Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five-digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four-digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four- and five-digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink  Traditionally, most species have been described using morphology, but morphological analyses cannot easily demonstrate isolation and so genetic analyses are generally required . StatistAdditional approaches can provide other important insights into speciation. Algorithms implemented within programs such as IMa2  the evidence for genetic species within C. mionecton, (ii) whether these reflect morphological differences, and (iii) the timing of divergence and degree of gene flow between these putative species.Chalcides mionecton were captured from 16 sites extending from the north to the south of its range during field trips carried out between June 2006 and June 2008 . We used individuals that had been collected for previous research projects  and Juan Grande in Gran Canaria, and C. coeruleopunctatus from Calera and Vallehermoso in La Gomera and from Valverde and Frontera in El Hierro.Canary Island skinks belong to the same Western ionecton . Divergets e.g., ; see Brots e.g.,  for moreExternal morphology of the 192 museum specimens was recorded. The numbers of digits was recorded from both the manus and the pes. The following 12 body dimensions were measured to the nearest 0.1 mm using digital calipers: snout–vent length (SVL), head length (HL), head depth (HD), head width (HW), jaw length (JL), snout–thorax (ST), snout–hind limb length (SHL), fore–limb length (FLL), rear–limb length (RLL), fourth toe on fore–limb length (FTFL), fourth toe on hind limb length (FTHL), and body width at thorax (BWT). The following three scalation characters were also recorded: the number of ventral scales rows between the rostral scale and the cloaca, the number of scales counted around the mid-trunk, and the number of infra-digital lamellae on the fourth toe of the hind limb.  Bilateral measurements were recorded from the right-hand side.10 body dimensions against log10 SVL were used to adjust each body dimension to its value at the overall mean log10 SVL. Discriminant function analyses (DFA) were computed on these size-adjusted values, with individuals grouped by sampling locality. This method provides functions that maximize among-group relative to within-group variation, and therefore allows investigation of whether body dimensions differ between regions. DFAs were also applied to the three scalation characters.Sexes were analyzed separately. The pooled within-group coefficients from the least-squares regressions of logcytb). Two nuclear gene fragments were also amplified: (i) 371 bp of the c-mos gene and (ii) 713 bp of the RAG-1 gene. Polymerase chain reaction (PCR) products were sequenced by GATC Biotech  and Source BioScience . Wherever possible, several individuals were sequenced from each site for the NADH  and c-mos (n = 84) fragments in order to gain insights into within-site genetic diversity. One or two individuals were sequenced per site for cytb (n = 21) and RAG-1 (n = 20).DNA was extracted from tail-tips using a standard phenol–chloroform procedure. Primers were designed or used from previous studies (Appendix S2). The following mtDNA fragments were amplified: (i) 737—795 bp (depending on primers used) of NADH dehydrogenase subunits 1 and 2 and three intervening tRNAs (this fragment will be referred to as “NADH”) and (ii) 655 bp from the cytochrome b gene . Multilocus genetic structure within mos and RAG-1 sequences . We obtained the multilocus maximum likelihood values of the per locus Watterson's estimator of population mutation rate  was used to specify upper bounds on uniform prior distributions. Specifically, population sizes were specified from the uniform distribution, U, splitting time, t (in generations) was specified from U, and migration rates per mutation (m) were specified from U. The rationale for using these prior upper bounds is outlined in the IMa2 documentation (http://lifesci.rutgers.edu/∼heylab/ProgramsandData/Programs/IMa2/Using_IMa2_8_24_2011.pdf). The mutation rate for the NADH sequence (per locus per year) was calibrated using a 1.1 Ma divergence between Chalcides from the Canary islands of La Gomera and El Hierro  approach implemented in the program IMa2  was usedrate see . This isμ and is the probability of migration in a specific direction, per gene copy per generation). A likelihood ratio test (LLR) (Probabilities of differences between population sizes and migration rates were obtained by pairwise comparisons of posteriors. Population migration rates were obtained as 2NM (where M = mst (LLR)  was used0) was assigned from the gamma prior G, while the other divergence time parameters were assigned from a Dirichlet prior  to G. Not surprisingly, this led to slightly different posterior estimates of these parameters, although the posterior probability for the two species model was unaffected. Analyses were run several times to check for consistency .The program BPP  was usedcytb sequences from C. mionecton from all sites (n = 21), together with the nine Canary Island Chalcides described above, were included in the analysis. Nuclear loci showed negligible diversity across the Canary Island taxa and so we used only the mitochondrial locus for the phylogenetic analyses  was used for robust estimation of divergence times between genetic clusters identified by BAPS . The metC. mionecton, (ii) Southern C. mionecton, (iii) C. sexlineatus (Gran Canaria), (iv) C. viridanus (Tenerife), (v) C. coeruleopunctatus (La Gomera), (vi) C. coeruleopunctatus (El Hierro). Time priors were placed on the species tree, with 1 time unit = 10 Ma. The general prior on divergence times was specified using a Birth–Death model, with the birth–death rate specified from the uniform distribution U and death rate relative to birth rate specified from U. The times of the most recent common ancestors (tMRCAs) of different species groups were constrained using geological information. The oldest emerged parts of the Canary island of El Hierro have been dated at 1.12 Ma with the island likely to have been colonized soon after its subaerial emergence  node. Two other nodes were also constrained albeit more loosely. The age of the root was specified from the gamma distribution G, and the ancestor of all Canary Islands sequences was specified from G, following previously published estimates (Species were defined as follows: (i) Northern genetic cluster of mergence . The gamstimates .Chalcides, and on each of the two forms of C. mionecton  distribution, and the standard deviation from the exponential distribution Exp(0.33), for all partitions. The HKY+G model was specified with kappa parameters assigned from G and alpha parameters from U, for all partitions. Monophyly constraints were imposed on sequences from the same island, on La Gomera and El Hierro together, on all Canary Island cton see .7 generations with a sampling interval of 1000, and the first 3000 samples were discarded as burn-in. Convergence diagnostics as well as posteriors on the gene and species trees were obtained using Tracer (v1.4.1) (MCMC chains were run for 3 × 10(v1.4.1)  and Tree(v1.4.1) .To date, it has been common to use Bayesian phylogenetic methods to date sequence divergence. In these analyses, time calibrations are applied to sequences and sequence divergence times are estimated. We also used this approach  for comparison with the *BEAST phylogenetic–coalescent analysis described above. The priors within the analysis were the same as for the *BEAST analysis, but applied to tMRCAs of the sequences within the relevant species.All individuals obtained from sites south of Cap Rhir (sites 11–16) were found to have five digits on the manus and the pes, except for one individual at site 16 which possessed four digits on the pes only . IndividFor female body dimensions, the first discriminant function (DF1) represented 35.4% of the total variation among sites, while the second function (DF2) represented 28.1%. Values were similar for males: DF1 = 36.2%, DF2 = 24.6%. Patterns of pooled within-group correlations between body dimensions and standardized DFs were quite similar between sexes . Howeverr = 0.76, P = 0.003) and female DF1  were significantly associated with digit number, but correlations were not significant for male DF1  or female DF2 . DFAs on males and female scalation characters revealed no differences between northern and southern forms (results are not shown for conciseness).The northern populations with one or more five-digit individuals were ostensibly intermediate between the remaining four-digit northern populations and the five-digit southern group . There wC. mionecton from different regions .For mtDNA, substantial numbers of mutational steps separate  regions A. The cybed here B and C. k = 5 genetic clusters by BAPS provided the highest log likelihood (lnL = −1507.5). Higher values of k produced decreasing log likelihoods . However, all analyses assigned all southern individuals to a single cluster, that is, an increase/decrease in the value of k simply increased/decreased the number of northern clusters  (k = 2. This produced a lower likelihood (lnL = −1914.1) but the two genetic clusters represented northern individuals from sites 1–10, and southern individuals from sites 11–16  . This ises 11–16 .P > 0.05). Population migration rates from the north to the south were also very low (P < 0.001). Marginal posteriors on current population size parameters (θ) were represented by single peaks. For the southern form, θS was small: posterior mean = 0.401, 95% HPD (0.125–0.731), compared with θN (northern form): posterior mean = 1.007, 95% HPD (0.390–1.730). The difference between θS and θN was significant (Pr [θ N> θ S] = 0.997). Various exploratory analyses with different priors indicated that the data were relatively uninformative about the ancestral population size parameter, θA, or the population splitting time.To facilitate interpretation, we will report on migration rates in the natural (forward) direction of time rather than coalescent time. Population migration rates from the south to the north are negligible A, with a, 0.158])A, but he0 were 0.0064 .The posterior probability of the north–south species delimitation was 1.00. As for IMa2, the posterior on Θ for the northern form indicated a larger population size , compared with the southern form . Note that BPP and IMa2 are parameterized differently and so parameters are not directly comparable between programs. The posterior mean and 95% HPD for τC. mionecton  species node (P = 0.98). The corresponding posterior mean speciation time was 1.43 Ma , which was approximately 1 Ma older than the posterior north–south speciation time within C. mionecton (see above). However, this does not represent a north–south split between sequences. The ancestral node that is common to the two mtDNA lineages, comprising sequences from northern (7–10) and southern sites (11–16), is quite well supported (P = 0.94). It has a posterior mean and posterior interval of 1.58 and  Ma, respectively, which is quite similar to that obtained under the phylogenetic–coalescent analysis.The *BEAST coalescent analysis provided strong posterior support for the –2.66 Ma)B. ComparSpecies delimitation can require a combination of genetic, morphological, and biogeographical information in order to be convincing. Here, coalescent analyses of genetic data alone demonstrate negligible gene flow between northern and southern groups of contiguously distributed populations, indicating that they form separate metapopulation lineages  is dated at 1.6 Ma which is not too different from the phylogenetic–coalescent date for divergence of northern and southern forms. While this highlights important differences between the dating approaches, detailed future comparisons between methods would be more informative in cases where it is appropriate to use a single topology for each analysis, rather than integrating over the range of possible topologies.Until recently, many phylogeography studies have used phylogenetic dating of sequence divergence times . We showic study  by an evC. mionecton, we find that digit number varies in a quite complex manner with five digits being more common throughout the far north of the range than previously thought  One hypothesis is that the four-digit morphology arose due to mutations that appeared in the northern form after N–S speciation. Gene flow between northern populations allowed these mutations to spread north, while restricted gene flow between species impeded southern movement. This scenario suggests that these morphological differences originated recently. However, it does not explain why the distribution of the four-digit morphology is interrupted by a northern area in which only five-digit individuals were found. It has been proposed that digit loss is evolutionary labile, with complete loss of digits over approximately 3.5 Ma in some Australian skink lineages  gene.It is interesting to speculate why some individuals from the most northerly populations should show morphological similarity with the most southern populations, despite delimitation of northern and southern individuals into discrete genetic clusters. The most parsimonious explanation is that the ancestral state in this species is five digits. Other morphological states found in the extreme north and south, such as a wider thorax, would also be considered ancestral but for clarity we focus our argument on digit number. The sister group to e digits  which woe digits  interestlineages . Rapid es remain . This seC. mionecton. However, it does provide information on current distributions and divergence times. There was no evidence of unsuitable habitat between the southernmost four digit sample from Cap Rhir and the adjacent five-digit sample from Taghazoute (separated by a distance of around 25 km), so it is probable that the two forms come into contact without significant hybridization. C. mionecton populations, would appear plausible.This paper does not aim to reconstruct the historical biogeography of C. mionecton therefore appear to represent good species under the unified species concept, but these species are not diagnosed by digit number or other external morphological characteristics.To summarize, we are able to examine key features of the speciation process, namely timing of divergence and gene flow between lineages, using only a small number of loci, two of which have low substitution rates. Bayesian methods provide strong statistical support for divergence of two lineages in the Pleistocene after which very few genes were exchanged. Previously described subspecies of


Background: We previously reported associations between organochlorines and behaviors related to attention deficit hyperactivity disorder among boys and girls at 8 years of age using a teacher’s rating scale for a birth cohort in New Bedford, Massachusetts (USA).Objectives: Our goal was to corroborate these findings using neuropsychological measures of inattentive and impulsive behaviors.p,p´-dichlorodiphenyl dichloroethylene  and attention and impulse control using a Continuous Performance Test (CPT) and components of the Wechsler Intelligence Scale for Children, 3rd edition (WISC-III). Participants came from a prospective cohort of children born during 1993–1998 to mothers residing near a PCB-contaminated harbor in New Bedford. Median (range) cord serum levels for the sum of four prevalent PCBs  and p,p´-DDE were 0.19 (0.01–2.59) and 0.31 (0–14.93) ng/g serum, respectively.Methods: We investigated the association between cord serum polychlorinated biphenyls (PCBs) and 4 had a higher rate of CPT errors of omission  and slower WISC-III Processing Speed . Weaker associations were found for p,p´-DDE. For girls, associations were in the opposite direction for the CPT and null for the WISC-III.Results: We detected associations between PCBs and neuropsychological deficits for 578 and 584 children with CPT and WISC-III measures, respectively, but only among boys. For example, boys with higher exposure to ΣPCBConclusions: These results support an association between organochlorines (mainly PCBs) and neuropsychological measures of attention among boys only. Sex-specific effects should be considered in studies of organochlorines and neurodevelopment. These lountries .Prenatal exposure to organochlorines, and to PCBs in particular, has been linked with behavioral impairments and functional deficits common to attention deficit/hyperactivity disorder (ADHD) , the mosp,p´-DDE levels and teacher-reported behaviors consistent with ADHD using a behavioral checklist  among 8-year-old children born to mothers residing adjacent to a PCB-contaminated harbor  near a PCB-contaminated harbor in New Bedford, Massachusetts, for at least the duration of pregnancy were recruited from a local hospital with approximately 2,000 births per year. Approximately 10% of mothers met study eligibility criteria and were available for recruitment during times when a study examiner was on site. Infants too ill to undergo neonatal examination or born by cesarean section were excluded from the study. Of the 788 mother–infant pairs enrolled at birth, 607 were followed up for neurodevelopmental testing when the child was approximately 8 years of age (78% of those eligible). Multiple births (n = 3 children) were excluded from the present analysis.Exposure assessment. Cord blood samples for organochlorine analyses were collected at the infant’s birth; the serum fraction was removed after centrifugation and stored at –20°C. All sample analyses were performed by the Harvard School of Public Health Organic Chemistry Laboratory . Laboratory personnel were blinded to health outcomes. Cord serum samples were analyzed for 51 individual PCB congeners and p,p´-DDE. Laboratory analytic methods and quality control procedures are described elsewhere  and was therefore measured for 12 randomly selected cord bloods from discarded, anonymous samples collected at the study recruitment site; values were reproducible (1.7 g/L ± 0.3).p,p´-DDE in serum was 0.07 ng/g (4)] was 3% and the between-batch CV was 20% over the 5 years of analysis, with similar performance for p,p´-DDE.The method detection limits (MDLs) for individual PCBs ranged from 0.002 to 0.04 ng/g serum, with most MDLs < 0.01 ng/g; the MDL for .07 ng/g . The fre.07 ng/g . We reta.07 ng/g . ReproduOutcome assessment. We analyzed components of a continuous performance test (CPT) and a psychometric test of intelligence that reflect attention and impulse control; both tests were administered at the 8-year exam. The Neurobehavioral Examination System 2 (NES2) CPT is a computer-assisted examination that measures response to visual cues in a continuous performance task (a) mean response time, b) response time variability (standard deviation of mean response time), c) total errors of omission, and d) total errors of commission. Outcomes were summed across the last three test blocks. Inattention was interpreted as a higher number of errors of omission and longer reaction time, and poor response inhibition was interpreted as a higher number of errors of commission. Higher reaction time variability or performance inconsistency is also thought to indicate fluctuations or lapses in attention , is a test that evaluates intellectual abilities . We focuStatistical analysis. We analyzed associations between attention and impulse control and two PCB congener groups: a) ΣPCB4, the sum of four prevalent PCB congeners with relatively high levels that were measured with less error, and b) the computed toxic equivalent (TEQ) for the sum of the five dioxin-like mono-ortho PCB congeners measured , computed on a lipid basis (1.7 g/L) and weighted with toxic equivalency factors . CPT errors of omission and commission were considered count data and initially modeled as Poisson distributed variables with log risk models; to correct for overdispersion (variance exceeded the mean), our final models were fitted using negative binomial regression.Covariate data came from multiple sources, including maternal and pediatric medical records (including lead screening) and questionnaires administered 2 weeks after birth and at the 8-year follow-up examiantion. The 8-year exam included an assessment of maternal intelligence and depression using the Kaufman Brief Intelligence Test  and the a priori considerations of covariate associations with exposure and outcome, model fit , and whether covariate inclusion materially affected the organochlorine exposure effect estimate. To check the sensitivity of our estimates to covariates that were not included in the model, we added each back to the final model to make sure they did not substantially change our final effect estimates. We also assessed differences in exposure–outcome associations across sex using stratified analyses and by including an interaction term between sex and exposure in the model.Inclusion of covariates in multivariable models was based on a) orientation, learning, and habituation (first 2 min), b) processing speed and selective focused attention (next 4 min), and c) sustained attention (last 4 min). Because our children were younger than those in the Based on recent literature suggesting that performance on the CPT may vary over the course of the test session   with unadjusted exposure–outcome associations in the subset of children with nonmissing covariate data.The study protocol was reviewed and approved by the Human Subjects Committees of Harvard School of Public Health and Brigham and Women’s Hospital  and Southcoast Hospitals Group . Written informed consent was obtained from all participating families before study evaluations.4 > 4 ng/g) that disproportionately influenced exposure–outcome associations was excluded from all analyses. Approximately half of the total PCB concentration  was attributable to ΣPCB4 (mean ± SD = 0.25 ± 0.26 ng/g serum). PCBs and p,p´-DDE were positively correlated, with Spearman correlation coefficients ranging from 0.56  to 0.88 (mono-ortho PCB TEQ and ΣPCB4).Exposure and outcome summary statistics are displayed in http://dx.doi.org/10.1289/ehp.1104372), presents the distribution of background characteristics and their unadjusted associations with the CPT and WISC-III outcomes. Briefly, 11% and 24% of mothers and fathers, respectively, did not complete high school; 20% had an annual household income < $20,000/year, and 41% were unmarried at the child’s 8-year examination, representing a socioeconomically diverse population. In addition, 31% of children were of nonwhite race/ethnicity, with 11% of Cape Verdean ethnicity, which is representative of the New Bedford population.Supplemental Material, 4, mono-ortho PCB TEQ, and p,p´-DDE, for all children  were very weak or null  increase in ΣPCB4: β = –5.8; 95% confidence interval (CI): –10.5, –1.0], the opposite association was observed for males, with longer mean reaction time with increasing exposure ; the ΣPCB4 × sex interaction was statistically significant using a likelihood ratio test (p = 0.02). Reaction time variability also differed by sex with lower variability among females and higher variability in males with increasing exposure to ΣPCB4, mono-ortho PCB TEQ, and p,p´-DDE. PCB-associated omission error rate was also higher among males than among females, with an 11% increase in the rate of an error of omission per IQR increase in ΣPCB4 among males and no association among females . This pattern is illustrated in 4 levels among males and the opposite association was observed for females. Errors of commission were not associated with organochlorines in males or females , whereas associations for females were null , as illustrated in p,p´-DDE–WISC-III associations were null.Sex-specific effects were also detected between organochlorines and WISC-III Processing Speed . Males hn = 45) did not change exposure–outcome associations. In addition, lead was not a confounder of the association between organochlorines and CPT or WISC-III measures reported here, which is consistent with the absence of an association between lead and organochlorines in our study . Finally, the unadjusted exposure effects were essentially unchanged when restricted to children with nonmissing covariate data , suggesting that excluding data from subjects with missing covariates data did not bias our estimates.Sensitivity analysis (data not shown) excluding children with a history of ADHD medication use  primarily for males. These findings were consistent with a previous study of methylmercury and NES2 CPT reaction time , which aOur study also found associations between PCBs and Processing Speed, a subscale of the WISC-III, among males only. Although associations between PCBs and Processing Speed have not been previously reported, associations were found with Freedom from Distractibility, another WISC-III subscale, in the Michigan study , with suSex differences in estimated PCB effects were not found for CPT outcomes in the Oswego study  and wereSexual dimorphism of effect has also been observed in animal studies in relation to PCB exposure, but results are inconsistent . Althouga) minimization of bias due to outcome misclassification, where categorization is often made at an arbitrary cutoff point that may change over time; b) detection of early or milder manifestations of a disorder that a clinical diagnosis could miss; and c) enhancement of power to detect an effect of an exposure ; this data point disproportionately influenced the association between ΣPCB4 and errors of omission. After omitting data for this child, the association between ΣPCB4 and errors of omission was null for males and females combined, although it did persist for males .A more conservative analytic approach may contribute to differences in our findings compared with other studies , 2005. FIn summary, our results support an association between organochlorines, particularly PCBs, and neuropsychological measures of inattention. Our observation of an association among boys only has not been consistently explored in previous studies and warrants further exploration of sex-specific effects of organochlorines on these outcomes. These findings contribute to a growing literature showing associations between PCBs and ADHD-related behavior.(135 KB) PDFClick here for additional data file.


Acetylation or deacetylation of histone proteins may modulate cytokine gene transcription such as TNF alpha (TNF). We evaluated the balance between histone deacetytlase (HDAC) and histone acetyltransferase (HAT) in patients with rheumatoid arthritis (RA) or ankylosing spondylitis (AS) compared to healthy controls (HC) and determined the influence of HDAC inhibitors (trichostatin A -TSA- or Sirtinol -Sirt-) on these enzymatic activities and on the PBMC production of TNF.ex vivo treatment of PBMC by TSA or Sirt. TNF levels were evaluated in PBMC culture supernatants in the absence or presence of TSA or Sirt.52 patients with RA, 21 with AS and 38 HC were evaluated. HAT and HDAC activities were measured on nuclear extracts from PBMC using colorimetric assays. Enzymatic activities were determined prior to and after Ex vivo treatment of PBMC by HDACi tended to decrease HDAC expression in HC, but Sirt significantly reduced HAT in RA. TNF production by PBMC was significantly down-regulated by Sirt in HC and AS patients.HAT and HDAC activities were significantly reduced in AS, while these activities reached similar levels in RA and HC. HAT and HDAC were disturbed in AS while no major changes were found in RA. HDACi may modulate HDAC and HAT PBMC expression, especially Sirt in RA. Sirtinol was able to down regulate TNF production by PBMC in HC and AS. An imbalance between HAT and HDAC activities might provide the rationale for the development of HDACi in the therapeutic approach to inflammatory rheumatic diseases. Active pro-inflammatory cytokines such as TNF alpha (TNF), IL-1beta or IL-6 are key players in the synovial inflammation of RA, and TNF is also considered to be a major cytokine driving inflammation in AS. Indeed, all these pro-inflammatory cytokines participate in a cascade of events, leading to synovial/sacroiliac cellular infiltration that causes pain, joint damage and progressive disability. Various factors may regulate cytokine production such as polymorphisms in the promoter region of cytokine gene, cytokine-cell interactions and therapeutic interventions. Accumulating evidence suggests that epigenetic modifications can modulate gene transcription and thus could regulate the production of pro-inflammatory cytokines ,3. These+ .Histone deacetylase enzymes target histone proteins but also non-histone proteins. Indeed, HDAC activities can interfere with inflammatory signalling pathways, transcription factors and tumor suppressor genes, regulating cellular activation, proliferation and differentiation. Thus, HDAC inhibitors (HDACi) have been developed in cancer therapy in an attempt to inhibit cell proliferation and to induce cell cycle arrest or apoptosis . HoweverHDAC/HAT activity in patients with RA and other forms of arthritis including AS. In this study therefore, we aimed to evaluate the HDAC / HAT ratio in the peripheral blood of patients with RA or AS and to analyze the influence of HDACi on the production of TNF by peripheral blood mononuclear cells (PBMCs).The equilibrium between HDAC and HAT activities could thus be considered as a relevant marker for gene regulation, including those coding for pro-inflammatory cytokines such as TNF. There are limited results on the ratio of We enrolled 52 patients with RA meeting the 1987 American College of Rheumatology criteria and 21 patients meeting the modified New York criteria for AS. They were all receiving follow-up at the department of Rheumatology in the University Hospital of Besançon, France. In RA, disease activity and functional impairment were evaluated using the DAS28 score and Health Assessment Questionnaire (HAQ), respectively. Low disease activity was defined by a DAS28 < 3.2, moderate disease activity by a DAS28 ≥ 3.2 and ≤ 5.1 and high disease activity by a DAS28 > 5.1. For AS, clinical activity was evaluated using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Ankylosing Spondylitis Disease Activity Score (ASDAS-CRP score) and the Bath Ankylosing Spondylitis Functional Index (BASFI). Erythrocyte sedimentation rate (ESR), C reactive protein (CRP) levels and circulating TNF were used as laboratory parameters to assess inflammation. Positivity for rheumatoid factors, anti-CPP antibodies and HLA-B27 status were also recorded. In the RA group, patients were receiving low dosage corticosteroids (< 10 mg prednisone daily) and/or traditional DMARDs . In the AS group, all patients received non-steroidal anti-inflammatory drugs (NSAIDs) and some of them had sulfasalazine or methotrexate. At the time of inclusion, no RA or AS patients were receiving TNF blocking agents or other biologics.The control group consisted of 38 healthy subjects without inflammatory conditions or treatment. All the patients and healthy controls (HC) gave their written informed consent to participate in the study according to the Helsinki declaration. The study protocol was approved by the local ethics committee .2, 1 mM EDTA). Cell pellets were then incubated on ice with cytoplasmic isolation buffer . Cytoplasmic extracts were collected by centrifugation and the nuclear pellets were washed twice in wash buffer, spun, and incubated for 15 min on ice with nuclear isolation buffer . Supernatants containing nuclear extracts were collected by centrifugation and stored at -80° C. Protease inhibitors  were added to all solutions. Protein concentration in nuclear and cytoplasmic extracts was determined by the Bradford method using a BioPhotometer .PBMCs were prepared from the peripheral blood of patients and HC and were cultured in RPMI-1640 medium supplemented with 10% (v/v) pooled AB human serum (Sigma), as previously described . IsolatiHAT/HDAC activities in PBMC nuclear extracts. Global HDAC activity was evaluated. These activities were measured prior to and after ex vivo exposure of PBMCs to the following HDACi: trichostatin A (TSA), a Class I and Class II inhibitor (1 microM), and sirtinol (Sirt), a sirtuin inhibitor (10 microM). The use of concentrations of TSA up to 1 microM has been previously reported in studying the effect of HDACi on macrophages and monocytoid cells [The EpiQuik™ HDAC Activity Assay colorimetric kit  and the HAT activity assay kit  were used to evaluate nhibitor  microM. id cells .Circulating TNF was evaluated in sera from patients and HC using an ELISA assay . PBMCs from HC and from patients with RA or AS were treated or not with TSA (1 microM) or Sirt (10 microM), and TNF production in supernatants was measured by ELISA after one and three days of culture.To assess the epigenetic regulation of the huTNF promoter, U937 cells (ATCC) were transiently transfected with 20 microg of a plasmid expressing the luciferase gene under the control of the huTNF promoter, pTNF-Luc   using el6/ml) were remained untreated or treated with TSA (1 microM) and Sirtinol (10 microM) for 24 or 72 h. The cells were washed in cold phosphate-buffered saline (1X PBS), supernatant was discarded and 106 PBMCs were resuspended in 1 ml 1X annexin binding buffer. 5 microl of FITC labeled annexin V  was incubated in 100 microl of resuspended PBMCs. After 15 min incubation at room temperature in the dark, 400 microl of 1X annexin binding buffer was added and 10,000 cells were acquired by FACSCalibur with CELLQuest software  [PBMCs  .6/well in triplicates in 96-well plates in a final volume of 100 microl medium containing TSA (1 microM) or Sirtinol (10 microM). Cell culture medium without cells was used as a blank. After 24 hrs or 72 hrs, 10 microl MTT reagent was added to each well and the plates were incubated for another 4 h in the cell culture incubator at 37° C. The plate was centrifuged at 500 g for 10 min. The cell culture media was aspirated and 100 microl crystal dissolving solution was added to each well and the absorbance was measured at 570 nm using Multiskan Ex .Cell viability was measured using the MTT assay kit . PBMCs were seeded at a density of 0.1X10HDAC/HAT ratio between RA and HC and between AS and HC. The Wilcoxon test was used for the paired comparison of spontaneous HAT or HDAC activity and enzymatic activity under HDACi exposure, and for comparing TNF production in PBMC culture with and without HDACi. Qualitative data (gender) were analyzed using the Chi-squared test. A Spearman’s r-test was used to calculate correlations between HAT and HDAC activities and indices of AS or RA disease activity. Values of p less than 0.05 were considered significant.Results were expressed as mean ± standard error of the mean (SEM). Non-parametric tests were used. Statistical analysis between the three groups  involved non-parametric analysis of variance (ANOVA) using the Kruskal-Wallis test. This test was used to compare age, ESR, CRP and circulating TNF. The Mann Whitney test was used for comparing HAT activity (with and without HDACi), HDAC activity (with and without HDACi) and Patients with RA had mild disease activity (DAS28 > 3.2) while disease was more active in AS patients (BASDAI > 4 and ASDAS > 2.1). Most of the laboratory parameters of inflammation investigated  were elevated in RA and AS compared to HC .vs 111.3 ± 15.5 ng/h/mg; p = 0.05) while it was higher in RA, but the difference was not significant   . ESR and CRP did not correlate with HAT or HDAC levels. However, TNF weakly correlated with HDAC activity in AS patients (p = 0.02). Interestingly, in RA patients with high disease activity and compared to patients with low disease activity, we observed a decreased HDAC activity  .HAT activity was lower in AS compared to HC (68.2 ± 8.1  > 0.05) . For HDA > 0.05) . When ex = 0.01) , an enhaex vivo treatment of PBMCs by HDAC inhibitors  .Results were given as spontaneous activities and after ex vivo addition of TSA or Sirt on PBMCs did not induce a change in HAT activity . Conversely, there was a reduction in HDAC levels that was more evident with Sirt (51.1% reduction), but the results did not reach significance (p = 0.07). In the RA group, TSA induced a non-significant increase in HAT, while there was a modest but significant decrease in this activity under Sirt (p = 0.01). In addition, a 37.7% reduction in HDAC was observed with TSA or Sirt without significance . In PBMCs from AS patients, neither HAT nor HDAC activities were influenced by HDACi exposure   .We then evaluated the influence of HDACi on TNF production by PBMC culture. In HC, TSA reduced TNF release but without significance at day one or three . Conversely, Sirt was more effective in reducing TNF production after one (p = 0.03) and three days (p = 0.002). In RA, these effects were less evident, with an increase in TNF at day one with TSA (p = 0.02) and a slight but non-significant decrease in TNF release at day three. Sirt had no effects on TNF production in RA. In AS, only Sirt induced a decline in TNF production at day one (p = 0.017) and three (p = 0.07) while TSA tended to induce TNF production at day one . In paraDetectionofapoptosisandcellviabilityinPBMCsfromhealthycontrols: Apoptosis was then assessed in HC PBMCs treated with TSA or Sirt using annexin-V assay. TSA and to a much lesser extent Sirt triggered apoptosis in HC PBMCs, mostly in peripheral blood lymphocytes (PBLs), after 1 and 3 days of treatment  but this may be explained by the parallel decrease in both HAT and HDAC activities. By contrast, RA patients were characterized by a non-significant increase in HAT activity and a parallel decrease in HDAC, resulting in a reduced HDAC/HAT ratio. Interestingly, when we studied sub-populations of RA patients taken into account their DAS 28 score, we observed that RA patients with high disease activity displayed significant decreased HDAC activity, with a parallel but non significant enhanced HAT activity and decreased HDAC/HAT activity ratio. Limited data are available on the levels of HAT or HDAC activities in PBMC extracts from patients with RA. Gillespie et al. evaluated HAT and HDAC activities in PBMCs in a limited series of patients with RA (N = 8) and found increased HDAC activity but no changes in the levels of HAT compared to HC [versus synovial fibroblasts). Low HDAC activity in RA synovial tissue was interpreted as a factor of hyperacetylation that can contribute to the activation of gene coding for pro-inflammatory cytokines and thus to the pathogenesis of RA [HAT/HDAC [Few studies have evaluated the equilibrium between HAT and HDAC activities in RA and there is no available data in AS ,6,14. Thed to HC . Besidesed to HC  or increed to HC ,18 in syis of RA . Alternais of RA ,17,18. Iis of RA . For insHAT/HDAC . In AS, ex vivo influence of HDACi on PBMC HAT/HDAC expression. Our results showed a decline, albeit not significant, in HDAC activity with TSA and Sirt in HC, while in RA, Sirt was able to reduce HAT. HDAC activity was also regulated in RA by TSA and Sirt, without reaching significance. By contrast, AS patients were not sensitive to TSA or Sirt. Trichostatin A is a pan-HDACi, inhibiting both Class I and Class II HDAC. We did not use specific HDACi in our study. HDAC1 and HDAC2 have been identified as the main HDAC Class I isoenzymes expressed within the synovium of patients with RA [HAT/HDAC in RA. Also, it would be more effective to target these specific HDAC activities by selective inhibitors in specific cell types, such as inflammatory macrophages.In our study we also evaluated the  with RA ,18. The  with RA . Additio with RA , making  with RA . The ree with RA . Thus, fE. Coli and S. Aureus [in vitro activation of immune cells might have given a more pronounced effect of HDACi on TNF production.It has previously been shown that HDACi may regulate cytokine production ,7,22–24.. Aureus ,31. Inhi. Aureus . TherefoIn contrast to our data, Sirt has been reported to increase TNF release in both Kupffer cells and macrophages . In our We observed in both AS and RA that TSA, but not Sirt, tended to induce TNF production at day one. These results are consistent with a preferential activation of the human TNF promoter by TSA in transfected U937 cells, indicating that TNF gene expression is repressed primarily by Class I and/or Class II HDACs. In fact, it has been reported that TNF gene expression is controlled by HDAC3, a Class I HDAC . Again, Ex vivo exposure of PBMCs to HDACi could modulate these activities, mainly in HC, but also in RA. TNF production could be regulated by HDACi treatment, especially with Sirt in the AS group. The mechanisms explaining the anti-inflammatory effects of these HDACi are complex and certainly not limited to histone modifications, and a better understanding of the regulation of HDAC and HAT activities in AS and RA could lead to new therapeutic approaches in the future.In conclusion, we observed a disturbed balance of HDAC and HAT activity in our patients with AS and, to a lesser degree, in RA.


More than 1.2 million new cases of colorectal cancer are reported each year worldwide. Despite actual screening programs, about 50% of the patients are diagnosed at advanced tumor stages presenting poor prognosis. Innovative screening tools could aid the detection at early stages and allow curative treatment interventions.A nine target multiplex serum protein biochip was generated and evaluated using a training- and validation-set of 317 highly standardized, liquid nitrogen preserved serum samples comprising controls, adenomas, and colon cancers.Serum levels of CEA, IL-8, VEGF, S100A11, MCSF, C3adesArg, CD26, and CRP showed significant differences between cases and controls. The largest areas under the receiver operating characteristics curve were observed for CEA, IL-8, and CRP. At threshold levels yielding 90% specificity, sensitivities for CEA, IL-8 and CRP were 26%, 22%, and 17%, respectively. The most promising marker combinations were CEA + IL-8 reaching 37% sensitivity at 83% specificity and CEA + CRP with 35% sensitivity at 81% specificity. In an independent validation set CEA + IL-8 reached 47% sensitivity at 86% specificity while CEA + CRP obtained 39% sensitivity at 86% specificity. Early carcinomas were detected with 33% sensitivity for CEA + IL-8 and 28% for CEA + CRP.Apart from CEA, IL-8, and CRP, the screening value of additional blood markers and the potential advantage of combining serum biochip testing with fecal occult blood testing needs to be studied. Multiplex biochip array technology utilizing serum samples offers an innovative approach to colorectal cancer screening. Detec-CSF) , Nicotin-CSF) , or C-re-CSF)  reached -CSF) , Calgizz-CSF)  and comp-CSF)  serum le-CSF) . HoweverBiochip array technology allows multiplex determination of multiple biomarkers from a single sample ,17. ThisThe aim of this study was to apply biochip array technology to colon cancer screening. For this purpose, a biochip array was designed and developed for the multiplex determination of nine serum markers allowing for low inter-analysis variability, decreased workload and faster processing time as well as lower costs due to high-throughput automation. The performance of the two biochip arrays for colon cancer screening was then evaluated in a training and a validation set consisting of 317 highly standardized, liquid nitrogen preserved serum samples.ColonBiomics being an integral part of the Surgical Center for Translational Oncology – Lübeck (SCTO-L), University of Lübeck, and the DKH e.V. funded network North German Tumorbank of Colorectal Cancer . Serum samples were collected adhering to the guidelines of the local ethical review board  and according to strict standard operation procedures. Serum samples of healthy control patients as well as cancer patients were both taken after bowl-preparation and prior to colonoscopy or oncologic resection. The 317 samples comprised 164 patients with histological confirmed colon cancer (96 men and 68 women), 34 patients with colon adenomas (18 men and 16 women), and 119 healthy controls (52 men and 67 women)  and is described in detail in Additional file All venous blood samples were obtained using serum gel-monovettes  and centrifuged at 1,550× g for 10 min at 4°C to separate the serum. Aliquots of serum samples were stored at −196°C within 30 min after venous puncture. Samples were thawed on ice before multiplex assessment on the newly designed biochips.Both chips were manufactured according to standards described ,17. TargOnce the biochip prototypes were manufactured, a pilot study of 400 serum samples . Due to different assay- and detection ranges of some biomarkers, the nine chosen serum markers had to be subdivided onto two biochip platforms: One biochip (CRCSI) comprised C3desArg, CD26 and CRP, and the other biochip (CRCSII) incorporated IL-8, CEA, VEGF, M-CSF, S100A11 and NNMT. Both biochips were manufactured according to standards described [Two biochip platforms were designed for the simultaneous quantitative detection of the nine-biomarkers biochip array in combination with the escribed , and assEvidence Investigator analyser. The system allows handling of up to 54 samples (6 x 9 wells) in biochip carriers .The analyser routinely assesses the quality of assay performance and generates calibration curves as described by FitzGerald et al.. The anaIBM Corporation, Somer, NY) and SAS version 9.1 , the serum levels of the individual markers in colon cancer cases, colon adenoma cases and healthy controls were described with respect to median levels, interquartile ranges and overall ranges. Nonparametric tests to compare median serum levels between different patient groups were applied. The correlation between age and sex and the serum levels of the markers as well as correlations between the single markers was assessed using Spearman’s correlation coefficient. Furthermore, receiver-operating characteristics (ROC) curves were constructed and the area under the ROC curves (AUC) with 95% confidence intervals (CI) for each single marker was calculated in relation to the discrimination between colon cancer cases and controls. We also calculated the AUC for the combination of all markers using logistic regression analysis and compared this AUC to the AUC of the single markers.Using SPSS Statistics version 19 , we selected markers showing the best discriminative power. The training set was then used to determine a threshold level (yielding a specificity of 90%) for each of these selected markers. The sensitivity of the marker was then calculated at this threshold level followed by an assessment of which marker combination could have a beneficial affect regarding test performance. Concerning the combination, individuals with at least one marker above the individual marker threshold were classified as positive. Marker combinations were considered as beneficial if they increased sensitivity compared to the single markers without substantially lowering the specificity. This approach reaches for a high specificity in order to keep the number of false-positive tests limited.Finally, this algorithm regarding threshold levels and promising marker combinations that were developed in the training set was applied to an independent validation set including early stage carcinomas and adenomas.Here we report a comprehensive evaluation of serum proteins in an effort to validate screening biomarkers for colon tumors. Two multiplex biochips (CRCSI and CRCSII) were manufactured consisting of altogether nine serum markers and evaluated their performance for detecting colon cancer in a minimally-invasive, patient-friendly and reliable fashion. Biochips and targets were simultaneously evaluated in a highly standardized serum collection of cancer and control samples stored at −196°C within 30 min after phlebotomy following strict standard operation procedures.The distribution of serum levels of all nine markers is described in Table In a first step, we assessed whether the combination of all markers indicates an advantage in terms of increasing the overall AUC compared to AUC of the single markers. The AUC of all markers combined as determined by logistic regression was 0.75, which was not statistically significantly different from the largest AUC among the single markers . While logistic regression modelling focuses on the overall AUC, we focused - in a next step – on the part of the AUC that is most relevant for the screening setting. We assessed whether there was a combination of markers that yields an increased sensitivity when focusing on levels of specificity that are typically required in a screening setting (≥ 90%). For that purpose, we selected CEA, IL-8 and CRP whose sensitivities were significantly different from the false-positive rate at these levels of specificities and developed a test algorithm for their combination as described below.Sera from 52 healthy controls and 81 patients with colon cancer were used to develop a test algorithm. We determined threshold levels yielding a specificity of 90% for CEA, IL-8, and CRP. For CEA the threshold was at 3.2 ng/mL, for IL-8 at 39.5 pg/mL, and for CRP at 14,600 ng/mL. Based on these threshold levels (yielding a specificity of 90%), CEA showed a sensitivity of 26% (95%CI: 17-37%), IL-8 of 22% (95%CI: 14-33%), and CRP of 17%  at a specificity of 83% (95%CI: 70-92%) and CEA + CRP obtained a sensitivity of 35% (95%CI: 24-46%) at a specificity of 81% (95%CI: 67-90%). The combination of IL-8 + CRP yielded both a lower sensitivity and a lower specificity compared to CEA + IL-8 and the combination of all three markers decreased the specificity to 73%, but increased the sensitivity by only 4% compared to CEA + IL-8.To exclude fortuitous separation of the colon carcinoma cases from healthy controls in the training set, the test algorithms described above were then applied to the validation set of 133 serum samples. The marker combination of CEA + IL-8 reached a sensitivity of 47% (95%CI: 36-58%) at a specificity of 86% (95%CI: 73-94%), while CEA + CRP obtained a sensitivity of 39% (95%CI: 28-50%) at a specificity of 86% (95%CI: 73-94%). Thus, the diagnostic performance for marker combinations of CEA, IL-8 and CRP proved to be slightly better in the validation set. In comparison, the performance of CEA alone would have reached a sensitivity of only 37% (95%CI: 29-44%) at the same level of specificity at 86%.The marker combinations of CEA + IL-8 and CEA + CRP each falsely determined seven of 52 healthy controls to be malignant. Out of these seven samples, four were falsely identified by both marker combinations. For colon carcinomas, the combination CEA + IL-8 correctly recognized 39 and CEA + CRP 32 of 81 patients correctly as malignant. A total of 31 cases overlapped between both combinations. Combining CEA + IL-8 with CEA + CRP analysis showed a minimal increased sensitivity of 48% as compared to 47% for CEA + IL-8 alone, while specificity decreased from 86% to 80%. Thus, combining both marker combinations did not present a relevant advantage regarding test performance  and late stage colon carcinomas , the median serum level for CEA was 8.00 ng/mL in early stage and 47.97 ng/mL in late stage carcinomas (P = 0.0051). The serum levels for IL-8 were 35.78 pg/mL in early stage and 52.76 pg/mL in late stage carcinomas (P = 0.0380). For CRP, median serum level was 3,366 ng/mL in early stage and 4,380 ng/mL in late stage carcinomas (P > 0.05)  than those afflicted with colon cancer (69.6 years) (P = 0.004). No differences were observed between adenoma cases and controls regarding the age of the patients (P = 0.144). Sex was distributed equally between all groups (P = 0.738). In order to explore whether the serum levels of the nine tested markers showed a correlation with age or sex, Spearman´s correlation coefficients were calculated. There was no relevant correlation between the nine tested markers and age or sex. However, serum levels of M-CSF correlated with age within the control-, adenoma- and carcinoma group  , which is an essential advantage concerning precious clinical samples. In addition, the throughput by a single operator is much higher for the multiplex assay than for ELISAs: in our set up 84 patient samples can be processed for nine analytes within four hours under highly standardized conditions. Furthermore, a scale up of the multiplex assay to even higher throughput is easily possible by using semi- or full-automation Evidence Multiplex Analyzers, which have the capacity to perform a test output in excess of 1,200 samples for nine analytes simultaneously per hour. Several independent studies already showed the validity of different Randox Evidence Multiplex assays -26. For The effectiveness of any screening program depends not only on economic and operational viability of the screening test but also on excellent diagnostic performance. It should be noticed, that the prevalence of colon cancer and adenomas in our study group is higher than in the general population. However, our results indicate that multiplex biochip based serum protein profiling validated certain biomarkers to discern sera from patients with and without colon cancer. The analyses revealed combinations of CEA + IL-8 and CEA + CRP to show the best screening performance for colon cancer reaching a sensitivity of 47% and 39% in the validation set, respectively. These combinations also proved to be useful when applied to early carcinoma detection with 33% and 28% sensitivity, respectively, using the combined training and validation set. Applying the test algorithm to an additional independent sample set of sera from patients with colorectal adenomas, we could show a sensitivity of 18% and 15% sensitivity, respectively (both at 86% specificity). It would be highly desirable to directly compare the performance of individual markers both used here in a multiplex fashion and as single marker assay reported in the literature. Unfortunately, this comparison can be severely biased, reaching from different antibodies applied up to different sample processing and storage conditions. We therefore focused our analyses primarily on our multiplex results and those markers with the highest performance either alone or in combination.et al.[et al. found that cytokeratins demonstrate a greater sensitivity than CEA in the diagnosis of colorectal carcinoma [et al. screened 148 patients regarding the expression of 74 putative biomarkers in plasma and reported among other markers CEA and IL-8 to be significantly elevated in CRC compared to healthy controls. They could hardly detect IL-8 by ELISA but by a proximity ligation-based multiplex assay [et al. analyzed a combination of CEA and CRP for preoperative staging of colorectal cancer, however, not for early diagnosis or screening [CEA is a well-known serum marker linked to CRC . It is tet al.. Howeveret al.. In linearcinoma . Lundberex assay . A combicreening . It has et al. reported a sensitivity of 25% at 98% specificity for detecting CRC [et al. reported 30 to 50% sensitivity at 84 to 96% specificity for FOBTs [Mortality from CRC can be reduced by early detection of cancer and removal of adenomas ,34. Baseting CRC . In 1997or FOBTs . iFOBT dor FOBTs  at specior FOBTs .Besides carcinomas, detection of early neoplasm like adenomas is favourable. For iFOBTs, sensitivities for detecting adenomas range from 4% to 63% for all adenomas, and 28% to 67% for adenomas of 1 cm or larger, both at a specificity of 89% . Among tThe effectiveness of any screening program depends not only on the diagnostic and economic performance of the screening test but also to a large extent on the compliance and general acceptance of the test by the public. Indeed, the compliance for colonoscopy is quite low compared to FOBTs . ColonosOur novel multiplex biochip revealed combinations of CEA + IL-8 and CEA + CRP to show the best screening performance for colon cancer with 47% sensitivity, for early carcinomas with 33% sensitivity, and adenomas with 18% sensitivity at an overall specificity of 86%. This performance could be improved by the addition of further promising markers. Since neither compliance nor diagnostic performance of FOBTs and serum markers alone seems satisfying for early colon cancer detection today, a combination of both methods may improve the performance of colon cancer screening.The authors declare that they have no competing interests.SB and KKG were responsible for study group collection, performed the biochip analyses and drafted the manuscript. MK, AC, VT, DM and SPF were responsible for chip development and validation. UH was responsible for the statistical data analysis. TG and TL assisted in drafting the manuscript. NP participated in the project design. UJR, JB, KF and HPB participated in clinical sample collection. HB, FE, JKH conceived and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript.The consortium “Colon Cancer Screening Chip” was generously supported by the German Federal Ministry of Education and Research (BMBF) within the Molecular Diagnostics funding scheme . The study was performed based on the serum collection of the biomaterial bank ColonBiomics being part of the Surgical Center for Translational Oncology – Lübeck (SCTO-L) and the “North German Tumorbank of Colorectal Cancer” network, the latter being generously supported by the German Cancer Aid Foundation .The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/393/prepubTable S1. Summary of clinical data of the pilot study group. a) Summary of clinical data of the pilot study group consisting of 400 serum samples from DKFZ: colon cancer patients, pre-malignant adenoma patients and healthy control patients. *All healthy patients received a full colonoscopy. b) Summary of clinical data of colon cancer patients.Click here for fileTable S2. Test performance of the multiplex-protein array. Assay ranges, intra- and inter-assay precision, accuracy and sensitivity of each analyte used on the CRCSI and CRCSII chip. *This is the full measuring range after dilution.Click here for fileFigure S1. A) 6 Carrier Holder for simultaneously processing of 54 samples B) One Carrier with nine Biochip-Arrays, C) Randox Evidence Investigator and D) Screenshot of Analysis Software.Click here for fileInstructions for use (IFU) for CRCSI and II.Click here for fileTable S3. Summary of diagnostic performances of biomarkers. Summary of diagnostic performances of biomarkers in single or combined fashion for detection of colon carcinomas. nd: not defined.Click here for fileTable S4. Spearman´s Correlation coefficient above 0.4. .Click here for file


Cushing's disease (CD) is a disorder in which chronic excess adrenocorticotropic hormone production is associated with multiple comorbidities and diminished quality of life. Postsurgical monitoring is important, and newer therapies are available for the management of surgical failure or disease recurrence. In this clinical case, we illustrate the importance of the nursing role in long-term management of CD, particularly as nurses may be the first point of contact for patients with CD. Alertness to disease signs and symptoms is crucial for timely diagnosis and improved outcomes. Successful therapy for CD requires careful monitoring of hormonal control, metabolic parameters, and therapy complications. Ongoing management requires lifelong monitoring of metabolic parameters, of side effects of treatment, and of signs of disease recurrence. Appropriate referrals may be required to facilitate overall outcomes and patient wellbeing. This patient was enrolled in a Phase III trial that was registered in the USA with clinicaltrial.gov. Cushing's disease (CD) is a disorder of chronic hypercortisolism secondary to excess adrenocorticotropic hormone (ACTH) production by a pituitary corticotroph adenoma . It is aTherapy aims at normalization of biochemical markers and the reversal of clinical features. Patients who relapse require second-line treatments including repeat surgery, radiation therapy, bilateral adrenalectomy, and/or medical therapy , 8. Thern = 162), prospective, randomized, Phase III trial (NCT00434148) of patients with CD showed that subcutaneous (sc) pasireotide 600 and 900 μg twice daily (bid) caused rapid and sustained reductions in urinary free cortisol (UFC) in the majority of patients, along with significant improvements in clinical signs and symptoms [The recently FDA-approved somatostatin analogue pasireotide  acts on the corticotroph tumor and decreases both ACTH and cortisol secretion. A large  with refractory hypertension (160/85 mmHg). His type 2 diabetes mellitus was being treated at presentation with glipizide; however, his fasting glucose at that time was 526 mg/dL.The patient had a history of osteoporosis with several nontraumatic vertebral compression fractures. Additional history included acid reflux, hiatal hernia, migraine headaches, depression, and anxiety. This formerly athletic teen of 100 kg had gained 68 kg body weight over 3 years, despite weight-lifting and several failed diets. He reported low back pain and had suffered a spontaneous compression fracture of his lumbar spine from sneezing the previous year.The patient reported difficulty sleeping, periodic nausea and abdominal pain, dizziness with standing, blurred vision, lower extremity cramping, alternating constipation and diarrhea, temperature dysregulation, fatigue, poor memory, feelings of social isolation, low libido, and increased thirst. His family history was negative for pituitary tumors but remarkable for hypertension, thyroid disease, diabetes type 2, alcoholism, and cardiovascular disease. He was married with a 7-month-old son. While he had a history of smoking for several years, the patient had quit prior to presentation.On examination, the patient demonstrated a Cushingoid habitus with central weight and relative extremity sparing, marked facial rounding and plethora, dorsocervical hump, and supraclavicular fat pad filling. Significant truncal obesity with distension and gynecomastia were present along with multiple 1–1.5 cm violaceous abdominal and bilateral flank striae. Moderate gynecomastia occurred with some tenderness on palpation. Skin examination was significant for scattered ecchymosis, some skin thinning, and numerous skin tags. Proximal muscle weakness was found along with 2+ pretibial and ankle edema. Medications at that time included lisinopril, furosemide, atenolol, potassium chloride (Klor-Con), escitalopram oxalate (Lexapro), and glipizide. μg/24 hr (reference range < 45 μg/24 hr). The patient had inappropriately low levels of follicle-stimulating hormone (1.0 mIU/mL), luteinizing hormone (1.5 mIU/L), with a low testosterone level (38 ng/dL). Prolactin (15 ng/mL), insulin-like growth factor-1 (271 ng/mL), and thyroid function were normal. A cavernous sinus sampling of ACTH levels after corticotrophin-releasing hormone (CRH) stimulation was elevated to 3650 pg/mL on the right side of the pituitary at 3 minutes and elevated to 12,368 pg/mL on the left at 5 minutes poststimulation.  Magnetic resonance imaging , which sought to assess the efficacy of pasireotide sc in patients with CD [μg/24 hr) and a positive dexamethasone/CRH test result . The patient was randomized to receive pasireotide 600 μg bid starting in December 2007.The patient consented to enrolment and treatment in a prospective, 12-month, multicenter, randomized, Phase III study ( with CD . The iniμg/24 h), and protocol dictated that his pasireotide dosage be increased to 900 μg bid.The patient showed a rapid response to pasireotide treatment. μg/24 h; morning ACTH, 33 ng/L; serum cortisol, 19 μg/dL), after which the patient lost 17 kg of body weight  and hip (+1.3%) measurements after 1 year of therapy. The Cushing's Quality of Life (QoL) questionnaire showed that pasireotide was associated with improvements in memory and healing, and with decreased irritability and bruising. Improvements in biochemical measures and clinical signs and symptoms of CD were progressive during the 2 years of pasireotide therapy.The patient reported mild AEs related to study drug, including mild pruritus and erythema at the injection site (resolved by study week 2), nausea (resolved when patient ate immediately postinjection), and transient fasting hyperglycemia (>300 mg/dL). All AEs were managed without pasireotide dosage adjustment.1c level persisted below 7.0% . The patient's HbAlow 7.0% . At the As part of the clinical protocol, patients received quarterly MRI scans. During the core study, asymptomatic gallstones developed. These may occur in conjunction with the disease or with the therapy. After the patient withdrew from the study, he developed symptomatic cholecystitis and required cholecystectomy. Because the patient wanted to pursue fertility options (a study protocol exclusion), he discontinued pasireotide after more than 2 years of therapy.Removal of a pituitary adenoma by transsphenoidal surgery is generally accepted as the first-line treatment for patients with CD. However, the 5- and 10-year recurrence rates for CD after successful surgical resection are reportedly as high as 25%  and 56% μg sc bid demonstrated a rapid and sustained reduction in UFC (~50% reduction in median UFC in both groups by month 2) in patients with de novo , persistent, or recurrent CD. Reductions in UFC were associated with significant improvements in clinical signs and symptoms of CD and health-related QoL [Pasireotide 600 and 900 ated QoL .μg sc bid. A rapid decrease in UFC level was observed following initiation of treatment with the patient achieving biochemical control (UFC ≤ ULN) at months 1 and 2; however, UFC rose > ULN following 3 months of treatment, leading to uptitration of the pasireotide dose. Despite significant variation, UFC levels progressively declined over the course of pasireotide treatment, with biochemical control (UFC < ULN) restored before completion of the 12-month core study. While it could be argued that the patient demonstrated a pattern of cyclic cortisol excess, he demonstrated progressive UFC control that was both dose related and associated with cumulative treatment.In this case study, a patient with a postsurgical recurrence of CD was enrolled in the Phase III study and randomized to receive pasireotide 600 The patient displayed progressive improvement in clinical signs and symptoms of CD over the 12-month core study. In addition, he reported improvements in QoL during the core trial and the extension phase.The safety profile of pasireotide was consistent with that of the somatostatin analogue class, except for the degree of hyperglycemia. The most common AEs were related to transient gastrointestinal discomfort. Importantly, all AEs were manageable without reduction of pasireotide dose.Hyperglycemia was effectively controlled by initiation of metformin treatment, which led to significant reductions in blood glucose level. Indeed, metformin dose was reduced and ultimately halted due to emergence of hypoglycemia-related AEs. Hyperglycemia associated with pasireotide treatment was reversed 1 month after discontinuation of study drug. These findings are in accordance with recent studies in healthy volunteers, whereby pasireotide-induced hyperglycemia was effectively managed using antihyperglycemic medication . As suchThis patient's care required ongoing management of clinical symptoms, disease-related parameters, blood sugar levels, cholecystitis, and drug AEs. A low-fat, low-glycemic index diet was recommended. The patient was referred to a local dietician and to a physical therapist for a low-impact, muscle-strengthening regime. To manage side effects, ice and heat were applied locally to the injection site. Nausea was avoided by offering a protein snack prior to each injection. The patient required counselling for depression early in the course of therapy and counselling for family planning. Collaboration with the patient's primary care provider was ongoing throughout treatment. This case illustrates the successful long-term management of a patient with recurrent CD using pasireotide. Biochemical control was achieved, along with clinical and QoL improvements and effective control of AEs. This case report demonstrates a patient with a progressive response to treatment over 2 years. In addition to monitoring biochemical parameters, clinical signs and symptoms along with QoL measures represent important endpoints in the treatment of CD. Anticipation and early treatment of common drug-related side effects improve patient tolerability and promote continuity of drug use. Treatment success is a composite of all measures.


Amblyomma maculatum is the primary vector for Rickettsia parkeri, a spotted fever group rickettsia (SFGR) and human pathogen. Cotton rats and quail are known hosts for larval and nymphal A. maculatum; however, the role of these hosts in the ecology of R. parkeri is unknown.R. parkeri (strain Portsmouth) grown in Vero cells to evaluate infection by R. parkeri in these two hosts species. Animals were euthanized 2, 4, 7, 10, and 14 days post-injection (dpi) and blood, skin, and spleen samples were collected to analyze by Vero cell culture and polymerase chain reaction (PCR). In a second trial, cotton rats and quail were inoculated with R. parkeri and nymphal A. maculatum ticks were allowed to feed on animals. Animals were euthanized on 14, 20, 28, 31, and 38 dpi and blood and tissues were collected for serology and PCR assays. Fed ticks were tested for R. parkeri by PCR and Vero cell culture.Cotton rats and quail were inoculated with low or high doses of Rickettsia parkeri was isolated in cell culture and detected by PCR in skin, blood, and spleen tissues of cotton rats in the initial trial 2, 4, and 7 dpi, but not in quail tissues. In the second trial, no ticks tested positive for R. parkeri by PCR or cell culture.R. parkeri rickettsiae can persist in the tissues of cotton rats for at least 7 days following subcutaneous inoculation of these bacteria; however, quail are apparently resistant to infection. Rickettsia parkeri was not detected in nymphal ticks that fed on R. parkeri-inoculated cotton rats or quail, suggesting an alternate route of transmission to naïve ticks.These studies demonstrate that viable  Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), is the best studied and most virulent of the SFGR [Spotted fever group rickettsiae (SFGR) are vector-borne organisms often causing disease in humans. the SFGR . The patthe SFGR -4 and ththe SFGR -7 have bR. parkeri in 1937 [R. parkeri, or a closely related SFGR [Despite the initial recognition of  in 1937 , studiested SFGR -12.R. parkeri is mainly limited to its occurrence in the primary tick vector, Amblyomma maculatum, commonly known as the Gulf Coast tick. Rickettsia parkeri has been detected in 12%-43% of questing adult Gulf Coast ticks collected across the southeastern United States [Rickettsia species is efficiently transmitted from the nymphal stage to the adult stage. It is unknown, however, if larval and nymphal A. maculatum acquire the microorganism predominantly by feeding on rickettsemic vertebrate hosts, through effective transovarial and transstadial transmission, or a combination of these transmission routes. Both larval and nymphal Gulf Coast ticks feed on small mammals such as cotton rats and ground-dwelling birds, including meadowlarks and northern bobwhite [Didelphis aurita) and cattle seroconverted when inoculated with R. parkeri. Some animals  also became transiently rickettsemic [R. parkeri, as described previously for R. rickettsii, the agent of RMSF [Our understanding of the natural history of d States -15, suggbobwhite -18. Adulbobwhite . Experimettsemic ,21. It i of RMSF -24.R. parkeri to cotton rats and bobwhite quail, two recognized vertebrate hosts for larval and nymphal stages of A. maculatum, and to investigate the ability of nymphal ticks to acquire R. parkeri from these R. parkeri-exposed hosts.This study was performed to assess the infectivity of Sigmodon hispidus) were purchased from Harlan Laboratories . Northern bobwhite quail were purchased from P & L Crowley Farm .Cotton rats (Candidatus Rickettsia andeanae” (100% of those tested), while ticks from the TAMU colony are not known to be positive for this organism . DNA was extracted individually from nymphal ticks obtained from both institutions and PCR amplified using primers 16S+2 and 16S-1 to target the 16S rDNA gene as confirmation that tick DNA had been extracted [rompA and “Ca. R. andeanae”-specific rompA gene fragments. The former was an assay using primers 190–70 and 190–701 for the primary reaction and primers 190-FN1 and 190-RN1 for the secondary reaction [Ticks were purchased from Texas A&M University (TAMU) and Oklahoma State University (OSU). Those from the latter institution, have previously been found to be infected with “xtracted . Extractreaction ; the latreaction .Rickettsia parkeri was grown in Vero cell culture with minimum essential media  supplemented with 10% fetal bovine serum. A low passage (P4 and P5) isolate of R. parkeri (Portsmouth) was used for all animal infections. Infected cultures were harvested when at least 90 percent of the Vero cells were infected, as determined by cell counts using 50 μl in a hemocytometer.R. parkeri (1000 infected Vero cells in 0.2 ml of culture media). Another set of five quail and five cotton rats were injected with a high dose of the organism (10 000 infected Vero cells in 0.2 ml). Percent infectivity of Vero cells was estimated by cytospin, and cell counts were performed using a hemocytometer. Animals were injected subcutaneously, at the nape of the neck on cotton rats and in the right leg of quail. One individual of each species served as a negative control and was injected with 10 000 uninfected Vero cells in a 0.2 ml volume. Four out of twenty animals—one low dose quail, one low dose rat, one high dose quail, and one high dose rat—were numbered and randomly selected from each group for euthanasia at 2, 4, 7, 10, and 14 days post injection (dpi). The controls were euthanized on 14 dpi. Animals to be euthanized were numbered and selected at random, within their dose assignment, and euthanized using carbon dioxide.The initial trial consisted of eleven quail and eleven cotton rats. All animals were pre-screened for SFGR antibodies via immunofluorescent antibody (IFA) testing (described in detail below). Five quail and five cotton rats received low dose injections of Upon euthanasia, blood was collected from the animals via intracardiac puncture. A 250 μl volume of whole blood from each animal was placed into individual flasks of confluent Vero cells. Skin from the injection site and spleen tissue samples were collected on necropsy. Half of each tissue sample was put into Vero cell culture (described in cell culture section below), and half was frozen at −20°C until DNA extractions and PCR assays could be performed.R. parkeri infected Vero cell culture (10 000 cells in 0.2 ml each). At 4 dpi, nymphal A. maculatum ticks were placed on each of the animals . Two cotton rats and two quail had OSU ticks (n=50), while all remaining animals (including controls) received TAMU ticks (n=65). Trays underneath each animal’s cage were examined daily for detached engorged ticks. All detached ticks were placed in humidity chambers (90% RH) and allowed to molt. Ticks were allowed to feed for 13 days, after which a blood sample was taken from the animals for IFA testing (dpi 17).The second trial consisted of eleven cotton rats and eleven quail. One cotton rat and one quail were injected with 10 000 uninfected Vero cells in 0.2 ml of culture media. The remaining ten individuals of each species received injections of Animals were euthanized on dpi 20, 24, 31, and 38. Upon euthanasia, a blood sample was collected via intracardiac puncture. At necropsy, tissues including skin from the original injection site, liver, spleen, kidney, and scrotal tissue  were collected and stored at −20°C until DNA extraction and PCR testing could be performed. All experiments were approved by the Institutional Animal Care and Use Committee at Mississippi State University (IACUC 10–067).Plasma from the blood samples was used to determine if SFGR IgG antibodies were present. Samples were screened at a 1:64 dilution. A 1:60 dilution of fluorescein isothiocyanate (FITC) anti-rat IgG (H+L)  was used as a secondary antibody for rat samples; FITC anti-chicken (H+L)  was used for the quail at a dilution of 1:275. Cotton rats and bobwhite quail known from previous IFA screening studies to be seronegative or seropositive for SFGR were used as controls.2, from animals in the first trial were triturated, using a sterile scalpel blade, into 250 μl of MEM + 10% FBS and added to 25 cm2 flasks of Vero cell culture. All flasks  received 10 μl penicillin-streptomycin (10000 U/ml penicillin and 10 mg/ml streptomycin).Tissues, of approximately 1cmR. parkeri-inoculated animals were pooled from each individual host. For example, all ticks that fed on quail 1 were put into one culture flask. Ticks were prepared for culture following a previously described protocol [2 culture flask along with any other triturated ticks that had fed on the same animal. Each flask also contained 100 μl of penicillin-streptomycin (10000 U/ml penicillin and 10 mg/ml streptomycin) and 5 μl of amphotericin B (250 μg/ml).In the second trial, ticks that successfully molted after feeding on protocol . BrieflyTwo days after tissues and ticks were placed in culture, flasks were emptied and fresh media was added. Flasks were then monitored weekly for three to six weeks for infection using cytospin preparations and acridine orange staining. Briefly, slides were allowed to air-dry and then were placed in methanol for 10 min. Slides were then flooded with acridine orange for 2-3 min.DNA was extracted from rodent blood samples using GE Healthcare’s illustra blood genomicPrep Mini Spin kit . DNA was extracted from quail blood using the QIAamp DNA Blood Midi kit . In all cases, 50 μl of blood was used for extraction following the kit protocols, as supplied by the manufacturer.DNA was extracted from cell culture and ticks halves using GE Healthcare’s illustra tissue and cells genomic Prep Mini Spin kit and following the manufacturer’s protocols . A 200 μl volume was extracted from all harvested cultures. Tick halves were triturated into extraction buffer using a new sterile scalpel blade for each individual sample. Final elution volumes were 200 μl.rompA) gene was used with primers 190–70 and 190–701 for the primary reaction and primers 190-FN1 and 190-RN1 for the secondary reaction [Rickettsia parkeri DNA extracts (Portsmouth) and non-template water controls were included in the assays.A nested PCR program targeting a segment of the rickettsial outer membrane protein A . Time of death allowed for blood to be obtained from only one of these rats. The control rat died on dpi 24, also from unknown causes apparently unrelated to the study; blood and spleen samples were collected and tested by PCR and found to be negative for SFGR DNA.In the series of experiments where All animals were seronegative on pre-screen by IFA. Controls remained seronegative throughout the study. On 17 dpi, 8 of 9 rats and 5 of 10 quail were positive for SFGR antibodies at a 1:64 dilution. No blood samples at this time-point tested positive by PCR. Two quails (one sampled 31dpi and the other on 38 dpi) were seropositive (1:64) at the time of euthanasia. All rats were seropositive (1:64) at the time of euthanasia. No blood samples or other animal tissues tested positive by PCR at any time point.Ca. R. andeanae” after feeding on animals.A total of 61 engorged ticks were recovered from the quail (0–12) and cotton rats (0–8), representing a range of 0–7 ticks per animal and resulting in 13 culture flasks . All cell cultures and ticks tested negative by PCR for rickettsial DNA. OSU ticks remained positive for “R. parkeri in various tissues for at least 4–7 days. Nonetheless, R. parkeri appears to be rapidly cleared by the immune system of cotton rats and even more quickly in bobwhite quail. Previous work with cotton rats indicated that R. rickettsii was cleared within 24 hours of infection [This study demonstrates that needle-inoculated cotton rats can maintain infection with nfection .Rickettsia honei occurred in 63% of Aponomma hydrosauri ticks collected (n=46), but not in lizard hosts (n=17) [A. maculatum with R. parkeri are also high. In Virginia, rates of infection with R. parkeri greater than 40% in A. maculatum have been reported [A. maculatum ticks sampled in Mississippi and Florida, with a maximum infection rate of 40% in Jackson County, Mississippi [A. maculatum collected throughout Mississippi [Although some rickettsiae have vertebrate reservoirs or amplifiers , in othes (n=17) . Infectireported ,30. One sissippi . Anothersissippi . Additiosissippi .R. parkeri is not dependent upon cotton rats or quail as reservoirs. While some feeding ticks may acquire the SFGR by feeding on recently infected animals such as cotton rats, there may be another mode of horizontal transmission occurring. Although our samples were small, our results suggest cotton rats and quail do not effectively transmit R. parkeri to naïve ticks. However, we also performed needle inoculations using cultured R. parkeri and laboratory-reared ticks, which may result in different infection dynamics than feeding naturally infected ticks on naïve animals. Transmission via co-feeding, from an infected tick to a naïve tick feeding nearby on the host, has been demonstrated with R. massilae and R. conorii[R. parkeri.It appears that the ecology of . conorii,34, suggR. parkeri in avian and mammalian hosts. Cotton rats may serve briefly as sources of infection for feeding ticks, or there may be other vertebrate species that have this potential. While cotton rats did not show substantial evidence of R. parkeri circulating in blood, they may still have the potential to infect naïve A. maculatum ticks indirectly (co-feeding) or during acute infection. With the increasing number of recognized R. parkeri cases in humans, it is important to identify potential sources of infection. Many questions remain and the ecology of this tick-borne rickettsia still needs to be examined thoroughly, both with surveys of potential vertebrate reservoirs and through experimental studies.This study adds valuable information to our limited knowledge of the dynamics of The authors declare that they have no competing interests.GMM contributed to study design, laboratory work, intellectual interpretation, and writing of the manuscript. JG was involved in study design, intellectual interpretation, and revision of the manuscript. CDP contributed valuable intellectual content and revised the manuscript critically. AVS supervised and designed the study, contributed intellectual content, and performed critical revisions of the manuscript. All authors read and approved the final version of the manuscript.The findings and conclusions are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.