ 30% of the data are below DLs.
Discussion
Results of our analysis of use of pesticide products in and around the home and pesticide residues in carpet dust and of the simulation study suggest that the method of imputation of missing environmental measurement data can substantially affect estimation of effects and statistical inference. The practice of inserting a single value, such as DL/2 or the conditional expected value E[Z|Z < DL] or by analogy DL/√2 , is ill-advised unless there are relatively few measurements below DLs. The use of a single imputation to fill in missing data is unbiased or minimally biased quite generally but suffers from inaccurate estimates of variance and, consequently, CIs that are too narrow, particularly when missing data exceed about 30%. The best protection against biased inference in the presence of nonignorable missing data is the use of multiple imputation, although with a high proportion of values below the DL, a large number of measurements are needed. It is worth reiterating, however, that multiple imputation is necessary only if explicit values are needed for measurements below DLs. If the purpose is to estimate regression parameters, then procedures for truncated data, such as Tobit regression, are nominal (Little and Rubin 1987).
In environmental monitoring, estimation of distributional parameters is often problematic because of limited numbers of measurements and an inability to evaluate distributional forms precisely. With 5–15 measurements, MLEs can be biased (Gleit 1985), suggesting the need for more robust approaches (Helsel 1990). With epidemiologic data, which usually include hundreds or thousands of measurements, MLEs are unbiased and fully efficient (Gilliom and Helsel 1986), and more detailed regression analyses are feasible.
When analyzing environmental data on pesticides, Moschandreas et al. used a fill-in imputation approach that applied the “best-fitting” probability distribution for values above a DL (Helsel 1990; Moschandreas et al. 2001a, 2001b), although Helsel and Hirsch (1991) had cautioned that the approach should be used primarily for estimating summary statistics. The approach we outline permits multiple DLs, incorporates regression parameters, and applies multiple imputation to account correctly for interval-measured data and to allow unbiased inference. However, our simulation study suggests that the fill-in approach may be quite adequate when measurements below the DL account for less than about 30% of the data.
The Tobit regression and multiple imputation approaches assume that the limits of detection are fixed and known in advance. In our example, we are justified in assuming DLs are fixed for type I missing measurements, but not for type II and III missing data where DLs depend on the amount of interfering compounds and are random variables. If the DL is not known, an estimate of its value is the minimum order statistic of the observed measurements—that is, the smallest measured value. Simulations suggest that for a random DL, estimates remain unbiased but variances are underestimated (Zuehlke 2003). Thus, CIs in Table 2 may be too narrow. However, relative to other sources of uncertainty that arise in the collection and handling of carpet dust samples, and the accuracy of questionnaire information, additional uncertainty induced by random DLs for type II and III missing values is likely small.
Environmental data are frequently well approximated by a log-normal distribution, and our data on concentrations of pesticide analyte in carpet dust are consistent with this assumption. Equations 1 and 2 remain valid for more general distributions, although estimation of parameters may be more problematic and necessitate potentially computer-intensive search algorithms. Validity of parameter estimates and their variances depend, of course, on the correct choice of error distribution. Our simulation study was based on a correct distributional form; however, misspecification of the probability model can lead to markedly biased results (Paarsch 1984). In the absence of knowledge about the error distribution, semiparametric and nonparametric methods have been proposed (Austin 2002a; Chay and Powell 2001; DiNardo and Tobias 2001). Bayesian approaches have also been suggested in the Tobit regression context (Austin 2002b). A reviewer suggested considering the set of measurements of a subject as a vector of multivariate outcomes, so that the covariance structure among the analytes could provide information for the imputation process. In our example, this requires the assumption that the logarithms of the measurements are multivariate normally distributed. The suggestion, however, adds complexity as the number of analytes increases, and additional work is needed to evaluate its practical feasibility.
The motivation for this work arose from the analysis of pesticide analytes in carpet dust and use of pesticide products in and around the home. However, data with DLs arise in a variety of settings, including upper DLs from health-care–related questionnaire data (Austin 2002a) and psychological profile scores, such as the Fagerstrom test for nicotine dependence (Fagerstrom and Schneider 1989; Heatherton et al. 1991) and lower DLs in radiation film badge measurements (Gilbert et al. 1996; Kerr 1994).
In summary, with epidemiologic data, our analyses indicate that unless there are very few measurements below DLs (< 5–10%), inserting DL/2, E[Z|Z < DL], or any single value to impute missing measurement data is not advisable. Further, inserting a randomly selected fill-in value is also inadvisable, unless the proportion of missing data is less than about 30%. Multiple imputation of missing data is the best approach of ensuring unbiased estimates of effects and nominal CIs.
Figure 1 Plots under a log-normal distribution of quantiles of environmental measurements of (A) propoxur and (B) carbaryl, and of regression residuals of measurements (Z) and predicted values (ZPred) after accounting for covariates for (C) propoxur and (D) carbaryl. The AMs, GMs, and GSDs are computed from imputed data. (A) AM = 456.6; GM = 65.6; GSD = 6.0. (B) AM = 1503.0; GM = 64.0; GSD = 14.0. (C) AM = 3.5; GM = 0.9; GSD = 2.0. (D) AM = 15.1; GM = 0.9; GSD = 2.6.
Table 1 Percentage of measurements below DLs or known only within bounds and AMs, GMs, and GSDs based on fill-in values from a single imputation (data on 478 control subjects).
Measurements known only within bounds
Type I
Type II
Type III
Dust (ng/g)
Insecticide Percent Range Percent Range Percent Range AM GM GSD
Propoxur 21.1 (0–46.0) 2.9 (0–65.0) 1.7 (21.1–75.7) 456.6 65.6 6.0
Carbaryl 37.9 (0–260.0) 11.1 (0–268) 18.0 (20.7–694.8) 1503.0 64.0 14.0
Chlorpyrifos 28.2 (0–77.4) 0.2 (0–20.9) 0.0 — 893.1 105.6 8.3
α-Chlordane 60.9 (0–44.7) 0.0 — 0.4 (20.8–29.1) 90.7 11.6 8.0
Types of missing measurements are as follows: no analyte detected and no interfering compound (I), no analyte detected but with an interfering compound present (II), and analyte and interfering compounds both present (III). The range for the DLs reflects the minimum of LBs and the maximum of UBs for the nondetected measurements.
Table 2 Proportional increase in analyte concentration in carpet dust (ng/g) for selected uses.
Crawling insects
Flying insects
Fleas/ticks
Termites
Lawn/garden insects
Insecticide, imputation approacha method Adjustment exp(β ) SE exp(β ) SE exp(β ) SE exp(β ) SE exp(β ) SE
Propoxur
DL/2 No 1.426b 1.167 0.987 1.144 1.231 1.153 1.145 1.219 0.756b 1.151
E[Z|LB < Z < UB] No 1.432b 1.170 0.986 1.147 1.231 1.156 1.135 1.223 0.751b 1.154
Fill-in No 1.459b 1.189 0.966 1.163 1.225 1.173 1.072 1.249 0.737c 1.171
Fill-in Yes 1.511b 1.182 1.030 1.157 1.251 1.166 1.209 1.239 0.687b 1.165
Multiple impute Yes 1.487b 1.196 1.016 1.165 1.247 1.170 1.082 1.244 0.704b 1.173
Direct estimate Yes 1.503c 1.276 0.994 1.235 1.245 1.250 1.090 1.363 0.714 1.249
Carbaryl
DL/2 No 0.853 1.201 0.661b 1.173 1.560b 1.185 1.129 1.266 1.660b 1.183
E[Z|LB < Z < UB] No 0.849 1.226 0.629b 1.194 1.703b 1.208 1.199 1.300 1.746b 1.205
Fill-in No 0.830 1.311 0.591b 1.265 1.812b 1.285 1.486 1.417 1.735b 1.282
Fill-in Yes 0.940 1.274 0.432b 1.235 2.337b 1.252 1.538 1.366 1.779b 1.249
Multiple impute Yes 0.826 1.338 0.508b 1.272 2.047b 1.313 1.326 1.490 1.950b 1.351
Direct estimate Yes 0.785 1.499 0.512c 1.413 2.180b 1.452 1.281 1.651 2.115b 1.444
Chlorpyrifos
DL/2 No 1.578b 1.209 0.779 1.181 1.264 1.182 1.581c 1.276 0.759 1.188
E[Z|LB < Z < UB] No 1.620b 1.218 0.771 1.188 1.300 1.190 1.613c 1.288 0.746 1.196
Fill-in No 1.917b 1.243 0.757 1.210 1.389c 1.212 1.669c 1.322 0.713c 1.219
Fill-in Yes 1.745b 1.244 0.740 1.210 1.383c 1.212 1.631c 1.323 0.731 1.219
Multiple impute Yes 1.770b 1.252 0.763 1.223 1.401c 1.223 1.689c 1.336 0.708 1.234
Direct estimate Yes 1.796c 1.378 0.740 1.323 1.392 1.327 1.698 1.492 0.702 1.338
α-Chlordane
DL/2 No 0.966 1.129 0.938 1.112 0.910 1.118 2.626b 1.168 1.091 1.117
E[Z|LB < Z < UB] No 0.954 1.153 0.925 1.132 0.894 1.140 3.031b 1.199 1.110 1.138
Fill-in No 1.060 1.230 0.828 1.198 0.868 1.210 3.110b 1.303 1.079 1.208
Fill-in Yes 0.762 1.206 0.927 1.177 0.908 1.188 3.898b 1.271 1.293 1.186
Multiple impute Yes 0.852 1.363 0.915 1.235 0.804 1.202 3.686b 1.290 1.169 1.270
Direct estimate Yes 0.858 1.379 0.919 1.316 0.803 1.339 3.666b 1.442 1.211 1.334
Entries are exponentials of parameter estimates (β) and their SEs from linear regression models of the logarithm of pesticide analyte on age, sex, race, education, study site, season, and pesticide use variables. Regression models also included year house was built (propoxur, carbaryl, α-chlordane), type of home (carbaryl), and presence of oriental rugs (α-chlordane).
a See “Materials and Methods” for a description of methods; adjusted imputation includes regression variables.
b 95% CI excludes 1.
c 90% CI excludes 1.
Table 3 Results of simulation study of imputation approachesa for log-normally distributed data with μ = 0 and σ2 = 1 with a DL (entries are means of 5,000 repetitions).
Sample size (no.) Percent < DL Complete data Tobit analysis Multi-impute using (μ̂, σ̂2) Single impute using (μ̃, σ̃2) Insert DL/2 Insert E[Z|Z < DL]
50
Estimate of μ 10.0 0.002 0.000 −0.003 −0.003 −0.020 0.007
30.0 0.002 −0.003 −0.003 −0.004 −0.017 0.032
50.0 0.002 −0.004 −0.003 −0.003 0.052 0.073
70.0 0.002 −0.006 −0.005 −0.002 0.229 0.143
Coverage of 95% CI 10.0 0.947 0.944 0.943 0.943 0.943 0.942
30.0 0.947 0.949 0.938 0.928 0.942 0.928
50.0 0.947 0.953 0.928 0.876 0.938 0.832
70.0 0.947 0.931 0.895 0.707 0.280 0.520
100
Estimate of μ 10.0 0.003 0.002 0.000 0.000 −0.019 0.009
30.0 0.003 0.001 0.000 0.000 −0.015 0.034
50.0 0.003 0.000 0.000 −0.001 0.055 0.076
70.0 0.003 −0.006 −0.004 −0.002 0.232 0.142
Coverage of 95% CI 10.0 0.944 0.945 0.940 0.940 0.943 0.942
30.0 0.944 0.949 0.938 0.929 0.942 0.914
50.0 0.944 0.948 0.922 0.870 0.910 0.781
70.0 0.944 0.940 0.904 0.721 0.036 0.440
200
Estimate of μ 10.0 −0.001 −0.002 −0.002 −0.002 −0.023 0.006
30.0 −0.001 −0.003 −0.003 −0.003 −0.019 0.031
50.0 −0.001 −0.002 −0.002 −0.002 0.052 0.074
70.0 −0.001 −0.003 −0.001 −0.002 0.229 0.142
Coverage of 95% CI 10.0 0.952 0.950 0.951 0.950 0.941 0.946
30.0 0.952 0.955 0.936 0.926 0.940 0.904
50.0 0.952 0.948 0.925 0.874 0.877 0.708
70.0 0.952 0.947 0.914 0.725 0.000 0.306
400
Estimate of μ 10.0 0.001 0.001 0.001 0.001 −0.021 0.008
30.0 0.001 0.000 0.000 0.000 −0.017 0.034
50.0 0.001 0.001 0.001 0.001 0.053 0.076
70.0 0.001 0.000 0.000 0.000 0.230 0.144
Coverage of 95% CI 10.0 0.954 0.954 0.952 0.951 0.931 0.949
30.0 0.954 0.948 0.938 0.928 0.941 0.874
50.0 0.954 0.954 0.927 0.880 0.776 0.545
70.0 0.954 0.947 0.914 0.723 0.000 0.128
a Parameter estimation using observed data with DLs (Tobit analysis), (μ̂, σ̂2) multiple imputation with allowance for uncertainty in model parameters using (μ̃, σ̃2), a single imputation using (μ̂, σ̂2), the insertion of DL/2, and insertion of the expected value conditional on being below the DL, E[Z|Z < DL].
==== Refs
References
Austin PC 2002a A comparison of methods for analyzing health-related quality-of-life measures Value Health 5 329 337 12102695
Austin PC 2002b Bayesian extensions of the Tobit model for analyzing measures of health status Med Decis Making 22 152 162 11958497
Bradman MA Harnly ME Draper W Seidel S Teran S Wakeham D 1997 Pesticide exposures to children from California’s Central Valley: results of a pilot study J Expo Anal Environ Epidemiol 7 217 234 9185013
Chay KY Powell JL 2001 Semiparametric censored regression models J Econ Perspect 15 29 42
Colt JS Lubin J Camann D Davis S Cerhan J Severson RK 2004 Comparison of pesticide levels in carpet dust and self-reported pest treatment practices in four US sites J Expo Anal Environ Epidemiol 14 74 83 14726946
Desrosiers M Schauer DA 2001 Electron paramagnetic resonance (EPR) biodosimetry Nucl Instr Meth B 184 219 228
DiNardo J Tobias J 2001 Nonparametric density and regression estimation J Econ Perspect 15 11 28
Efron B 1979 Bootstrap methods; another look at the jack-knife Ann Stat 7 1 26
Fagerstrom KO Schneider NG 1989 Measuring nicotine dependence—a review of the Fagerstrom tolerance questionnaire J Behav Med 12 159 182 2668531
Garland M Morris JS Rosner BA Stampfer MJ Spate VL Baskett CJ 1993 Toenail trace-element levels as biomarkers—reproducibility over a 6-year period Cancer Epidemiol Biomarkers Prev 2 493 497 8220096
Gilbert ES Fix JJ Baumgartner WV 1996 An approach to evaluating bias and uncertainty in estimates of external dose obtained from personal dosimeters Health Phys 70 336 345 8609025
Gilbert RO 1987. Statistical Methods for Environmental Pollution Monitoring. New York:Van Nostrand Reinhold.
Gilliom RJ Helsel DR 1986 Estimation of distributional parameters for censored trace level water quality data. I. Estimation techniques Water Resour Res 22 135 146
Gleit A 1985 Estimation for small normal data sets with detection limits Environ Sci Technol 19 1201 1206 22280138
Haas CN Scheff PA 1990 Estimation of averages in truncated samples Environ Sci Technol 24 912 919
Hatch M Thomas D 1993 Measurement issues in environmental epidemiology Environ Health Perspect 101 49 57 8206042
Heatherton TF Kozlowski LT Frecker RC Fagerstrom KO 1991 The Fagerstrom test for nicotine dependence—a revision of the Fagerstrom tolerance questionnaire Br J Addict 86 1119 1127 1932883
Helsel DR 1990 Less than obvious—statistical treatment of data below the detection limit Environ Sci Technol 24 1766 1774
Helsel DR Hirsch RM 1991. Statistical methods in water resources. In: Techniques of Water-Resources, Book 4. Reston, VA:U.S. Geological Survey. Available: http://water.usgs.gov/pubs/twri/twri4a3/ [accessed 13 August 2004].
Hornung RW Reed LD 1990 Estimation of average concentration in the presence of nondetectable values Appl Occup Environ Hyg 5 46 51
Kerr GD 1994 Missing dose from mortality studies of radiation effects among workers at Oak-Ridge-National-Laboratory Health Phys 66 206 208 8282563
Lewis RG Fortune CR Blanchard FT Camann DE 2001 Movement and deposition of two organophosphorus pesticides within a residence after interior and exterior applications J Air Waste Manage Assoc 51 339 351
Lewis RG Fortune CR Willis RD Camann DE Antley JT 1999 Distribution of pesticides and polycyclic aromatic hydrocarbons in house dust as a function of particle size Environ Health Perspect 107 721 726 10464072
Ligman B Shaughnessy R Kleinerman R Lubin J Fisher E Wang ZY 2004. Indoor air pollution characterization of underground dwellings in China, 1997. Blacksburg, VA:Virginia Polytechnic Institute and State University Press, 51–56.
Little RJA Rubin DB 1987. Statistical Analysis with Missing Data. New York:John Wiley & Sons.
Moschandreas DJ Karuchit S Kim Y Ari H Lebowitz MD O’Rourke MK 2001a On predicting multi-route and multimedia residential exposure to chlorpyrifos and diazinon J Expo Anal Environ Epidemiol 11 56 65 11246803
Moschandreas DJ Kim Y Karuchit S Ari H Lebowitz MD O’Rourke MK 2001b In-residence, multiple route exposures to chlorpyrifos and diazinon estimated by indirect method models Atmos Environ 35 2201 2213
Nigg N Beier RC Carter O Chaisson C Franklin C Lavy T 1990. Exposure to pesticides. In: The Effects of Pesticides on Human Health, Vol 18 (Baker SR, Wilkinson CF, eds). Princeton, NJ:Princeton Scientific, 35–130.
Paarsch HJ 1984 A Monte-Carlo comparison of estimators for censored regression models J Econ 24 197 213
Persson T Rootzen H 1977 Simple and highly efficient estimators for a type I censored normal sample Biometrika 64 123 128
Rubin DB 1987. Multiple Imputation for Nonresponse in Surveys. New York:John Wiley & Sons.
Schafer JL 1997. Analysis of Incomplete Multivariate Data. New York:Chapman & Hall.
Sim M 2002 Case studies in the use of toxicological measures in epidemiological studies Toxicology 181 405 409 12505343
Singh A Nocerino J 2002 Robust estimation of mean and variance using environmental data sets with below detection limit observations Chemometr Intell Lab 60 69 86
Singh AK Singh A Engelhardt M 1997. The Lognormal Distribution in Environmental Applications. Washington, DC:U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response.
Tobin J 1958 Estimation of relationships for limited dependent variables Econometrica 26 24 36
Travis CC Land ML 1990 Estimating the mean of data sets with nondetectable values Environ Sci Technol 24 961 962
Woodward A Al Delaimy W 1999 Measures of exposure to environmental tobacco smoke—validity, precision, and relevance Ann NY Acad Sci 895 156 172 10676415
Zuehlke TW 2003 Estimation of a Tobit model with unknown censoring threshold Appl Econ 35 1163 1169
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PMC1253661
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2021-01-04 23:40:52
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no
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Environ Health Perspect. 2004 Dec 13; 112(17):1691-1696
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utf-8
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Environ Health Perspect
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10.1289/ehp.7199
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oa_comm
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