Traditionally the rapid origin of megadiverse species flocks of extremely closely related species is explained by the combinatory action of three factors: Disruptive natural selection, disruptive sexual selection and partial isolation by distance. However, recent empirical data and theoretical advances suggest that the diversity of complex species assemblages is based at least partially on the hybridization of numerous ancestral allopatric lineages that formed hybrids upon invasion of new environments. That reticulate speciation within species flocks may occur under sympatric conditions after the primary formation of species has been proposed but not been tested critically.Pungu maclareni.We reconstructed the phylogeny of a complex cichlid species flock confined to the tiny Cameroonian crater lake Barombi Mbo using both mitochondrial and nuclear (AFLP) data. The nuclear phylogeny confirms previous findings which suggested the monophyly and sympatric origin of the flock. However, discordant intra-flock phylogenies reconstructed from mitochondrial and nuclear data suggest strongly that secondary hybridization among lineages that primarily diverged under sympatric conditions had occurred. Using canonical phylogenetic ordination and tree-based tests we infer that hybridization of two ancient lineages resulted in the formation of a new and ecologically highly distinct species, Our findings show that sympatric hybrid speciation is able to contribute significantly to the evolution of complex species assemblages even without the prior formation of hybrids derived from allopatrically differentiated lineages. The scenario may either involve secondary localized hybridization, i.e. hybridization of parapatric  lineages within the geographical range of the primary radiation, or alternatively hybridization of sympatrically diverged lineages. Distinguishing between these two alternatives is central for the understanding of the processes that lead to the evolution of megadiversity. The first alternative predicts that increased species richness due to hybridization is dependent primarily on the spatial scale and the accompanied possibility to establish localized metapopulations. The second alternative predicts that hybridisation can aid the build-up of diversity even under fully sympatric conditions. Several studies, some published, some in preparation support the hybrid swarm origin hypothesis for some species assemblages endemic to comparatively large areas [Recent empirical data and theoretical advances suggest that the diversity of complex species assemblages is based at least partially on the hybridization of numerous ancestral allopatric lineages that formed hybrids upon invasion of new environments -5. A groge areas -5,8 but However, despite increasing evidence for sympatric speciation, uncontested examples remain rare and rarely go beyond the formation of single species-pairs ,16. In aSarotherodon galilaeus. Because the lake's conical basin is only 2.15 km in diameter, because there are no migration-barriers along the shore, and because the lake is isolated from nearby river systems by cataracts of its outflow, allopatric scenarios for the origin and diversification of the flock were ruled out. However, in the light of the aforementioned methodological drawbacks of mitochondrial phylogenies, both the monophyly-hypothesis for the Barombi flock and the relationships among its 11 endemic species are worth to be reevaluated.Until recently the phylogeny of mitochondrial lineages of the cichlid species flock of crater lake Barombi Mbo (Cameroon)  was consThe ability to score thousands of amplified fragment-length polymorphisms (AFLPs) has created a powerful possibility for the phylogenetic reconstruction of rapidly originated species flocks. It has been successfully applied to a limited number of taxa belonging to the Lake Malawi and Lake Victoria haplochromine species flocks and revealed previously undetectable phylogenetic patterns including those supporting the hybrid swarm origin hypothesis ,18,19. IS. galilaeus populations (one to two samples per population). 2191 sites of the alignment were constant, 198 variable characters were parsimony-uninformative and the number of parsimony-informative characters was 164. Empirical base frequencies in this data set were A = 0.2721; C = 0.3271; G = 0.1268; T = 0.2740. Bootstrapped Maximum Parsimony (MP), Maximum Likelihood (ML) and Neighbour Joining (NJ) trees all recovered identical 50%-majority rule consensus-trees  [Pungu haplotypes from Konia took place, while all other clades radiated into several species much later. A 94 sample data set with only cytochrome b and partial proline tRNA sequences  confirmed the previous findings for the Barombi flock and suggests that lineage sorting between the four large clades is complete (for a Neighbour Joining Tree see Pungu, Myaka, Konia ssp, S. lohbergeri and S. steinbachi), but not within Stomatepia ssp., S. caroli and S. linnellii.We obtained a DNA sequence-alignment with 2553 bp including two complete mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome b (cytb), partial proline tRNA as well as from part of the control region from all Barombi species (two samples per species) and relevant Stomatepia and Konia clades , Myaka is sistergroup to the other Sarotherodon subclade consisting of the pelagic species S. caroli and S. linnellii. The Konia clade is resolved as the sistergroup to the rest of the flock. In contrast to the unresolved basal topology in the mitochondrial tree, the two species from Lake Ejagham are resolved as a monophylum which is the sistergroup to the geographically closest S. galilaeus population from the river Cross. An additional data set with 3 selective amplifications but 80 samples recovered only three supraspecific nodes with moderately high bootstrap support, the monophyly of the Barombi flock, Konia and the node containing the rest of the Barombi flock without Pungu. The latter was placed intermediate between the two well supported nodes.The phylogenetic reconstruction based on the same individuals and using 22 restrictive primer combinations with 3489 AFLP size fragments (3004 variable) confirmed the monophyletic origin of the Barombi Mbo flock and the monophyly of the s figure . HoweverPungu maclareni, is the result of speciation by hybridization. By identifying the clades which contain taxa with discordant phylogenies we hypothesized that traces of three ancient hybridization events are still detectable in the multilocus AFLP data. To test for the presence of the respective phylogenetic signal for these three hypothetical ancient syngameons in the large AFLP data set, we used the recently developed method of Canonical Phylogenetic Ordination (CPO) [Both the Shimodaira-Hasegawa and Templeton's test confirmed significantly the difference for the alternative tree topologies for each sequence and AFLP data sets, respectively figure . These don (CPO) . In addiS. galilaeus and lake taxa without S. gal multifasciatus and S. gal "Niger" and (2) the node of the AFLP based hypothesis of the monophyly of the Lake Ejagham sister pair. Of the three hypothetical syngameons, the one uniting Pungu with its potential ancestor clades represented now by the Konia ssp and S. steinbachi and S. lohbergeri contributed significantly to the variation in the AFLP data set, whereas the other two did not.All phylogenetic groups detected either by the mitochondrial or the AFLP data set, or formulated according to the three hypothetical syngameons were coded as phylogenetic variables and their explanatory value for the variance in the AFLP data set was tested table . As a rePungu, a tree-based method as outlined in Seehausen [Pungu resulted in far outlying higher support values for the respective nodes, whereas all other removals resulted in much lower values  based on the well supported phylogeny of mitochondrial haplotypes in the lake, the primary radiation in Barombi Mbo resulted into the almost instantaneous split into four distinct lineages approximately one million years ago. The accumulation of numerous apomorphic characters that support these mitochondrial lineages suggests strongly that they represented reproductively isolated species at that time. Only in an advanced stage of species flock formation and after considerable time had elapsed, their cohesion was broken partially by hybridization events between these lineages. However, according to the chronogram the ancestral mitochondrial clades that contributed, for example, to the hybrid origin of Pungu maclareni, represents an ecologically highly specialized ecotype. Both its peculiar dentition and the accompanying hypertrophic jaw-muscles are unique not only in Barombi Mbo but in cichlids in general [Konia dikume, ranks among the most unusual cichlids as it is the single species which is able to exploit chironimid larvae in the almost oxygen-free deep water due to its extremely high haemoglobin concentration in its blood [Traditionally the rapid origin of megadiverse species flocks of extremely closely related species was explained by the combinatory action of three factors: Disruptive natural selection, disruptive sexual selection and partial isolation by distance -24. Alth general . Accordits blood . In the Sarotherodon galilaeus populations and from all species endemic to crater lakes Barombi Mbo and Ejagham, which are related to S. galilaeus. Oreochromis niloticus and Sarotherodon melanotheron were used as outgroups based on published information and pilot-study data confirming their outgroup status with respect to S. galilaeus and the investigated species flocks.We obtained genetic data from relevant Sarotherodon caroli and S. linnellii. Black adult breeding males of the S. caroli/S. linnellii-phenotype from the deepwater were identified as S. caroli, whereas golden males from the shallow inshore area were identified as S. linnellii. Fin-clips were taken from the right pectoral fin in the field directly after collection and preserved in 96% Ethanol p.A.. Samples from additional specimens belonging to different populations of Sarotherodon galilaeus and outgroups were either collected by UKS in the field in Cameroon or donated by others. Vouchers are deposited in the ichthyological collection of Bavarian State Collection of Zoology (ZSM). Of few S. galilaeus-samples, only photographs of the specimens are available and are deposited in the ZSM, too. Informations about specimens and their species identifications, geographic origin, accession numbers and information about which specimens were sequenced and AFLP typed in different data sets are provided in Adult specimens from Lake Barombi Mbo were collected by UKS during field visits to the lake in 2001 and 2002 and from Lake Ejagham in 1994. Identification to species is according to Trewavas et al.  and was 2 fin tissue with the DNeasy™ Tissue Kit (Qiagen). DNA-quality was visually inspected under UV-light on a 0.8% agarose-gel stained with ethidium bromide. For subsequent AFLP-analysis only samples with a clearly visible high-molecular band were used. DNA-concentration was determined using the VersaFluor-Fluorometer-System (BioRad) using the stain Picogreen® dsDNA Quantitation Kit (Molecular Probes). All samples were adjusted to 60 ng/μl.DNA samples were isolated from approx 10 mm7 for the 5'-end of the control region. PCR was performed by using a PTC 220 DYAD thermocycler (MJ Research) in a 25 μl reaction volume using the Expand PCR system (Roche Diagnostics) with 25 pmol of each primer, 20 pmol of dNTPs, 12.5 pmol MgCl2 and 0.88 units of Taq polymerase. PCR parameters were 94°C for 4 min, 35 cycles with 94°C for 1.5 min, 55°C for 1 min, 72°C for 1.5 min, followed by a final elongation at 72°C for 3 min. PCR products were cleaned by using MinElute PCR purification kit (QIAGEN) and their DNA-concentration adjusted to 100 ng/μl. PCR-Products were then used as templates for cycle-sequencing reaction using the "Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit" (Applied Biosystems) with each of the PCR primers or internal primers. Cycle parameters were the following: 94°C, 2 min; 25 cycles of 94°C, 20 s; 52°C, 10 s; 60°C, 4 min. The sequenced product was filtered through Sephadex-G50 fine (Fluka) packed spin columns (Amersham) to remove unincorporated dye terminators, primers, and salts, and finally dried in a speed-vac. These products were resuspended, electrophoresed and analysed with an ABI PRISM™ 377XL-96 automated sequencer using a 4.25 % polyacrylamid gel (BioRad). Electrophoretic information was transcribed to sequence data using the program Genescan (PE Applied Biosystems).Two different data sets were assembled, one long data set with approx. 2550 bp including the complete NADH dehydrogenase subunit 2 (ND2), the complete cytochrome b (cytb) gene and part of the proline tRNA, and finally, one part of the control region. For this long data set only two samples per species were used, but a short data set with more individuals but only the cytochrome b and the partial proline tRNA genes was generated, too. ND2 was PCR-amplified using the primers "ND2Met" 5'-CATACCCCAAACATGTTGGT-3'"ND2Trp" 5'-GTSGSTTTTCACTCCCGCTTA-3'; a second fragment containing the cytb, proline and threonine tRNAs and the 5'-end of the control region was amplified with the primers "L14725" 5'-TGACTTGAAAAACCATCGTTG and "H16498" 5'-CCTGAAGTAGGAACCAGATG  ; internIndividual sequence files were edited and contigs assembled using Sequence Navigator™ (PE Applied Biosystems). Homologous protein-coding regions (ND2, cytb) were aligned manually and confirmed by translating DNA data into amino acid sequences in BioEdit . The shoDYE): TC-CAFAM, AT-CAFAM, AC-CCNED, TA-CAFAM, AA-CTFAM; AA-GGJOE; AC-CAFAM; AA-CAFAM; AG-CAFAM; AA-CGJOE; AC-CTFAM; AG-CTFAM; AT-CTFAM; AG-GGJOE; TC-CTFAM; TA-GGJOE; AG-ACNED; AT-ACNED; AG-CCNED; AC-ACNED; AT-CCNED; TA-CGNED. For the short data only the five primer pairs TC-CAFAM, TA-GGJOE, AC-CAFAM, AC-CCNED, AT-CAFAM were used but typed for 80 individuals  with an ABI PRISM™ 377XL-96 sequencer. Fluorescent signals were detected using the GENESCAN software (Applied Biosystems) with internal size standard . The fluorescent threshold was set to 50 units and the correct identification of ROX-marker bands by GENESCAN was checked for all electropherograms.. The program generates aligned spreadsheets from GENESCAN-sized AFLP-data by assigning each sized fragment to a size-class of user-defined distance to the next size-class. Using a spreadsheet routine, fragments were inferred in a second step to be homologous if they differed by no more than 1.00 bp, and if the scoring procedure identified the same size-classes whether scored from small to large size-fragments (forward) or vice versa (backward). Size-classes with inconsistent allocation of fragments according to forward and backward scoring were excluded, as well as adjacent size-classes differing by less than 0.35 bp, which corresponds to the double standard deviation of 0.15 bp of the sequencer [Bands between 100.5 bp and 499.5 bp were scored in a first step for presence or absence using the software Binthere (developed by N. Garnhart and available through the T. Kocher laboratory equencer . As a reMrModeltest 1.1b , a simplth character). Non-parametric bootstrapping with 1000  or 100 (ML) pseudoreplicates was used for testing the robustness of the inferred trees. Tree topologies using the HLRT settings under ME were not different from topologies gained with AIC settings (data not shown).The AIC settings were subsequently used for Maximum Likelihood (ML) analyses and to estimate ML distances for minimum evolution (ME) analyses in the program PAUP* 4.0b1.0 (PPC/Altivec)  and for 2 = 701.0154, df = 32, p = 0.001). To date cladogenetic events in the absence of rate constancy, the nonparametric rate smoothing (NPRS) method [A LRT  as imple) method  as imple) method  was usedSarotherodon galilaeus sensu lato (excluding Oreochromis niloticus and Sarotherodon melanotheron). "Pruned" data matrices using only those parsimony informative sites were constructed for Principal Canonical Ordination and homoplasy excess tests (see below) in order to account for noise in the data potentially introduced by the distant outgroup. Pairwise genetic distances were calculated from the binary data-matrix with two different algorithms: One developed by Link et al. [We used PAUP*  to calcuk et al.  as implek et al. , which ik et al. . Trees wk et al.  with unck et al. . Non-parAlternative phylogenetic hypotheses produced as described by tree topologies based on mtDNA and AFLP data were compared with each other and statistically evaluated using either the Shimodaira-Hasegawa LRT  for mitoIn order to test for the presence of a phylogenetic signal that possibly reflects reticulate events in the AFLP-data, two methods were applied.Sarotherodon galilaeus-clade  provided the data-matrix to be tested. Phylogenetic hypotheses derived from mtDNA- and AFLP-analyses as well as hypothetical syngameons as derived from the conflict between the two data-sets were translated into a phylogenetic matrix by assigning binary indicator variables, each coding for the membership of investigated samples to phylogenetic groups  [First, a canonical correspondence analysis (CCA) was performed using CANOCO 4.0 . This megameons) ,54. 9999Sarotherodon steinbachi and S. lohbergeri in the 2355 loci data with n = 16 experiments (each taxon removed once). Analogous support values for the node uniting the Konia eisentrauti and K. dikume were computed with the 530 loci data set with n = 14 experiments, because bootstrap support in the larger data set was always larger than 98.85% and identical runs yielded values differing by more than 1.15 % (data not shown). By reducing the number of loci but increasing the number of samples we obtained a meaningful distribution of bootstrap support values for that node.Second, a tree-based method as outlined in Seehausen  was perfUKS designed the study, carried out the field work, part of the lab work, the major part of the data analysis and wrote the manuscript. BK carried out the major part of the lab work and carried out parts of the data analysis. Both authors read and approved the final manuscript.Sarotherodon melanotheron (not shown)Neighbour Joining phylogram of 94 specimens Neighbour. Joining phylogram calculated from complete sequences of the mitochondrial cytochrome b gene (1141 bp) and the partial proline tRNA gene (71 bp) sequences for 94 specimens based on the GTR+ Γ model and rooted with Click here for filea. The removal of Konia dikume resulted in an increased bootstrap support for the clade uniting Konia and Pungu in the analysis of the 530 loci data; set. b. The removal of S. caroli resulted in increased outside values for the bootstrap support of the b. the Myaka/S. linnellii node and the c. the clade that unites all Barombi taxa with the exclusion of Konia; d. the exclusion of both Myaka and K. eisentrauti increased the bootstrap support for the S. linnellii/S. caroli node; e. Finally, the removal of S. mariae increased the support for the S. pindu/S. mongo split strongly. f. Among the riverine populations of S. galilaeus, the removal of S. gal. "Meme" increased strongly the value for the clade uniting the S. gal. "Cross" specimen with the two endemic species from Lake Ejagham ("Cross clade"); in addition, the exclusion of S. lohbergeri increased the bootstrap value for the same node, albeit weakly.Additional box-plots of bootstrap supports in taxon removal experiments. Box-plots of the distribution of %-bootstrap support values for nodes that yielded additional higher outside or far outside values in taxon removal experiments , Genbank-Accession numbers and representation of specimens in two AFLP-data sets.Click here for file


Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease.93ASSDSSNTE101 and 39DMGHGLRLIHYSYDVNSTEKG59).The present study in the Lewis rat used a conventional idiotypic immunization based on antigenized antibodies expressing selected peptide sequences of the Vβ8.2 TCR (68GSLPQKSQRSQDENPVVHF88 in complete Freunds' adjuvant (CFA). Antigenized antibodies induced an anti-idiotypic response against the Vβ8.2 TCR, which was detected by ELISA and flowcytometry. No evidence was obtained of a T cell response against the corresponding Vβ8.2 TCR peptides.The study demonstrates that vaccination with antigenized antibodies markedly attenuates, and in some instances, prevents clinical EAE induced with the encephalitogenic peptide The results indicate that antigenized antibodies expressing conformationally-constrained TCR peptides are a simple means to induce humoral anti-idiotypic immunity against the TCR and to vaccinate against EAE. The study also suggests the possibility to target idiotypic determinants of TCR borne on pathogenetic T cells to vaccinate against disease. Experimental allergic encephalomyelitis (EAE) is an experimentally induced autoimmune disease mediated by T cells. It can be induced in susceptible animals either by immunization with myelin basic protein (MBP) or proteolipid protein PLP, or by immunization with synthetic peptides from the MBP sequence . EAE can+ T lymphocytes bearing an antigen receptor (TCR) variable (V) regions belonging to a limited set of TCR V region gene families [EAE is mediated by MHC class II -restricted, MBP-specific CD4families ,6 and refamilies . Severalfamilies ,9, antibfamilies  and T refamilies -14. Actifamilies ,16 and vfamilies -20. Togein vivo. However, limitations exist to methods available for TCR vaccination. Vaccination using attenuated encephalitogenic T cells requires that these are specifically expanded in vitro and can only be used in an autologous system. Synthetic peptides, albeit successful in several instances [e.g., worsening of disease [Approaches to directly target the TCR of pathogenetic T cells are an attractive direction for therapy and immunointervention as well as an opportunity to further understand the immunological events involved in protection nstances -20, offe disease ,22. Simi disease .i.e., Ig molecules in which foreign peptide sequences are conformationally-constrained and expressed in the complementority-determining region (CDR) loops [39DMGHGLRLIHYSYDVNSTEKG59 (CDR2) and 93ASSDSSNTE101 (CDR3, VDJ junction), both reported to confer protection against EAE in the Lewis rat [In previous work from this laboratory we demonstrated the induction of anti-receptor immunity using immunoglobulins (Ig) expressing discrete peptide portions of human CD4 . We refeR) loops . Immunizewis rat -20 when at libitum.Eight week old, weight-matched female Lewis rats were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed (three rats per cage) in the animal facility of the Universitiy of California, San Diego. Food and water were provided 93ASSDSSNTE101 and 39DMGHGLRLIHYSYDVNSTEKG59 were engineered into the CDR3 loop of the murine VH62 gene [H was then ligated in plasmid vector containing a human γ1 constant (C) region gene. Transfection of the plasmid DNA was performed on murine J558L cells, a H-chain defective variant of myeloma J558, carrying the rearrangement for a λ1 light (L) chain [4)2SO4. Antibodies were purified by affinity chromatography on a Protein A-Sepharose column (Pharmacia-LKB, Alameda, CA) equilibrated with 3 M NaCl/1M glycine, pH 8.9. Elution was performed using glycine 0.1 M- HCl/0.5 M NaCl pH 2.8. The eluted fractions were neutralized using 1 M Tris-HCl, pH 8.0, and dialyzed against 0.15 M phosphate-buffered saline (PBS) pH 7.3. The purity of the antibodies was assessed by electrophoresis on a 10% Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel (PAGE).The peptide sequences H62 gene  accordinH62 gene . The antL) chain . The resL) chain  engineerL) chain . Culture3 peptide of Plasmodium falciparum parasite [Synthetic peptide GSLPQKSQRSQDENPVVHF corresponding to amino acid residues 68–88 of guinea-pig MBP , DMGHGLRparasite  were allparasite .2O2. After 30 minutes, the plates were read in a micro-plate reader (Vmax, Molecular Devices) at 492 nm. Tests were done in duplicate. Antibodies to TCR peptides were detected in ELISA on 96-well polystyrene microtiter plates coated (10 μg/ml) with the Vβ8.2 synthetic peptides 39DMGHGLRLIHYSYDVNSTEKG59 and 93ASSDSSNTE101 in 0.1M carbonate buffer, pH 9.6, by overnight incubation at +4 C. After blocking unreactive sites, sera (1:25 dilution in PBSA) were added to plates and incubated overnight at +4°C. The bound antibodies and reactive peroxidase were detected as detailed above. Ig reactive with synthetic peptide 21KKSIQFHWKNSNQIKILGNQGSFLTKGPS49 of human CD4 were determined on 96-well polystyrene microtiter plates coated (2.5 μg/ml) with peptide 21–49 in 0.9% NaCl by drying at 37 C as previously established [Serum antibodies against antigenized antibodies and their control were determined by ELISA on 96-well polystyrene microtiter plates (Costar, Cambridge, MA) coated (5 μg/ml – 50 μl/well) with γ1TCR-I, γ1TCR-II, γ2bTCR-I proteins in 0.9% NaCl by drying at 37°C. The wells were blocked with a 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), and then incubated overnight at +4°C with individual rat sera diluted in PBS containing 1% BSA and 0.05% Tween 20 (PBSA). After washing, the bound antibodies were detected by adding peroxidase-conjugated goat antibodies to rat IgG (γ specific) (Biomeda, CA) at 1:500 dilution in PBSA for 1 hour at room temperature. After washing, the bound peroxidase was measured by adding o-phenylenendiamine (100 μl/well) and Hablished . Briefly+ TCR were sought by flowcytometry on the S23B1E11 T cell hybridoma [+ CD4 T lymphoblasts specific for MBP with the murine TCR α/β- BW1100.129.237 thymoma cell line [6 hybridoma T cells in 100 μl of RPMI-1640 containing 1% egg albumin, 0.01% NaN3 and 10 mM Hepes, were incubated with rat sera (1:10 dilution) for 90 minutes at +4°C. Cells were washed three times with cold RPMI-1640 and subsequently incubated with a fluorescein-isotyocianate (FITC)-conjugated goat antibody (0.5 μg/106 cells) to rat Ig (H+L)  for 20 minutes at +4°C. After incubation, the cells were washed twice, resuspended in 1% paraformaldehyde, and analyzed in a FACS Scan (BD Biosciences). To stain for dead cells, 20 μl of propidium iodide in PBS were added to unfixed cells before FACS analysis. R-phycoerythrin conjugated mouse monoclonal antibody R78 (IgG1, k) specific for the rat Vβ8.2, the kind gift of Pharmingen (San Diego, CA), was used to control for the expression of the Vβ8.2 TCR on S23B1E11 hybridoma cells.Autoantibodies reactive with the Vβ8.2ybridoma , derivedell line . For FAC5 cells/well in the presence of various  amounts of antigen in 200 μl of RPMI containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 0.1 mM non-essential aminoacids, 1 mM sodium pyruvate and 0.5 μM 2-β mercaptoethanol. Cultures were incubated for 72 hours in a 10% CO2 atmosphere. The evening before harvest 1 μCi/well of [3H]-thymidine was added to each well. Cells were harvested onto glass fiber filters and counted on an automatic Beckman LS 6000IC β-counter.Poplyteal, inguinal and paraortic lymph nodes were removed from immunized animals at different times, dissociated and washed in RPMI-1640. Lymph node cells were plated in round-bottom 96-well plates at 2.5 × 1068GSLPQKSQRSQDENPVVHF88 (30 μg/rat) in the anterior paws (25 μl each) in CFA (H37RA 10 mg/ml). Control rats were similarly injected with transfectoma antibody γ1WT or γ2bWT. Rats inoculated with Freunds' adjuvant only served as additional control. Serum samples were collected from the retro-orbital sinus on day 0 before vaccination, day 21 before booster injection, day 28 before EAE induction, and day 50 after recovery from disease. Sera were stored at -20 C until use.Animals were vaccinated with antigenized antibodies (100 μg/rat) in complete Freunds' adjuvant (CFA) divided equally between the posterior paws (25 μl each) and two points in the back subcutaneously. A booster injection (50 μg/rat) in incomplete Freunds' adjuvant (IFA) was given subcutaneously on day 21. EAE was induced on day 28 by immunization with MBP peptide EAE was monitored daily by two operators for clinical signs using the following scale: grade 0 = no appreciable symptoms; grade 1 = tail atony; grade 2 = paraparesis; grade 3 = paraplegia; grade 4 = paraplegia with forelimb weakness, moribund state. Typically symptoms of disease began to appear on day 11–13 from the injection of the encephalitogenic peptide. The Disease Index was calculated according to the formula: [(Maximum Score) × (Duration of Disease) × (Incidence)].Statistical analyses was performed using the Fisher's test.93ASSDSSNTE101 and CDR2 39DMGHGLRLIHYSYDVNSTEKG59 sequences, and were termed γ1TCR-I and γ1TCR-II, respectively  used as a control. Anti-TCR (anti-idiotypic) antibodies were tested using two approaches. In the first case, sera of immunized animals were tested on Vβ8.2 synthetic peptides by ELISA. A weak but distinct response was detected in both instances starting on day 21 or 28 , we engineered an antibody with the same V region (γ2bTCR-I). Rats vaccinated with γ2bTCR-I and subsequently immunized with MBP peptide, were protected only partially compared to rats vaccinated with γ1TCR-I (10.2 vs. 1.9)  Table . All ser-5 M (26). It also binds antigen:antibody complexes formed at molar equivalence with an affinity about 100 fold higher [93ASSDSSNTE101, we would have found similar results. As shown .+ and CD8+ T cells [In this report we demonstrate that the severity of EAE in the Lewis rat can be greatly attenuated, and in some instances completely prevented, by active immunization with antigenized antibodies expressing amino acid sequences of the rat Vβ8.2 gene product. Immunity against synthetic peptides of the TCR has been shown to be effective in preventing or reducing the severity of EAE in the rat -20, sugg T cells -38 as we T cells . A preva T cells  and in t T cells  idiotypi T cells ,42,43 or T cells  have beeHere we show that immunity against idiotopes of antibodies engineered to express TCR peptides is effective in generating anti-idiotypic immunity directed against rat Vβ8.2 TCR gene product. Importantly, this type of immunity protected from EAE. The new approach used herein to induce anti-TCR immunity is based on conventional idiotypic immunization in which antigenized antibodies mimic the immunogenic properties of soluble TCR functioning as a surrogate internal image  in much + T cells suggests that cross-reactive anti-idiotypic antibodies may be very low titer. Alternatively, they may be adsorbed on T cells in vivo precluding their detection in the serum. The first possibility is consistent with the self nature of TCR peptides and a predicted paucity of self reactive clonotypes within the natural B cell repertoire. Interestingly, we noted that the anti-idiotypic response against a non-self peptide expressed in an antigenized antibody [93ASSDSSNTE101 sequence developed flowcytometry-reactive autoantibodies could reflect difference in conformation once the two peptides are embedded in the CDR3 loop of an antigenized antibody. For instance, 93ASSDSSNTE101 could be better surface exposed and more stably expressed 39DMGHGLRLIHYSYDVNSTEKG59. A computer-assisted comparison of hydrophilicity profiles [93ASSDSSNTE101 peptide in the parental. TCR Vβ8.2 gene product and in the antibody V region shows that in both instances the peptide is highly hydrophilic  of cases . We conc93ASSDSSNTE101 but not 39DMGHGLRLIHYSYDVNSTEKG59 reacted immunologically with a synthetic peptide of human CD4 previously described to bind Ig [93ASSDSSNTE101 serum antibodies underscores qualitative differences of the immune response between rats immunized with γ1TCR-I and 1TCR-II, respectively. Thus, it appears as if 93ASSDSSNTE101 induced a different immune response than 39DMGHGLRLIHYSYDVNSTEKG59. Furthermore, since vaccination with γ1TCR-I also promoted greater protection from EAE, it is tempting to speculate that a component of the anti-idiotypic response against γ1TCR-I is associated with protection.Interestingly, rats immunized with the antigenized antibody expressing  bind Ig . BecauseIn conclusion, three points have emerged from this study. First, antigenized antibodies expressing conformationally-constrained loops of the Vβ8.2 TCR can be used as vaccines in the prevention of EAE in the Lewis rat. Our new approach to generate anti-TCR immunity, confirms the relevance of anti-idiotypic regulation in controlling rat EAE ,18,20. SThe authors declare that they have no competing interests


Hypertension is a growing worldwide problem associated with an increased risk of cardiovascular morbidity and mortality. However, the rates of prevalence of hypertension are higher in some populations than others. Although ethnic and genetic factors have been implied in the past to explain this, the environmental influence and psychosocial factors may play a more important role than is widely accepted. Examining the non-genetic influences in future hypertension research may be necessary in order to clearly define the local blood pressure demographics and the global hypertensive disease burden. Hypertension is a common problem, with a consistent and continuous risk of cardiovascular disease and stroke associated with rising blood pressure levels . FurtherThe consequences of poor blood pressure control are huge. As high blood pressure is the most important risk factor for cardiovascular disease, it has been calculated that by achieving the target of 140 mmHg, there would be a reduction of 28–44% in stroke and 20–35% in ischaemic heart disease depending on the age. This would prevent approximately 21400 stroke deaths and 41400 ischaemic heart disease deaths each year – and these translate to approximately 42800 strokes and 82800 ischaemic heart diseases saved, making a total of 125600 events saved per year in the United Kingdom alone [Nonetheless, what is more intriguing and perhaps still not fully explained, is why some populations seem to have a much higher population prevalence of hypertension as compared to others. For instance, the prevalence and incidence of hypertension differs between the non-westernised and westernised populations. Even within the western world, the Afro-Caribbean or African-American black population has a higher prevalence of hypertension and target organ damage related to it, as compared to white Europeans or Americans . DiffereBMC Medicine, address the issue of whether there is a truly genetic predisposition or perhaps an environmental influence is to blame for higher rates of prevalence of hypertension seen in some of these ethnic populations. In a well-designed pooled analysis, incorporating eight studies involving 8 white and 3 black populations from the North American, European and African populations – a dataset of nearly 85,000 patients – Cooper et al [Cooper et al , writinger et al  examinedper se together with the unhealthy lifestyle and diet in the western world increase the risk of hypertension, compared to the rural, 'low stress', healthier lifestyle and dietary habits in Africa? Perhaps the genotype of black subjects was not idealised for the 'pro-hypertension' environment of the western world, leading to the greater risk of developing hypertension amongst blacks in the western world. This 'genetic predisposition' of certain ethnic groups, coupled with the 'wrong' environment, leads to an unhealthy combination that predisposes to cardiovascular disease [Does this take us back to the drawing board? Perhaps environmental factors do play a more major role in developing hypertension than is widely accepted. Indeed, does urbanisation  disease . However disease . Therefo disease . SimilarCan this 'wrong genotype in the wrong environment' hypothesis be applied to hypertension in black African-Americans? Efforts have already been made to understand the reasons behind the higher prevalence of hypertension in African Americans, with the underlying assumption that there may a genetically determined predisposition as compared to their white counterparts; however, no convincing data are available . This maNumerous potential explanations for the higher prevalence of hypertension in blacks have been proposed. Genetic mechanisms have been used to explain familial aggregation of hypertension in Jamaican blacks and the intra-class correlation of systolic blood pressures amongst black twins ,14. Low Notwithstanding the shortcomings of a retrospective, pooled study, with varying criteria for inclusion, and the intra-, inter- and cross-study observational errors, Cooper et al  have shoEvidence that environmental and particularly psychosocial factors are important in the development of hypertension also comes from a series of epidemiological studies conducted in the early part of the last century, which have shown that urbanisation and adoption of a westernised life style leads to blood pressure rises. Many of these studies have been conducted in Africa, where the rural populace has a relatively low prevalence of hypertension. For example, primitive black populations living in more frugal circumstances in rural Africa have been shown to have low blood pressures. Evidence suggests that there are some populations that exist who are naïve to hypertension and related morbidity. Interestingly, their blood pressures hardly rise with increasing age, a common response to age in many other (urbanised?) communities -18. The Migration, not withstanding the complexity of the studies in these populations, has been shown to significantly affect blood pressures. A study examining the migrant islanders into New Zealand showed raised population blood pressures, as well as an increased slope of the age-blood pressure relationship . The KenThe issue of the influence of colour of the skin to blood pressures is even more complex. On the one hand, studies in America have shown relationship between the dark skin and blood pressures, leading to some suggesting that the link is genetic. In contrast, some argue that it is a manifestation of the stress and social pressure of having a dark skin that causes this. In Cuba, for example, where communist principles are considered to have broken the racial barriers, the ethnic differences in blood pressures were shown to be small, supporting the latter argument . In addiThus, the process by which a society becomes more economically advanced or "developed" seems to be associated closely with rates of hypertension prevalence. Indeed, lifestyle and dietary changes related to the so-called "development" seem linked to the prevalence of hypertension. In the INTERHEART population case-control study , which wThere remain many uncertainties to the relative importance and contribution of environmental versus genetic influences on the development of blood pressure – there is more than likely an influence from both. However, there is now evidence to necessitate increased attention in examining the non-genetic influences on blood pressure, a neglected area of hypertensive research but perhaps a goldmine for establishing causal influences. As stated earlier, the future of hypertension research should focus on more standardised and comparable protocols, with comparable designs in data collection. Multi-centric data collection with a view to establishing local or national blood pressure demographics is crucial for the formal assessment of the global hypertension burden and the implementation of cost-effective primary preventive measures.The pre-publication history for this paper can be accessed here:


This method has been shown to produce reliable results on simulated data and on human data from the TAP2 region. Strikingly, however, it finds very little support for recombination rate variation at TAP2 in the western chimpanzee data. Moreover, simulations suggest that there should be stronger support if there were a hotspot similar to the one characterized in humans. Thus, it appears that the human TAP2 recombination hotspot is not shared by western chimpanzees. These findings demonstrate that fine-scale recombination rates can change between very closely related species and raise the possibility that rates differ among human populations, with important implications for linkage-disequilibrium based association studies.Recent experiments using sperm typing have demonstrated that, in several regions of the human genome, recombination does not occur uniformly but instead is concentrated in “hotspots” of 1–2 kb. Moreover, the crossover asymmetry observed in a subset of these has led to the suggestion that hotspots may be short-lived on an evolutionary time scale. To test this possibility, we focused on a region known to contain a recombination hotspot in humans, TAP2, and asked whether chimpanzees, the closest living evolutionary relatives of humans, harbor a hotspot in a similar location. Specifically, we used a new statistical approach to estimate recombination rate variation from patterns of linkage disequilibrium in a sample of 24 western chimpanzees  The human TAP2 recombination hotspot is absent from the homologous region in western chimpanzees, with important implications for association studies, the HapMap project and understanding fine-scale variation in recombination rates Understanding recombination is also of practical importance for evolutionary inference and human genetics . UnfortuOne way to learn about finer-scale recombination rates in males is sperm typing . In thisD. melanogaster and D. simulans, two sibling species that differ in their recombination landscape , another Old World monkey. For more closely related species, nothing is known. However, observations in yeast . The two species are thought to have had a common ancestor five to six million years ago and differ at approximately 1.2% of base pairs on average  but not western (P. t. verus) chimpanzees , consistent with previous findings  and Pr(λ > 10); these are the posterior probabilities of a hotspot of any kind and of a hotspot of intensity at least ten times the background rate, respectively.The LD data are summarized in t, λ see . Within sian see  so, as aλ > 1) = 1 and Pr(λ > 10) = 0.982. When the same method was applied to the human genotype data (i.e., ignoring the information about the phase of multiple heterozygotes), we estimated Pr(λ > 1) = 1 and Pr(λ > 10) = 0.992. The results are almost identical, suggesting minimal loss of information with the use of genotypes. Interestingly, the point estimate of λ using either haplotypes (28.4) or genotypes (32.1) is higher than the corresponding estimate from sperm typing (11). This difference may reflect error in the estimates; alternatively, it may point to a more intense hotspot in females than in males  = 0.200 and Pr(λ > 10) = 0.006, reflecting tepid support for a hotspot of any kind and almost no support for a hotspot similar to the one observed in humans. Indeed, the latter figure represents very strong evidence against a hotspot of moderate intensity and rules out the possibility that the chimpanzee polymorphism data are simply uninformative, because of, for example, insufficient sample size or diversity.Next, we applied the same method to the genotype data collected from western chimpanzees. The estimate of the background rate of recombination, ρ^ype data . Howeverλ = 11 and then tabulated the proportion with posterior probability estimates as low or lower than that observed , the probability of obtaining Pr(λ > 1) ≤ 0.200 is p = 0.010 and the probability of obtaining Pr(λ > 10) ≤ 0.006 is p = 0.005. With a lower ρ value  ≤ 0.200 is p = 0.020. In other words, we can reject the null hypothesis that there is a hotspot in western chimpanzees similar to the one in humans, because we would expect to see more support for a hotspot in these data if one were there. It appears that western chimpanzees do not harbor a hotspot in the same location as humans.To assess how likely we would be to obtain such weak support if there were in fact a hotspot in western chimpanzees similar to the one in humans, we generated 200 simulated genotype data sets under a model with a hotspot of intensity rved see . We tookρ and λ . This in turn raises a number of more general questions. Are hotspots frequently born de novo or do they tend to migrate within circumscribed regions of the genome? Are particular sequence motifs sufficient to produce recombination hotspots, or are larger-scale requirements, such as chromatin accessibility, required for their formation ? The sysComparative studies of hotspot regions will also increase our understanding of the determinants of mutation rates. As noted by In conclusion, these analyses demonstrate that fine-scale recombination rates can change between closely related species. Together with the observations that crossover frequencies can depend on specific haplotypes  and thatThe extent to which local recombination rates vary among human populations influences the degree of similarity of LD patterns among them, with important consequences for the design of efficient LD-based association studies (including, for example, the choice of appropriate “haplotype tagging SNPs”  in diffe(Pan troglodytes verus) that were wild caught or known to be unrelated based on recent pedigrees. Twelve samples (Annaclara, Frits, Hilko, Liesbeth, Louise, Marco, Oscar, Regina, Socrates, Sonja, Yoran, and Yvonne) are from the collection stored at the Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany, while 12 other samples (NDH0311G1, NDH0312G1, NDH0313G1, NDH0314G1, NDH0317G1, NDH0320G1, NDH0321G1, NDH0322G1, NDH0325G1, NDH0326G1, NDH0328G1, and NDH0329G1) were kindly provided by P. Morin and the Primate Foundation of Arizona.We used DNA from 24 western chimpanzees We amplified 9,491 bp from the TAP2 region, corresponding to base pairs 113102–122585 of the sequence from 2, PCR buffer , 0.5 U of Taq DNA polymerase , and 10 pmol of each primer. We used 50–100 ng of DNA in each 30 μl PCR. Amplification conditions for all regions were the following: incubation for 3 min at 94 °C, 35 cycles (45 s at 94 °C, 1 min at 45–62 °C, and 1 min at 72 °C) and a final elongation of 5 min at 72 °C. A nested PCR was performed to obtain regions 6 and 7 by using the product of the primers Tap2 5 5′ and Tap2 8 3′ as a template. PCR products were separated from primers and unincorporated dNTPs by treatment with a solution of 10% PEG 8000/1.25 M NaCl followed by centrifugation. PCR products were then air dried and resuspended in 10–15 μl of H2O.DNA amplification reactions contained 250 μM of each dNTP, 1–2 mM MgClSequencing reactions consisted of 1 μl of ABI Prism BigDyeTM Terminators version 2.0 , 8–10 ng of purified PCR product, and 1 μl of 2.5 μM primer (the same primers used for PCR) in a volume of 7 μl. Cycling conditions were 96 °C for 2 min and then 35 cycles of 96 °C (20 s), annealing temperature (30 s), and 60 °C (4 min). Isopropanol-precipitated cycle sequencing products were run on an ABI 3730 DNA analyzer. Base calling was done with ABI Prism DNA Sequencing Analysis version 5.0 and ABI Basecaller. BioEdit version 5.0.6 was used for sequence analysis and alignment.In total, 2-fold coverage of a 9,370 bp sequence was obtained for each individual; these are available from GenBank see . Most ofθ = 4Neμ (Ne is the diploid effective population size and μ is the mutation rate per generation), using Watterson's estimator, θW  to allele frequencies in western chimpanzees. Specifically, we ran 104 coalescent simulations of the standard neutral model with the same number of chromosomes and base pairs as in the actual data, with θ equal to θW, and with the population recombination rate equal to the estimated value (see below). We then tabulated the proportion of simulated runs with a Tajima's D value as or more extreme than that observed.We estimated the population mutation rate, tor, θW , based o, θW  model of H given the underlying recombination parameters α . This can be used directly to estimate α from H in situations where haplotypes have been experimentally determined . To do so, we start with an initial guess for the haplotypes, and iterate the following steps: (i) estimate a new value for α, using the current estimate of H and (ii) estimate a new value for H, using the genotypes G and the current value for α. Step (i) is performed using the PAC-B model of α described below. Step (ii) is performed by using the method for haplotype inference described in i = ziρ/(j+ ziρ)q in their Appendix A with i =1−exp(−ziρ/j)q.) Both the PAC-B model and the Fearnhead and Donnelly conditional require the specification of a mutation parameter, θ, and a mutation process. In each case, we used the value of θ given in More formally, the model of ed e.g., . HoweverH, α | G) from which we wish to sample. Provided that the algorithm is run for sufficiently long, the estimates of α obtained each iteration provide a sample from the distribution Pr(α | G), and thus allow α (i.e., the underlying recombination process) to be estimated directly from G, taking full account of the fact that the actual underlying haplotypes are not known. The algorithm is implemented within the software package PHASE version 2.1, which is available online at http://www.stat.washington.edu/stephens/software.html.This iterative scheme defines a Markov chain whose stationary distribution is the distribution Pr(λ. Crossovers occur as a Poisson process (i.e., there is no interference) of constant rate r (per base pair) outside the hotspot and of constant rate λr inside the hotspot; gene conversion is not explicitly modeled. In the first version, we assumed that, if present, the hotspot is at the same location as estimated by sperm typing in humans (4180–5417). (This location is not precisely the same as the one used by ρ (= 4Ner, where Ne is the effective population size) and λ. We assumed a priori that a hotspot exists with probability 0.5 and that, if the hotspot exists, λ is between one and 100. Specifically, we assumed that λ = 1 with probability 0.5 and otherwise that log10(λ) is uniformly distributed on (0, 2). The prior on ρ is uniform on log10(ρ) in the range (−8, 3), which covers all plausible values.We considered two versions of the simple hotspot model of λ and ρ. In this case, we assumed a priori that the hotspot exists with probability 0.18 (corresponding to an assumption that a hotspot occurs roughly once per 50 kb of sequence), that the center of the hotspot is equally likely to be anywhere along the length of the sequence, and that the width of the hotspot is between 200 and ∼4,000 bp . Priors on ρ and on λ  are as in the first version.In the second version, we assumed that the location and width of the hotspot are unknown and to be estimated along with To allow for potential problems with convergence of this Markov chain Monte Carlo algorithm, we ran the algorithm ten times for each analysis, using different seeds for the pseudorandom number generator. For each run, we obtained a point estimate of the parameters (using sample posterior medians) and posterior probabilities. The reported estimates are the median of the estimates obtained from the ten runs.λ = 11, and θ = θW. Haplotypes were randomly paired to form genotypes and phase information was ignored. The data were masked to mimic the actual data structure, i.e., they included a gap of 487 bp in the same position. We then counted the proportion of simulated data sets for which our estimate of the posterior probability was as low as observed or lower .Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for the human TAP2 region of The GenBank (


Atrial fibrillation (AF) has been recognised as an important independent risk factor for thromboembolic disease, particularly stroke for which it provides a five-fold increase in risk. This study aimed to determine the baseline prevalence and the incidence of AF based on a variety of screening strategies and in doing so to evaluate the incremental cost-effectiveness of different screening strategies, including targeted or whole population screening, compared with routine clinical practice, for detection of AF in people aged 65 and over. The value of clinical assessment and echocardiography as additional methods of risk stratification for thromboembolic disease in patients with AF were also evaluated.The study design was a multi-centre randomised controlled trial with a study population of patients aged 65 and over from 50 General Practices in the West Midlands. These purposefully selected general practices were randomly allocated to 25 intervention practices and 25 control practices. GPs and practice nurses within the intervention practices received education on the importance of AF detection and ECG interpretation. Patients in the intervention practices were randomly allocated to systematic (n = 5000) or opportunistic screening (n = 5000). Prospective identification of pre-existing risk factors for AF within the screened population enabled comparison between high risk targeted screening and total population screening. AF detection rates in systematically screened and opportunistically screened populations in the intervention practices were compared to AF detection rate in 5,000 patients in the control practices. Atrial fibrillation (AF) has been recognised as an important independent risk factor for thromboembolic disease, particularly stroke with which it is associated with a five fold increase in risk . There aScreening for AF in the elderly fulfils many of the Wilson-Jungner criteria for a screening programme . It is aOne UK study has compared systematic nurse-led screening with prompted opportunistic case finding for AF in primary care . This smopportunistic case finding, where a health care professional took the opportunity to feel a patient's pulse during a consultation. If the pulse is irregular, they might make a clinical diagnosis of AF, or request/perform an electrocardiogram (ECG) as a confirmatory test. However, opportunistic case finding is likely to miss a significant proportion of people who would otherwise have benefited from treatment. For example, detection of hypertension in general practice was traditionally detected in an opportunistic way until the introduction of health checks with the 1990 GP contract. The Health Survey for England shows that in 1991, 42% of the population over the age of 75 had hypertension for which they were not taking any medication [The simplest strategy was dication . This fitargeted screening programme. Cardiac failure, hypertension and rheumatic heart disease are important precursors of AF [One possible approach was to screen patients who are at higher risk of AF – a rs of AF . AF is mrs of AF . Most gewhole population screening programme.Another approach was to screen everyone 65 and over (65+) for AF – a A modelling exercise using decision analysis to inform on the methodology for this study indicated that there were not sufficient primary data available to recommend which of these (targeted or whole population) would be the optimum policy12-lead ECG is recognised as the gold standard test, but this test is time consuming (taking at least 15 minutes to perform in an outpatient setting). Therefore, it is important to consider simpler tests. This study assessed simpler methods compared to the gold standard, both in terms of accuracy, time taken and patient acceptability. These include taking the pulse, and simpler ECGs.Cardiologists offer the most accurate readings of ECGs, but can satisfactory interpretations be obtained by the GP, the practice nurse, or computerised diagnostic software? This study assessed the accuracy of these different approaches to interpreting the ECG.The main treatment options to reduce risk of stroke in patients' with AF are currently warfarin or aspirin. Aspirin is much less effective than warfarin – it achieves a barely significant 21% reduction in stroke risk . HoweverThis study, by providing answers to these questions, allowed the optimum strategy for introducing a screening programme for AF in the over 65s to be determined. However, before a decision is made as to whether to institute a screening programme, not only must the question of the best strategy be considered, but also, the question of whether any screening programme at all should be introduced. This study provided data to assist in answering this fundamental question by providing:i) An accurate estimate of the community prevalence and incidence of AF in over 65s;ii) An assessment of the health economic implications of screening for AF;iii) An assessment of the service provision implications of implementing such a programme;iv) An assessment of the impact on patient quality of life and anxiety after various screening methodologies.Although the cost effectiveness of different approaches to screening is often put in terms of the average cost per case detected, such an approach ignores the sensitivity and specificity of the screening test. This is because average cost per case detected focuses entirely on true positives, paying no attention to false positives, false negatives and true negatives. False positives and false negatives impose costs on patients and health services which would be neglected if the focus was confined to true positives . An unduThis study compared the incremental cost per case detected for different methods of AF screening. This refers not to the average cost but rather approximates the incremental cost per case detected in moving from one of the screening options to another. Use of incremental cost per case detected by option shows how the cost per additional case detected is likely to increase as the intensity of screening increases. This method has been used to deal with similar uncertainties about the cost effectiveness of screening for other diseases, including breast and colorectal cancer and has been recommended by the US guidelines .• To determine baseline prevalence and the incidence of AF based on a variety of screening strategies and in doing so to evaluate the incremental cost-effectiveness, in terms of cost per case identified, of the different screening strategies (targeted or whole population screening) compared with routine clinical practice for detection of AF in people aged 65 and over.• To evaluate the relative cost-effectiveness of screening methods for AF diagnosis, comparing 12 lead ECG (gold standard) with pulse taking, lead II rhythm strip from standard ECG limb leads alone and single lead thoracic placement ECG.• To evaluate the most cost-effective method of test interpretation, comparing cardiologist (gold standard), with GP, practice nurse, or computerised diagnostic software.• To assess the differing combinations of screening strategies and procedures in terms of patient acceptability and impact on patient quality of life, including any psychological effects of screening.• To determine the community prevalence of AF in people 65+.• To evaluate the value of clinical assessment and echocardiography as additional methods of risk stratification for thromboembolic disease in patients with AF.• To evaluate the service provision implications should screening for AF become a national programme, and identify the optimum screening algorithm for identification of patients with AF.• The incidence of AF according to a variety of screening strategies• The associated costs providing an incremental cost per case detected. The cost data was collected from an NHS and patient perspective. It has focused on resources required to establish screening, time taken to complete screening and the cost of the equipment.• Cost effectiveness of 4 different methods of screening for AF. The cost data focused on the difference in the cost of the equipment and the time taken for each of the different methods of screening to be completed. This was from both an NHS and patient perspective.• Cost effectiveness of 4 different methods of ECG interpretation. The cost data focused on the difference in the cost of the grade of staff interpreting the ECG and the accuracy of their interpretation.• Overall community prevalence and incidence of AF• Patient acceptability to AF screening was measured using an adapted version of the screening specific questionnaire used in the Colorectal Screening Programme . Patient• Modelling techniques were used to identify the implications of AF screening on health service provision nationally. This included the effect on echocardiography and anti-coagulation clinic provision.This was a multi-centre randomised controlled trial. The study schema is shown in figure 50 computerised general practices within the West Midlands were recruited through MidReC (Midlands Research Practices Consortium). This was undertaken by writing to all practices in the West Midlands and surrounding counties explaining the study and asking whether they were interested in participating. Practices showing an interest were given further information about the study and invited to attend an investigators meeting. Following the investigator meetings sixty practices interested in participating in the project were randomised (stratified based on Townsend score and practice list size): 25 as intervention, 25 as control practices with 10 reserve practices.1/3rd of the total population of patients 65+ in this group), and 5000 from the control practices  were identified.A computerised list of all patients aged 65+ was obtained from each practice, and from this a random sample of 10,000 patients from the intervention practices , radial pulse and a 12 lead ECG. The 12 lead ECG was performed using an electronic ECG machine which allowed print-out of single lead thoracic placement ECG and a rhythm strip of lead 2 using limb leads from standard ECG.All 12 lead ECGs were sent to two cardiologists for reporting (GL, MD). Where there was disagreement over the diagnosis a third cardiologist was used to decide. The cardiologists were asked to state whether the ECG showed AF or not, and to state whether there were any other significant abnormalities. Patients were informed of the result within two weeks. Patients with normal ECGs were informed of this, patients with any abnormality were asked to make an appointment with their GP. At the GP appointment patients with AF aged 65 – 74 were offered echocardiography. GPs were asked to make a clinical decision as to thromboprophylaxis both before and after the echocardiogram. Patients with other ECG abnormalities were managed as clinically indicated.At the end of the screening process, GPs and Practice Nurses from both intervention practices (who had received education on ECG interpretation) and control practices (who had received no education) were sent ECGs to interpret for the presence or absence of AF. All ECGs recorded within the study were printed off as either 12 lead, single lead thoracic placement or limb lead recordings. Allocation to ECG type was random and resulted in three equal ECG groups. In order for each interpreter to read all three types of ECG, batches of ECGs were collated with the same numbers of each type of ECG. Allocation to a batch was also random. In total, there were 25 batches of ECGs to match the number of practices in each arm. The GP and Practice Nurse from the same practice read the same batch of ECGs and each batch was read by one control practice and one intervention practice. Therefore each ECG was read by two GPs and two Practice Nurses. All ECGs were anonymised, and practices did not receive any ECGs from their own practice. The interpreters were given a sheet to fill in to indicate for each ECG the presence or absence of AF. All ECGs (as 12 lead) were also analysed by the specific software package accompanying the electronic ECG and results recorded.Patient acceptability and quality of life for different screening strategies were established using EuroQol (EQ-5D) combined together with the Speilberger 6-item Anxiety Questionnaire. EQ-5D allowed the measurement of broad aspects of quality of life. The shortened Speilberger anxiety questionnaire also has proven validity and is more specific to anxiety than is the SF-12 . An adapA random sample of 750 patients  were sent postal versions of the psychological instruments (EQ-5D and Spielberger) on entry to the study (i.e. before the intervention group has received their invitation to attend for screening). One reminder was sent a month later to non-responders. The same questionnaires were sent to the same groups plus those patients who had screened postive at the end of the screening period, approximately 17 months later. This allowed a non-randomised comparison between the effects on quality of life and anxiety in screen positive and screen negative patients.In addition, all patients who were screened were asked to complete the acceptability and Spielberger questionnaire immediately after screening. The patient acceptability questionnaire was also administered to all patients who proceeded for echo.The value of clinical assessment and echocardiography in risk stratification were determined in patients aged 65–74. This compared GP assessment based on the Birmingham guidelines for thromboprophylaxis in AF with any changes in recommendations for treatment once echocardiography results were available to the GP.The assumptions for the power calculations were that patients aged 65 and over represent 17% of the total population; that 40% of study population will be in the high risk group.Also assuming that:1. Minimum worthwhile change in detection rate was 1% for targeted screening versus routine practice. It is estimated that this change would equate to £10,000 per life year gained. This is based on the following assumptions:a) 60% of new cases of identified AF would be suitable candidates for warfarinb) Annual risk of stroke in this population was 5%, reduced by 60% to 2% if treatedc) Costs: £25 to screen a patient; £100 to treat with warfarin pa; £6,000 NHS costs to treat a stroke2. 50% of patients with AF will be already known to their general practitioner (estimates range from 30%  to 76% )3. Community prevalence of AF in this population was 6% See figure All sample size calculations were for 90% power and 5% significance levels unless otherwise stated.a) To detect a 1% difference in detection rate between intervention (GP educated) and control practices (B2-A1) vs (C2-C1). This requires 1,236 patients. However, since this is a difference based at the practice level of randomisation, it needed to be inflated by the design factor. Based on AF prevalence data from the EcHoES (Echocardiographic Heart of England Screening) Study [actices B-A1 vs  and control practices (A3-A1) vs (C2-C1)b) To detect a 1% difference in detection rate between intervention  and control practices (A2-A1) vs (C2-C1)c) To detect a 1.8% difference in detection rate between intervention  required fewer patients to detect the expected difference (2%) stated in the assumptions, it would be possible to detect differences as low as 1%, should the detection rate not be as high as expected.The a), b) and c) comparisons are all at practice level randomisation.d) To detect a 1% difference in detection rate between high risk screening strategy and routine practice prompted by education (opportunistic arm) (A2-A1) vs (B2-A1).This requires 1,236 patients in both the high risk systematic screening and the GP educated (opportunistic) screening arms of the intervention practices assuming the high risk screening detects a 1% increase and opportunistic screening detects 0% increase. Should the increased detection rates be higher in each arm (1.7% in the high risk arm and 0.7% in the opportunistic arm) then this could require 2,686 patients in each arm. However, since there is a ratio of 2:5 patients in these arms there will be sufficient patients as only 1,880 are needed in the high risk arm and 4,700 in the opportunistic arm to be able to detect this 1% difference.e) To detect a 1% difference in detection rate between total screening strategy and routine practice prompted by education (opportunistic arm) (A3-A1) vs (B2-A1).This requires 3,300 patients in both the total screening and the GP educated (opportunistic) screening arms of the intervention practices.f) To detect a relative risk (RR) of 2 (1% detection rate difference) between total population and high risk screening (A3 vs A2).It was assumed that 40% of the study population fall into the high risk group, and the prevalence of undetected AF is 3%. This meant that 1,434 patients would be needed in each of the two risk groups to detect a two fold difference in risk (i.e. RR of AF in high risk as compared to low risk group is 2). This RR of 2 equates to an increase in AF detection rate from 3% in the total population arm to 4% in the high risk arm. Since there was a 40:60 split in the two risk groups unequal sample size calculations only require a minimum of 1,200 patients in the high risk group and 1,800 in the moderate/low risk group. This was achievable with a screening arm of 5,000 patients, as there would actually be 1,320 in the high risk group and 1,980 in the moderate risk group if a 66% screening acceptance rate was assumed.Although some of the variances are from North American populations we have no reason to suspect that the variation will be different in a British population since data from the ECHOES study on the SF36 gives variations very similar to the North American norms.The shortened (6-item) version of the Spielberger state anxiety questionnaire has been validated and used in populations different from that under consideration in SAFE, namely it tends to have been used in young and mostly female populations ,21,22. TThe VAS variance as reported for an elderly population aged 75 and over was 365  but for This was reported in different ways in the Johnson  and DormIntention to treat analysis will be used.Any previously known Atrial Fibrillation cases will be subtracted from the totals obtained at the end of the study to ensure there is no double counting in the incidence figures.Chi-squared, independent t-tests and log-linear models will be used to describe demographic data. If there are differences between the groups this may need to be adjusted for in later analyses.Proportions and rates will be used as the measures of prevalence and incidence.The independent t-test and ANOVA with random effects (as appropriate) will be used to examine the detection rate differences between the intervention and control screening strategies. Should the data be strongly non-normal a non-parametric equivalent will be used.a) to assess patient acceptability and impact on QoL of different screening strategiesThe independent and related t-test and ANOVA will be used to examine the differences between the intervention screening strategies on the Spielberger and EuroQol EQ5D. Should the data be strongly non-normal a non-parametric equivalent will be used. Chi squared tests will be used on the screening tool.b) to assess the value of echocardiography in risk stratification for thromboembolic disease in patients with AFMcNemar's test will be used to see whether there is any significant change in the doctor opinion on risk of CVA and treatment decision before and after echo screening.c) to evaluate the most cost effective method of test interpretationThe cost effectiveness will be covered in the economic section. However, the use of sensitivity, specificity, Cohen's κ and conditional logistic modelling will allow for comparison of the various methods for detecting AF between the GPs, nurses and consultants.d) to evaluate the most cost effective method of screeningThis will be covered in the economic analysis section.Multivariate and logistic modelling analyses will be undertaken in order to determine which markers might be the best predictors of the presence of AF. This will act as confirmatory analysis for the risk factors used in the screening strategy to define a high risk patient.This trial evaluated a large number of alternative screening scenarios for identifying atrial fibrillation . The study has been powered to detect a difference in the targeted versus population arms, and in systematic screening versus routine clinical practice. However, the economic analysis will compare the cost-effectiveness of all alternative screening approaches using a modelling framework, whereby data will be drawn from the trial (where appropriate and available) and from external sources.The use of a modelling approach allows the timescale for the economic analysis to be extended beyond the follow-up period allowed for in the trial. The use of such extrapolation will enable estimation of the incremental cost-effectiveness ratios (ICERs) for each approach:Incremental cost per case detectedIncremental cost per life year gainedIncremental cost per quality-adjusted life year (QALY) gainedThe use of EQ-5D allows the measurement of broad aspects of quality of life. EQ-5D allows changes in health status to be measured but also valued, using the University of York Measurement & Valuation of Health general population survey tariff .The cost analysis adopted a broad perspective to include costs incurred within the health sector and by patients and carers. Data collection was undertaken on all trial patients in order to allow a stochastic cost analysis to be conducted. The focus of the data collection will be upon the key cost drivers which will include:a) the resources required to establish screening (invitation to patient, follow ups, communication of results),b) the time taken to carry out the various tests, andc) the cost of the equipment (expressed as cost per test).a), b) and c) will be based on data collected in the study.The analysis adopted an incremental approach such that data collection concentrated on resource use differences between alternative screening scenarios. The process of collecting data on resource use was undertaken separately from data collection on unit costs. Resource use data on the screening process was principally collected within the trial. Unit costs were collected from published sources and a representative sample of NHS providers in order to increase generalisability. The methods used in collecting data will include patient questionnaires (see above) and review of patient records . Data on private costs were collected from a survey of a sub-cohort of the trial population.The plan for the analysis is:1. Report a cost consequence analysis, which will involve providing a full description of all important results relating to costs and consequences.2. Conduct both a cost-effectiveness analysis and a cost utility analysis (using data on true positive cases detected as the measure of effect and data on EQ-5D to estimate QALYs).An incremental approach will be used in order to compare the large number of alternative screening strategies.We are interested in comparing the mean costs per patient since our concern is with predicting overall programme costs. However, the data on costs are likely to have a skewed distribution. Therefore, the plan for the analysis of costs is:1. To explore the nature of the distribution of costs2. If required, to use non-parametric comparison of means (e.g. bootstrapping)3. If the distribution of the data is approximately normal, parametric methods will be used.This approach is in line with recent recommendations .If missing data are a problem at the economic analysis stage, then imputation techniques will be employed.Longer term costs and consequences will be explored by extrapolating beyond the end of the trial using a modelling framework using data from a range of trial and non-trial sources. The precise form of modelling is yet to be determined, but is likely to be either Markov or Discrete Event Simulation, depending upon the extent to which the Markov assumptions are justified. An advantage of using such an approach is that it will allow the additional costs of increasing survival to be explicitly incorporated into the analysis. In particular, modelling will provide estimates of the optimal frequency of screening for AF, based on the estimates of incidence and prevalence from the trial.Identifying untreated patients with AF will have implications for service provision. These will depend upon the prevalence of atrial fibrillation, the screening mechanism employed, the use made of echocardiography, and the additional requirements for anticoagulation monitoring. The separate parallel study, BAFTA, will provide empirical data that will allow such implications for service provision to be assessed. The modelling exercise, which will draw on both SAFE and later BAFTA results, will combine best estimates of both screening and anticoagulation options.The robustness of the results of the economic analysis will be explored using sensitivity analysis . This wiPatients aged 65 years or over (65+).Patients who were terminally ill.Randomisation of practices and patients was performed by statisticians from the Department of Primary Care and General Practice at The University of Birmingham. Cluster randomisation of practices to intervention or control was stratified by Townsend quartiles and practice size. Computer searches were carried out to identify cases of known AF, within the sample of patients identified above, using a published strategy . The ranFollowing initial sampling of the total population the list of patients from each practice were returned to the practices who were asked to remove any patients who had died, moved or were terminally ill. Patients removed following this process were replaced with patients from a reserve list, which had been randomised at the same time as those on the initial lists.Patients aged 65+ who were selected to the systematic screening arm received an information sheet with an invitation letter to attend the ECG clinic. Entry to the trial was discussed with the practice nurse at the clinic. The practice nurse then obtained written consent from those patients who were willing to participate.Study patients found opportunistically to have an irregular pulse were given an information sheet and invited to attend the screening clinic.GPs and other members of the primary health care team in the intervention practices attended investigator days at which they were given educational materials informing them of the importance of detection of AF, and the treatment options that are available. The materials encouraged them to consider opportunistic screening of patients.Members of the primary health care team in control practices received no educational input from the research staff.Practice nurses attended an ECG training day prior to starting the ECG screening clinics. Training included how to perform an ECG (using the Biolog) to ensure a standardised high quality tracing and basic ECG interpretation .Computer searches were carried out to identify cases of probable AF in the 15,000 study patients using a published strategy [Case notes of patients identified as 'known' or 'probable' AF in any of these computer searches were reviewed for mention of a diagnosis of AF. AF diagnosis were drawn from hospital letters stating the existence of the condition or ECG recordings from the last 5 years.An additional 5% random sample of case notes of patients not identified as 'known' or 'probable' AF by computer searching were reviewed  to estimate how many other patients who are known to have AF were not identified by the computer searches. If this had revealed a significant number of extra cases of known AF, then the sample size for manual searching would have been increased to allow a precise estimate of the baseline rate of known AF. Unidentified extra AF cases were not found to be significant so no additional note search was required.The same computer searches on both intervention and control practice patients notes were performed prior to, and 12 months after, commencement of screening.All patients in the systematic screening arm and those found to have an irregular pulse in the opportunistic screening arm of the study were invited to attend an ECG screening clinic. At the clinic the practice nurse explained the aims of the study and answered any questions about the study. Written informed consent to participation in the study was obtained from the patient. The nurse then recorded baseline information on age, sex, present smoking and alcohol status and past medical history, including previous diagnosis of AF, and any treatment the patient may be receiving for AF. Radial pulse rate, and whether regular or irregular, was noted. A 12 lead ECG, the gold standard by which other traces were compared, was then recorded using the Biolog machine, which was also able to produce a trace corresponding to the single lead thoracic placement and a rhythm strip of lead II. Finally, the patient was asked to complete an acceptability questionnaire.This study will identify the most cost-effective strategy for identifying atrial fibrillation in patients aged 65 and over. The policy implications will be dependent on the findings and one of the strengths of the current study is the utilisation of modelling techniques to investigate the implications of different screening strategies and frequency of screening within different health care environments. The initial draft of the report has been submitted to the Department of Health and publication of the results should be expected later this year (2004).None declared.RH and DF were principal investigators. EM and SJ were project managers. DS collected data and prepared the first draft of the paper. JM, EM, JR, SB, MD and GL all contributed to the project design. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Pilot studies are often used to transition therapies developed using animal models to a clinical setting. Frequently, the focus of such trials is on estimating the safety in terms of the occurrence of certain adverse events. With relatively small sample sizes, the probability of observing even relatively common events is low; however, inference on the true underlying event rate is still necessary even when no events of interest are observed. The exact upper limit to the event rate is derived and illustrated graphically. In addition, the simple algebraic expression for the confidence bound is seen to be useful in the context of planning studies. While pilot studies may vary in the fundamental objectives, many are designed to explore the safety profile of a drug or a procedure ,2. Oftenα (the Type I error rate) and β (the Type II error rate) may be inappropriate since the objective of the research is not to provide definitive support for one treatment over another [In the context of pilot studies, traditional levels of  another . For exaβ) is of less practical importance in a single arm, non-comparative  pilot study since the results would almost always require confirmation in a controlled trial setting. Shih et al [Similarly, power % confidence interval is to be generated for π and an estimate of the sample size, n, is desired. Denote X as the number of patients sampled who experience the adverse event of interest. Then, the probability of observing x events in n subjects follows the usual binomial distribution. Namely,For ease of presentation, assume the pilot study will involve πu as the upper limit of the exact one-sided 100 × (1 - α)% confidence interval for the unknown proportion, π [πu is the value such thatDenote rtion, π . Then πux = 0), equation (1) reduces toA special case of the binomial distribution occurs when zero events of interest are observed. In pilot studies with relatively few patients, this is of practical concern and warrants particular attention. When zero events are realized (i.e., πu)n = α.(1 - α)% confidence interval for π isAccordingly, the upper limit of a one-sided 100 × (1 - πu = 1 - αn1/.     (2)α)% one-sided confidence interval is (0, 1 - αn1/).The resulting 100 × % confidence that the true rate did not exceed a pre-specified π, say π0, given that zero adverse events were observed. Using (2), it follows that:Furthermore, one can consider using (2) in other clinically important manners. For instance, an investigator may be planning a pilot study and want to know how large it would need to be to infer with 100 ×  declare that they have no competing interests.RC and RW contributed to the conceptualization, writing and editing of this manuscript.


Although significant response time deficits (both reaction time and movement time) have been identified in numerous studies of patients with Parkinson's disease (PD), few attempts have been made to evaluate the use of these measures in screening for PD.Receiver operator characteristic curves were used to identify cutoff scores for a unit-weighted composite of two choice response tasks in a sample of 40 patients and 40 healthy participants. These scores were then cross-validated in an independent sample of 20 patients and 20 healthy participants.p < 0.025) with the motor score of the Unified Parkinson's Disease Rating Scale (UPDRS).The unit-weighted movement time composite demonstrated high sensitivity (90%) and specificity (90%) in the identification of PD. Movement time was also significantly correlated (r = 0.59, Measures of chronometric speed, assessed without the use of biomechanically complex movements, have a potential role in screening for PD. Furthermore, the significant correlation between movement time and UPDRS motor score suggests that movement time may be useful in the quantification of PD severity. Nevertheless, a misdiagnosis of PD can cause psychological trauma and unnecessarily expose patients to PD drugs. Additionally, as new, and possibly neuroprotective, drugs become available for the treatment of PD, early and accurate diagnosis will become increasingly important. As the diagnosis of PD is usually based on subjective clinical assessment of overt symptomatology , the neemography ), but thMontgomery et al. ,4 have pThe global slowing that is consistently demonstrated by PD patients suggests that measures of cognitive or motor speed are logical methods for obtaining quantitative measurements of PD severity. As reaction time (RT) and movement time (MT) have repeatedly been demonstrated to show substantive and significant deficits in PD populations , these The goal of this study, therefore, is to evaluate the diagnostic properties of a choice reaction time task that uses a simple external response console (i.e. a "button box"), similar to other similar response time tasks extant within the PD literature.M = 62.13, SD = 9.59) and 40 healthy participants (Age: M = 65.02, SD = 8.84), and the second consisting of 20 PD patients (Age: M = 64.50, SD = 10.88) and 20 healthy participants (Age: M = 62.65, SD = 12.02). To ensure that no baseline ability differences existed between groups, Wechsler Adult Intelligence Scale (WAIS) full scale IQ estimates were computed for all participants, using the National Adult Reading Test (NART) [M = 24.49, SD = 9.79), and the second sample (M = 22.73, SD = 7.66), with no significant severity differences demonstrated between samples. The spectrum of motor severity within the clinical group is graphically depicted with an area graph in Figures Two independent samples were drawn for this study, the first consisting of 40 PD patients (Age: t (NART) . PatientThe response time tasks used in this study started with an instruction to watch a fixation point (asterisk) in the centre of the computer screen, while depressing the home key (measuring 1.905 cm × 1.905 cm) in the centre of the response console. For the 'uncued' task, participants were not given any advance information concerning the location of the upcoming stimulus. For the 'cued' task, an arrow appeared in place of the fixation point (i.e. in the center of the screen) for a period of 2 seconds, immediately following the disappearance of the fixation point, and correctly cued the location of the upcoming stimulus on all trials. The visual stimulus to which the subject responded was presented on the right or left side of the monitor, at a random interval (between 500 and 1500 ms) following the fixation point ('uncued') or the arrow ('cued'). Participants responded to the stimulus by moving the index finger of their dominant hand from the home key to a response key (measuring 1.905 cm × 1.905 cm) placed 3.175 cm to the left or right of the home key, as directed by the stimulus placement on the screen. The time measured between the onset of the visual stimulus and a participant's movement from the home key was defined as reaction time (RT), and the time measured between a participant's lift from the home key and depression of the response key was defined as movement time (MT). Each task consisted of 10 practice trials, and 40 experimental trials. A participant's RT and MT was computed as the unit-weighted average of scores on the 'cued' and 'uncued' choice response time tasks. This testing apparatus is described in further detail by Johnson et al. .All patients involved in the study were asked to remain drug-free overnight, and to delay taking their morning anti-Parkinsonian medications until after the testing. To avoid any confounding effects resulting from different levels of caffeine intake among participants, all participants were asked to have a normal caffeine-free breakfast prior to testing. None of the participants reported any acute physiological conditions that may have precluded them from putting forth their best effort during the testing session. All procedures and materials were approved by the Health Sciences Research Ethics Board at the University of Western Ontario.In the first sample , separate receiver operator characteristic (ROC) curves were generated for the composite RT and MT scores. The best prediction (i.e. the largest area under the ROC curve) was achieved using the composite MT score. The cutoff score was identified as the point on the curve that maximized sensitivity, with a specificity of at least 70%. This cutoff score was determined to be 230 ms . To control for the possibility that classification success in the first sample was the result of a capitalization on sample-specific variability , this clp < 0.025) were positively correlated with the UPDRS, suggesting that these response time tasks are good predictors of the severity of Parkinsonian symptoms, particularly when considering the MT component of response time.To identify the extent to which response time predicted disease progression, correlations were computed between the UPDRS and the aggregate RT and MT scores. As the samples demonstrated no significant differences on the UPDRS, correlations were computed across all data collected in both samples. Both RT (r = 0.23, n.s.) and MT (r = 0.59, This study confirms previous research that has shown significant movement time (MT) differences between PD patients and healthy participants . The resStandardized objective test batteries will be diagnostically useful in two general scenarios: (a) as an adjunct to the physical examination performed by a specialist (to improve diagnostic accuracy), and (b) as a standardized preliminary screening tool, for situations in which a movement disorders expert is unavailable for the physical examination. The latter situation is more important than the former, as primary care physicians are often the first point of contact for these patients. Given the waiting times to see a movement disorders specialist, patients that are considered likely to have PD (based on a screening measure) might be assigned a higher priority in their wait for an initial appointment. This assumes, of course, that primary care physicians will have access to the appropriate motor performance testing devices – and while this is currently prohibitive from a logistical standpoint, it is technologically feasible for the simple tasks described herein to be packaged in smaller (perhaps handheld), less expensive devices.A MT battery may also allow for the communication of results in a "common metric" – without relying on subjective clinical judgments, thereby complementing other clinical tools. Aside from its diagnostic utility, MT batteries may also be useful in tracking a patient's progress as he/she undergoes treatment. At present, motor evaluations conducted during the clinical exam are the only method for tracking change, and this is considerably more qualitative than the MT measures described herein. Along similar lines, MT batteries could make useful adjuncts to clinical drug trial protocols, as they provide good quantitative measures of motor skill that may be used to gauge the effectiveness of the medication under study – in the present study, MT was able to explain 34.81% of the variability in UPDRS motor scores.It should, of course, be noted that the present research was only used to separate PD patients from healthy participants, and so it has not been demonstrated to have any differential diagnostic capabilities . Future research in this area should, therefore. investigate the differential diagnostic power of response time batteries – it may be that the MT battery administered in this study are detecting a general 'impairment' factor, and is not useful as a standalone instrument for the diagnosis of Parkinson's disease. Extending the study base to include patients with disorders such as progressive supranuclear palsy would provide important information concerning appropriate norming that may be done to maximize diagnostic utility.At the very least, however, these results suggest that the use of simple response time batteries may serve as a useful adjunct to other clinical assessment batteries, and may also open interesting avenues of exploration into the consideration of the biological underpinnings of reaction time, and its relationship to movement disorders in general.None declared.AJ conceived and designed the study, collected all data in the norming sample, supervised data collection in the cross-validation sample, supervised data analysis, and contributed to the writing of the paper. PV supervised data collection in the norming sample, assisted in the development of the response time tasks, contributed to the data analysis, and to the writing of the paper. QA assisted with data collection in the cross-validation sample, and contributed to the writing of the paper. LG did all clinical testing on patients in the norming sample. RS assisted with data collection in the cross-validation sample. MJ provided patient diagnoses for participants in the norming sample and the cross-validation sample, and contributed to the writing of the paper.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here: