Staphylococcus aureus, a major human pathogen, exacerbates allergic disorders, including atopic dermatitis, nasal polyps and asthma, which are characterized by tissue eosinophilia. Eosinophils, via their destructive granule contents, can cause significant tissue damage, resulting in inflammation and further recruitment of inflammatory cells. We hypothesised that the relationship between S. aureus and eosinophils may contribute to disease pathology. We found that supernatants from S. aureus (SH1000 strain) cultures cause rapid and profound eosinophil necrosis, resulting in dramatic cell loss within 2 hours. This is in marked contrast to neutrophil granulocytes where no significant cell death was observed . Supernatants prepared from a strain deficient in the accessory gene regulator (agr) that produces reduced levels of many important virulence factors, including the abundantly produced α-hemolysin (Hla), failed to induce eosinophil death. The role of Hla in mediating eosinophil death was investigated using both an Hla deficient SH1000-modified strain, which did not induce eosinophil death, and purified Hla, which induced concentration-dependent eosinophil death via both apoptosis and necrosis. We conclude that S. aureus Hla induces aberrant eosinophil cell death in vitro and that this may increase tissue injury in allergic disease. Eosinophils and their products are major components of allergic inflammation in tissues, in particular in atopic dermatitis, rhinitis and asthma, where their abundance in the tissues and circulation correlates with disease severity S. aureus, exacerbates the pathology of allergic diseases that are typically characterised by tissue eosinophilia, in particular atopic dermatitis S. aureus produces a number of virulence factors pertinent to its survival in the host, notably cytolysins which include the hemolysins  and Panton-Valentine leukotoxin (PVL). It is unclear, however, how the relationship between S. aureus and eosinophils may contribute to disease and the effects of S. aureus upon eosinophil viability had not been examined. In these studies we show S. aureus mediates rapid eosinophil cell death and that the cytolysin Hla is a major contributory factor in eosinophil death.The important human pathogen, Staphylococcus aureus wild type (WT) SH1000 strain and its corresponding agr derivative were used hla strain was created from DU1090 S. aureus was grown on LB plates (supplemented with tetracycline 5 µg/ml for agr and erythromycin 5 µg/ml and lincomycin 25 µg/ml for hla) from which single colonies were picked and inoculated into 5 ml LB broth for an overnight shaking culture (18 hr). For complementation of SH1000 hla to yield the strain hla+hla, the hla gene plus an 812-bp sequence upstream of the open reading frame was amplified using primers F-ApaI and R-BamHI  at 37°C/5% CO2.Granulocytes were isolated from fresh whole blood from healthy human volunteers by dextran sedimentation and plasma-percoll centrifugation Light microscopy: Eosinophils display characteristic morphological nuclear changes during apoptosis that can be visualised by light microscopy Flow cytometry: Eosinophils were studied in mixed granulocyte populations using antibodies to CD66c-PE (BD Biosciences) as a positive marker for neutrophils which highly express CD66c compared to eosinophils which dimly stain for CD66c. This was verified by showing both that the CD66c-PE low events were also CCR3-FITC positive (when used), and that they fit a FSC-SSC profile consistent with that of eosinophils  [28]. CeUsing this staining protocol it was possible to measure total eosinophil cell number and to assess the percentage of cells that were either apoptotic  or necrotic (ToPro3+ve).Statistical analysis was performed using PRISM v5  and the tests indicated in the figure legend.S. aureus. Cells were treated with cell-free bacterial supernatant prepared from overnight growth of S. aureus in LB broth. Initial experiments to establish dilution ranges and time points revealed a potent and rapid pro-death effect of the supernatant on eosinophils and this was characterized using flow cytometry. Mixed granulocytes were cultured with media alone or with S. aureus supernatant  for time points up to 2 hours followed by staining with CD66c-PE and ToPro-3 and measurement of absolute cell loss using fluorescent beads. S. aureus supernatant induced significant eosinophil necrosis by 15′, with an increase in ToPro-3 positive events  or low staining  was of a proteinaceous nature, supernatant was heat-treated  or digested for 2 hours with proteinase K and the effect on eosinophil cell death examined . AlthougS. aureus mutant deleted for the agr locus which controls the expression of key virulence factors agr derivative were grown simultaneously and CFU counts shown to be comparable. Supernatant prepared from the agr deficient strain did not induce eosinophil ToPro-3 positivity  and eosinophil cell death assessed after 30 and 120 mins. hla supernatant did not cause ToPro-3 positivity (hla supernatant induced moderate eosinophil cell death in these assays (3.7±2.3% ToPro-3 positivity in control vs 12.6±5.9% in hla supernatant diluted 1 in 100 after 30 mins) that was comparable to the profound cell death seen with substantially more diluted WT supernatants (data not shown). In genetic complementation experiments, the hla mutant was genetically reconstituted with a plasmid expressing Hla to further demonstrate the eosinophil killing abilities of Hla in bacterial supernatant. hla+hla supernatant results in a significant increase in ToPro-3 positivity and significant decrease in eosinophil number respectively, therefore restoring the death-inducing capacity of the Hla deficient supernatant. To provide further confirmation that Hla was a potent eosinophil lysogen, granulocytes were incubated with a concentration range of purified Hla for 1  and in support of this, Hla has been shown to have a high tolerance to heat In these studies we describe the ability of S. aureusS. aureus strains with alternative toxin profiles may also have a role in eosinophil-induced cell toxicity however, based on the findings in this study, we show that the critical factor in S. aureus-induced eosinophil cell death is Hla.Hla is a clinically important pore forming toxin which is produced by the vast majority of clinical strains of S. aureus supernatants nor purified Hla induced death of neutrophils granulocytes. In support of this, published data confirm neutrophils are remarkably resistant to cell death mediated by Hla S. aureus supernatant was able to induce modest but statistically significant levels of neutrophil ToPro-3 positivity at late time points. This could be due to the fact that neutrophils are relatively, rather than absolutely resistant to Hla, or attributable to the effects of high levels of eosinophil lysis within the mixed granulocyte populations  rather than being a direct effect of Hla. It is yet to be elucidated whether the eosinophil cell death is a host- or pathogen- mediated process. The rapid and pro-necrotic death suggests a pathogen-mediated phenomenon, both since death by apoptosis would be a preferable mechanism for the host and since in vivo disease models have shown Hla mutants have reduced virulence S. aureus skin infections, where Hla deficient strains result in smaller skin lesions compared to wild type strains and that vaccination against Hla reduces disease severity in vivoS. aureusin vivo is that high local concentrations of Hla at the site of infection, in combination with other potentially cytolytic factors, would lead to the necrotic death of tissue eosinophils, with consequent release of injurious granule contents causing damage to the surrounding cells and perpetuating inflammation S. aureus disease.Interestingly, we showed neither S. aureus colonisation of allergic disease.These data are the first example of a pro-necrotic effect of Hla on human eosinophils, and may in part explain tissue inflammation seen in


There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.Drug resistant pathogens are one of the key public health challenges of the 21 Drug-resistance is a major public health problem. Conventional wisdom on resistance management is to use aggressive chemotherapy to kill pathogens as rapidly as possible so as to prevent them from acquiring resistance. This is the reason why physicians frequently exhort patients to finish drug courses even after they no longer feel sick. However, this approach is based on the notion that we need only prevent new resistant mutants from arising. We hypothesize that in the situation where such mutants are already present at the time of treatment, more aggressive chemotherapy will select for these the fastest by rapidly killing all sensitive competitors. Here we demonstrate in a rodent malaria model that such selection indeed occurs more intensely following aggressive treatment than following less aggressive treatment, without any benefit to host health or infectivity. This suggests that aggressive chemotherapy will not be the best way to retard resistance evolution in some - perhaps many - circumstances. We suggest that an evidence-based approach across a wide range of infectious diseases is needed to manage resistance evolution. It is why physicians frequently exhort patients to finish drug courses even after they no longer feel sick At the end of his 1945 Nobel Prize lecture, Alexander Fleming warned of the dangers of drug resistance and proclaimed that “if you use penicillin, use enough” and the strength of selection de novo. But in an infection which already contains drug-resistant pathogens, either by de novo mutation, lateral transfer, or by transmission from other hosts, aggressive chemotherapy will rapidly eliminate drug-susceptible competitors, thus powering the very evolution it is designed to inhibit.Yet the rate of adaptive evolution is determined by the available genetic variation de novo or from other people. The question then becomes: among the wide range of drug doses, inter-dose intervals and treatment durations that can achieve the required clinical outcomes, which patient treatment regimen best retards the evolution of resistance? Here we present the first empirical data that shows that these need not be treatment regimens which remove susceptible pathogens as fast as possible.Quite how these opposing evolutionary forces combine to affect the rate of resistance evolution in any particular host-parasite system is unclear. Yet without understanding that, it is impossible to determine whether Fleming's rule  reducing drug pressure must reduce the extent of competitive release of resistant parasites, with the consequence that, 2) less aggressive treatment reduces the onward transmission of resistant pathogens. However, resistant management strategies are only of interest if they also achieve clinical and public health gains. Therefore, we would also require that 3) less aggressive treatment regimens do not increase host infectiousness, and 4) that less aggressive treatment regimens generate improvements in host health which are as good or better than those generated by aggressive chemotherapy.in vivo. Here we report such a test using Plasmodium chabaudi in laboratory mice. The results show that in this biological model, lighter touch regimens better contain already existing resistant parasites than aggressive treatment, without compromising host health.So far as we are aware, these four requirements have never been tested simultaneously for any infectious agent 1∶106 resistant to susceptible parasites was administered. To mimic the rarity of a de novo mutational event, a ratio of close to 101∶109 was established in experiment 2 by seeding ∼25 resistant parasites at peak susceptible parasitaemia . As expected, parasite densities of the susceptible parasites were inversely related to the drug pressure  as well as the density of resistant transmission stages . The probability of resistant parasites infecting mosquitoes was negligible following light touch treatment and increased 162-fold following aggressive treatment . Susceptible parasites did not contribute substantially to the overall post-treatment infectiousness in the higher dose treatments, but dominated in the light touch treatment and untreated infections .SP, RP and N track the daily densities of drug-susceptible parasites, drug-resistant parasites and host red blood cells. The functions Sf and Rf describe the process of red blood cell invasion by parasites as well as the production of progeny parasites within an infected cell. The functions Sh and Rh account for the loss of red blood cells due to infection. In both cases, the subscripts denote the fact that while the functions are the same, they take on unique parameter values for each parasite strain , the density of drug-susceptible parasites on the next day is a simple linear function of the current density. Thus, SP (t+1) = 0.06 SP (t), while the rest of the system remains unaltered. Using this approach, we were able to qualitatively capture the outcome of competition experiments between drug-susceptible and drug-resistant malaria parasite clones in the presence of drugs, with conventional drug treatment (a dose of 8 mg/kg for 4 days starting at the onset of disease symptoms) and with different initial ratios of susceptible to resistant parasites. This model was used to predict the effects of different drug regimens on the success of the drug-resistant parasite clone within a host. The treatment regimens which were predicted to result in substantially different infection dynamics and the best potential for suppressing the resistant parasites were included in the experiment  in approximately 8 week old female C57Bl/6 laboratory mice (Charles River Laboratories). Experimental methods are described elsewhere 6 susceptible parasites and were also either left untreated, or treated with pyrimethamine beginning on day 6 PI. Mice in one half of experiment 2 were inoculated with 106 susceptible parasites on day 0 and subsequently received ∼25 resistant parasites five days later, the remaining control mice were sham-injected with uninfected blood on day 0 and subsequently inoculated with ∼25 resistant parasites on day 5. Such extremely unequal starting conditions were chosen to generate what we knew from our previous work would be considerable (experiment 1) and overwhelming (experiment 2) competitive suppression of resistant parasites in the absence of chemotherapy CG1 assay was used ama-1 assay.Two experiments  were inidrug treatment regime (aggressive/moderate/light) was used. For overall infectiousness, also untreated infections were included.The geometric mean parasite density over the whole infection period was calculated for clone R. As a measure of transmission potential, the predicted infectiousness was calculated for clone R from day 10 onwards using gametocyte densities in the density-infectivity function for clone R as derived from previous infection experiments Experiment 1: Six mice failed to become infected with clone R (determined with the absence of detection by qPCR during the entire course of infection), presumably as a result of stochastic loss due to the low inoculum size . Experiment 2: Seven mice died or were euthanized during the course of the infection . Kinetics of infections in all mice is shown in Several mice were excluded from the analyses due to preterm death or failure to establish an infection. Figure S1Parasite dynamics of resistant parasites in single clone infections of experiment 2 following aggressive treatment (dark blue line), moderate treatment (orange line), light treatment (green line) and untreated infections (light blue line). Different drug treatments do not affect the parasite dynamics. Sample sizes as in (EPS)Click here for additional data file.Figure S2Asexual parasite dynamics of individual mice in experiment 1 with mixed infections of susceptible (black lines) and resistant (red lines) parasites in untreated infections (A–H), and infections that received a light (I–P), moderate (Q–X) and aggressive (Y-AF) drug treatment Click here for additional data file.Figure S3Gametocyte dynamics of individual mice in experiment 1 with mixed infections of susceptible (black lines) and resistant (red lines) parasites in untreated infections (A–H), and infections that received a light (I–P), moderate (Q–X) and aggressive (Y-AF) drug treatment Click here for additional data file.Figure S4Asexual parasite dynamics of individual mice in experiment 2 with mixed infections of susceptible (black lines) and resistant (red lines) parasites in untreated infections (A–J), and infections that received a light (K–T), moderate (U-AD) and aggressive (AE-AN) drug treatment Click here for additional data file.Figure S5Gametocyte dynamics of individual mice in experiment 2 with mixed infections of susceptible (black lines) and resistant (red lines) parasites in untreated infections (A–J), and infections that received a light (K–T), moderate (U-AD) and aggressive (AE-AN) drug treatment Click here for additional data file.Figure S6Mean asexual parasite dynamics  and gametocyte dynamics  of the susceptible clone under aggressive (dark blue lines), moderate (orange line), light (green line) or no treatment (light blue line) for experiment 1 (left panels) and experiment 2 (right panels). Data are means (± s.e.m.) with sample sizes as in (EPS)Click here for additional data file.Figure S7Mean total gametocyte dynamics (susceptible plus resistant gametocytes) in mixed infections under aggressive (dark blue lines), moderate (orange lines), light (green lines) or no treatment (light blue lines) for experiment 1 (A) and experiment 2 (B). Data are means (± s.e.m.) with sample sizes as in (EPS)Click here for additional data file.Figure S8Probability of infection from the susceptible clone gametocyte density for experiment 1 (A) and experiment 2 (B) based on an established gametocyte density – infectivity relationship see  during t(EPS)Click here for additional data file.Figure S9Drug regimen model simulations (left panels) and experimental data from experiment 2 (right panels) of mixed infections of susceptible (black lines) and resistant (red lines) parasites under aggressive , moderate  and light  treatment regimens. Data in right-hand panels are means (± s.e.m.) with sample sizes as in (EPS)Click here for additional data file.


We tested these predictions in an intertidal mudflat characterized by a two-state mosaic of hummocks (humps exposed during low tide) dominated by the sediment-stabilizing seagrass Zostera noltii) and hollows (low-tide waterlogged depressions dominated by the bioturbating lugworm Arenicola marina). In contrast to expectations, seagrass recolonized both natural and experimental clearings via lateral expansion and seemed unaffected by both clearing size and lugworm addition. Near the end of the growth season, however, an additional disturbance (most likely waterfowl grazing and/or strong hydrodynamics) selectively impacted recolonizing seagrass in the largest (1 m2) clearings (regardless of lugworm addition), and in those medium (0.25 m2) clearings where lugworms had been added nearly five months earlier. Further analyses showed that the risk for the disturbance increased with hollow size, with a threshold of 0.24 m2. Hollows of that size were caused by seagrass removal alone in the largest clearings, and by a weaker seagrass removal effect exacerbated by lugworm bioturbation in the medium clearings. Consequently, a sufficiently large disturbance increased the vulnerability of recolonizing seagrass to additional disturbance by weakening seagrass engineering effects (sediment stabilization). Meanwhile, the counteracting ecosystem engineering (lugworm bioturbation) reduced that threshold size. Therefore, scale-dependent interactions between habitat-mediated facilitation, competition and disturbance seem to maintain the spatial two-state mosaic in this ecosystem.Recovery of an ecosystem following disturbance can be severely hampered or even shift altogether when a point disturbance exceeds a certain spatial threshold. Such scale-dependent dynamics may be caused by preemptive competition, but may also result from diminished self-facilitation due to weakened ecosystem engineering. Moreover, disturbance can facilitate colonization by engineering species that alter abiotic conditions in ways that exacerbate stress on the original species. Consequently, establishment of such counteracting engineers might  One of the most studied but also debated issues in ecology is the relative importance of factors affecting how organisms and ecosystems respond to disturbance exceed engineers both spatially and temporally, i.e. extends beyond engineer patch edges and outlive engineers Theory and observation suggests scale-dependent ecosystem shifts can also be caused by increased abiotic stress on recolonizing individuals, when the removal of an “ecosystem engineering” species simultaneously removes the self-facilitation required for recovery. Such disturbance responses should be most common in abiotically stressed environments, where communities depend on facilitation from ecosystem engineers. These are species that via presence and/or function change abiotic conditions, concentrate resources and/or alleviate local stress, which induces positive organism-environment feedback increase stress on recolonizing members of the original community. There are many examples of how contrasting engineering effects speeds up competitive exclusion of “counteracting” engineers via “habitat-mediated competition”, which contributes to the formation of spatial mosaics of engineered patches reduce the threshold disturbance size associated with increased risk for changes in recovery and shifts to alternative states increases. This is because counteracting engineering should exacerbates stress on recolonizing individuals, and thereby slow down recovery.Neither of these mechanism takes into account that those inferior competitors that may recruit into a disturbed area are often ecosystem engineers that modify abiotic conditions in other ways than the original community. Establishment of such engineers may actually counteract the spatial facilitation from un-impacted individuals in surrounding areas, and hummocks) dominated by the sediment-stabilizing seagrass Zostera noltii Hornem (from here on seagrass), alternating with low tide water-logged pools or depressions (hollows) dominated by the sediment-destabilizing lugworm Arenicola marina L. (from here on lugworms). Seagrasses can facilitate themselves by stabilizing sediments and reducing erosion and turbidity through attenuation of water flow We tested these predictions using a field survey and a removal-addition experiment in the eastern Dutch Wadden Sea (Netherlands). The study area was a near-shore intertidal mud flat characterized by a striking mosaic of two alternating habitat types : low tid2, we predicted that (i) the strength of seagrass ecosystem engineering and rate of recovery decreases with increasing size of disturbance, (ii) lugworm establishment success increases with size of disturbance on seagrass due to weakened spatial influence from surrounding seagrass, and (iii) lugworm engineering in disturbed areas reduces the threshold disturbance size needed to slow down seagrass recovery by exacerbating erosion caused by the removal of seagrass.After experimentally clearing seagrass and/or adding lugworms from areas ranging in size from 0 to 1 mZostera noltii still occurs and has expanded The study was conducted on the intertidal mudflats at Emmapolder ; one of a few areas in the Dutch Wadden Sea where Zostera noltii) patches occurred primarily on hummocks, whereas lugworms occurred mainly in gaps (distinct unvegetated areas) which occurred primarily in waterlogged hollows  hummocks and (ii) hollows . Third, average size of gaps in the seagrass beds were assessed by noting the position of gap edges along 50 m transects randomly placed parallel to the shore (n = 3 transects per site).Seagrass . “Time” was treated as fixed , and “Site” (two levels) was included as random offset, since (i) site effects were not of specific interest but (ii) should be incorporated to reduce within-treatment and reduce the risk for type 1-errors associated with the repeated measures. Test assumptions were first assessed , and if necessary, data was transformed (square root or log). The full models were fitted down to minimal adequate models using stepwise deletion of non-significant factors (α = 0.05) followed by model fit comparisons using information theory .Hypotheses were tested using statistical modeling in the R environment 2 intact plots), (ii) “Small” (0.0625 m2 clearing), (iii) “Medium” (0.25 m2 clearing), and (iv) “Large” (1 m2 clearing). To experimentally remove only seagrasses (and not the sediments they stabilized), plywood frames were hammered 20 cm deep along plot edges during low water. Next, the frame was filled with water and all seagrass shoots, roots and rhizomes were removed using a hand rake. After allowing suspended particles to settle, the water was slowly released and the frame gently removed. Lugworm  were added on half of the plots (lugworm addition) in densities corresponding to 32 ind. per m−2. This relatively high density was intentional, as lugworm densities quickly self-regulate to local carrying capacity due to interspecific competition A field experiment was conducted between June and November 2009 (the seagrass growth season), testing the relative and interactive effects of size of seagrass clearing  and lugworm addition on ecosystem recovery. In each of the two sites (see Field survey), 24 quadratic plots were established within large (>5 m wide) seagrass patches. Plots were placed in a row parallel to the shore, >2 m apart and >2 m from natural gaps to avoid edge effects. Experimental quadratic clearings in plots were conducted in four sizes, mimicking natural disturbances (e.g. waterfowl grazing or uprooting caused by hydrodynamic stress): (i) “Control” . This was assessed as % weight loss of plaster dissolution balls; a reliable method when – as here – used in similar water-flow environments per se, facilitated lugworm establishment . Therefore, relative rates of recolonization will decrease with clearing size even if the absolute rhizome expansion rate is constant, simply because the distance from the edge to the center increases. However, our “stress-induced shifts” model predicted that absolute recovery rates should decrease with increasing clearing size due to weaker engineering effects (see −1). This resulted in five monthly estimations of daily gap contraction rates (from start of June until start of November). During days with low water and clear skies, plots were photographed (with a Konica Minolta Dimage ×50 at 5MP resolution) at a 90° angle from 1.5 m height (on the same dates as lugworm counts). The sizes of gaps were estimated to the closest 0.0001 m2 using the freeware image analysis program ImageJ gcr, in mm day−1) over the foregoing month as:t1x and t2x are gap sizes at start and finish of that month, respectively (in mm2), and Δt is the number of days between start (t1) and finish (t2). We also calculated the seagrass relative recovery at the start of each month as “percent of the gap size at start” (in June).Lugworm fecal casts were counted in plots at the start of each month (six times) to assess if (i) the lugworm addition treatment remained over time (high vs. low densities), (ii) seagrass Clearing size, rather than seagrass removal ment see , and (iiects see . We therTreatment effects were explored using linear mixed effects modeling (see above) with the fixed factors Clearing size, Lugworm addition and Time , and their respective interactions, included in full models. “Plot” (n = 48) was included as a random factor, and effects of first order autoregressive and compound symmetry correlation structures were compared to account for effects of repeated measures Effects of Clearing size and Lugworm addition on flow-induced weight loss of dissolution balls were tested with a linear mixed effects model with “Site” (two levels) as a random factor  were highly non-normal and zero-inflated. We therefore modeled effects of Clearing size, Lugworm addition, Site and Time using a generalized linear mixed model with a Poisson distribution, using a Bayesian approach. The initial density was modeled as a function of site, clearing size and lugworm addition, with all interactions. Subsequent densities were modeled with additional terms for months, and an autoregressive term for the previous lugworm abundance (i.e. the expected cast density), which varied by month. The time effect and autoregressive terms were given Normal priors with mean 0, and variance 10. The main effects, and first and second order interactions were modeled as random effects, with a Jeffreys prior on the variance (i.e. a uniform prior on the log variance): if interactions are small, this shrinks their effects towards zero Finally, effects on seagrass recovery (daily gap contraction rate) were evaluated by fitting a linear mixed effects model with “site” as a random offset, after excluding no clearing (Control) plots .what local conditions triggered the additional disturbance, we gathered plot-specific data on five potentially important variables serendipitously collected just before the additional disturbance (early October). The variables were (i) seagrass gap size and (ii) lugworm fecal cast density (reflecting the two treatments); (iii) seagrass ground cover (%) in recolonized areas, which may influence self-facilitation , which may influence exposure, and (v) the size of hollows (water-logged low-tide depressions) encircling the gaps created by the seagrass clearing; a measure of the spatial extent of sediment erosion . As “gap size” and “hollow size” were highly correlated (Pearson rho = 0.88), we removed “gap size” (regarded less important because the disturbance occurred outside the gaps but in the hollows). The minimal adequate model was identified as described above.To our surprise, gaps in three treatment combinations expanded on average instead of contracting during October; in Large clearings with and without lugworm addition, and Medium-sized clearings with lugworm addition see . This ef6 = 12.41, P<0.001). Seagrass ground cover  higher than hollows  and sediment erosion (as relative gap height) indicated that seagrass ecosystem engineering strength in the center of plots depended on the on balls  was highon balls  also depLugworm fecal cast densities were mainly affected by the main effects of Clearing size (explaining 55% of variation), Lugworm addition (20%) and Time (15%); cast densities increased with the size of initial clearing and with lugworm addition, but decreased over time in all treatments . The thrhigher in Medium and Large than Small clearings . This was explained by a much faster relative recovery in Small than Medium and Large plots in June and July , and absolute recovery rates shifted from positive to negative (2), and recovery rates shifted from positive to negative . The threshold hollow size (where disturbance risk = 50%) was ∼0.24 m2 . Hollows in Large plots were 19 and 5 times larger than those in Small and Medium plots, respectively, and Lugworm addition increased hollow size by 45% across clearing sizes , local environmental conditions shifted from sediment stabilizing to de-stabilizing over the course of the experiment regardless of lugworm addition, most likely because of weakened (i) water flow attenuation and (ii) sediment stabilization (the latter caused by a slower relative recovery rate). This essentially caused the formerly seagrass-dominated low-tide hummocks to switch into low-tide hollows during the autumn. However, this scale-dependent response was not due to simple slowing down of recovery due to lack of facilitation 2. Consequently, counteracting engineering reduced the threshold size of disturbance necessary to cause shifts in ecosystem as predicted, but via a mechanism that was largely unexpected.Here, we show that habitat-mediated self-facilitation and habitat-mediated competition may interactively result in scale-dependent ecosystem responses to disturbance. In the largest seagrass clearings (1 mZ. noltii in the Wadden Sea Branta bernicla) and Wigeon (Anas penelope) were observed feeding daily on Zostera noltii from mid September to November . Importantly, the grazing was spatially restricted to seagrasses in hollows, while seagrass-dominated hummocks covered 50% of the area . Such hollow-specific waterfowl grazing has been observed elsewhere, and is explained by the low-tide standing water in hollows, which facilitates feeding while reducing ingestion of silt persistentThe scale-dependent seagrass recolonization was – in contrast to our expectations – not caused by slower recolonization, but by increased risk for disturbance on recolonizing seagrasses during autumn. Since the study was not designed to identify the additional disturbance or the underlying cause(s) to it, we can only speculate on which was the disturbance. Visual observations in the field strongly suggested that waterfowl grazing – a well-known seasonal disturbance on Habitat-mediated competition can alone maintain spatial mosaics if counteracting engineering is strong enough to exclude competitors In conclusion, our study supports the notion of biota as an important factor for disturbance responses and as drivers of ecosystem heterogeneity Figure S1Zostera noltii ground cover and seagrass shoot density, above-ground biomass and total biomass. To test how accurately estimations of Zostera noltii ground cover predicted Z. noltii shoot density and biomass, we in the field placed a 0.25 m2 frame in random points ranging in seagrass cover from 5 to 90% (n = 6), photographed the frame (see the article for details), and excavated all seagrass material in the center of the frame using a 10.5 cm wide corer (to 20 cm depth). Seagrass ground cover in each frame was estimated with the same digital photography analysis as in the experiment . In the laboratory, we counted all shoots, separated and cleaned the above- and below-ground material in seawater, and dried both fractions until constant dry weight (at 60°C). Results from multiple regressions showed that estimated cover was a reasonable predictor of shoot density , above-ground biomass  and total biomass .Relationship between (EPS)Click here for additional data file.Figure S2Arenicola marina density and lugworm fecal cast density. Counts of Arenicola marina fecal casts is a commonly used and rapid method to assess worm densities . To ensure the reliability of the method in our study area, we compared counts of fecal casts in randomly placed 0.25 m2 frames (both within and outside of Zostera noltii patches) with actual densities of worms in the sediment, by excavating all worms to 50 cm depth within the frame (n = 10). A linear regression analysis showed that the number of fecal casts accurately predicted worm densities .Relationship between (EPS)Click here for additional data file.Figure S3A–D. Five predictor variables used in multiple logistic regression analysis, as effects of Clearing and Lugworm addition .(EPS)Click here for additional data file.


Human and animal studies provided controversial data on asymmetric cortical representation of cardiac function, which may partially be due to different study designs and inter-individual variability. Here, we investigated whether seizure-related changes in heart rate (HR) and cardiac repolarization depend on the side of seizure-activity in people with mesial temporal lobe epilepsy (mTLE).To account for inter-individual variability, EEG and ECG data were reviewed from patients with medically refractory mTLE undergoing pre-surgical video-EEG telemetry with at least 2 seizures arising from each hippocampus as assessed by bilateral hippocampal depths electrodes. RR and QT intervals were determined at different timepoints using a one-lead ECG. QT intervals were corrected for HR (QTc) using 4 established formulas.Eighty-two seizures of 15 patients were analyzed. HR increased by ∼30% during hippocampal activity irrespective of the side (p = 0.411). QTc intervals were lengthened to a significantly greater extent during left hippocampal seizures . Abnormal QTc prolongation occurred in 7 of 41 left hippocampal seizures of 4 patients, and only in 2 of 37 right hippocampal seizures of 2 patients.Seizure-related modulation of cardiac repolarization, but not of HR, appears to depend on the side of ictal activity, strengthening the hypothesis of asymmetric cerebral representation of cardiac function. The clinical relevance of this is unclear, but may indicate an increased risk of abnormal ictal QT prolongation in people with left mTLE. Cardiac activity is dynamically modulated by the autonomic nervous system to ascertain sufficient blood and oxygen supply to all organs in response to various physiologic and pathophysiologic conditions. The major players consist of the sympathetic and parasympathetic branches which are regulated through cortical and subcortical neuronal networks including brain stem, insular cortex, amygdala, and hippocampus Taken together, cardiac function appears to be asymmetrically represented in the brain with potential clinical relevance according to the side of cerebral affection. To date, however, it is unclear whether transient disturbances of brain function e.g. by epileptic seizures have also a hemispheric-specific effect on cardiac function. Previous studies have produced controversial results, maybe due to variable interindividual patterns of cardiac regulation Here, we have asked whether seizure-related changes in HR and cardiac repolarization depend on the side of seizure-activity in people with medically refractory mesial temporal lobe epilepsy (mTLE) undergoing pre-surgical video-EEG telemetry using intracranial electrodes. We included patients with bilateral hippocampal depths electrodes and, to account for intrinsic differences in autonomic function and to allow intraindividual comparison, only those who had at least two seizures arising from each hippocampus independently from each other during video-EEG telemetry.We reviewed patients with medically refractory mTLE undergoing presurgical video-EEG monitoring during a 12 years period from January 2000 to December 2011 in the Department of Epileptology at the University Hospital Bonn (Germany). This study is a retrospective audit of EEG and ECG data which have been collected during standard clinical procedures, and has been approved as such by the local medical ethics committee (“Ethikkommission an der Medizinischen Fakultaet der Rheinischen Friedrich-Wilhelms-Universitaet Bonn”). Informed patient consent was not required because of the retrospective design and the anonymization of patient-related data for analysis and publication. Inclusion criteria were presence of bilateral hippocampal depth electrodes and the occurrence of at least two seizures with independent onset from each hippocampus. Two to 4 seizures per hemisphere per patient were analyzed. Seizures were selected according to the side of seizure-onset and the interpretability of simultaneous ECG traces. If more than 2–4 seizures per patient and hemisphere were eligible for analysis, seizures were selected in chronological order of occurrence. The correct position of the electrodes was controlled with the help of MRI after implantation. In 13 patients, hippocampal depths electrodes  were implanted via a posterior approach as displayed in Standard presurgical assessment included cerebral MRI (1.5 or 3 Tesla), non-invasive video-EEG telemetry using scalp EEG  prior to invasive video-EEG telemetry and neuropsychological testing. Video-EEG telemetry is the simultaneous acquisition of a video film  and EEG recordings  in order to correlate the patients’ behaviour with the seizure activity. In all included patients, invasive video-EEG monitoring was performed with hippocampal depth electrodes (see above) and in some patients with additional neocortical subdural strip or grid electrodes . All eleEEG data acquisition was performed with a Stellate Harmonie digital video-EEG system  using up to 128 channels, a 200 Hz sampling rate and a 16 bit analogue-to-digital converter. Data were band pass filtered between 0.016 and 70 Hz. We determined the timepoint and localization of seizure-onset according to the intracranial EEG recordings.A modified lead-I ECG (adhesive electrodes placed below the clavicles of either side) was recorded simultaneously with EEG. RR intervals were determined manually at different timepoints . QT intervals were manually measured from the start of the QRS complex to the end of the T wave (defined by the intersection with the isoelectric line). QT and preceding RR intervals were determined from 3–5 successive ECG complexes and QT intervals were corrected for variable RR intervals. All correction formulas tend to over- or underestimate QTc 1/2(1) Bazett: QTc = QT/RR1/3(2) Fridericia: QTc = QT/RR(3) Framingham: QTc = QT +0.154×(1-RR)(4) Hodges: QTc = QT +1.75×(HR –60)In formulas (1)–(3), QTc, QT and RR interval values are expressed in seconds. In (4), QTc and QT intervals are in milliseconds and HR is in beats per minute. To reduce a bias error of putatively pathologic QTc intervals (due to higher HR), we have used modified normal limits for QTc intervals as proposed by Luo and colleagues see  [17].Statistical differences of HR and QTc values between seizures with left- and right-sided ictal activity were assessed pairwisely using a mixed linear regression model adjusted for variable seizure numbers per patient and individual patient effects . P-values <0.05 were regarded as statistically significant, based on the null hypothesis that there is no hemispheric side effect on (i) ictal HR and (ii) QT intervals. All data are given as means ± S.E.M.Fifteen  of 205 patients with medically refractory mTLE fulfilled the inclusion criteria . ThirteeA total of 82 seizures with unilateral hippocampal onset were analyzed . A repreQTc intervals, however, were prolonged to a significantly greater extent with ongoing hippocampal seizures on the left side after adjustment with all 4 correction formulas  215O-PET scans) and simultaneously recorded ECG features during a subtraction task with or without time restriction  and isometric handgrip squeeze under specific conditions . The major findings are that proarrhythmic abnormalities of cardiac repolarization were positively correlated with an asymmetric increase in regional blood flow in the midbrain on the right side, and, at least in one feature of QT abnormality, also in the right parahippocampal gyrus, supporting the notion of an asymmetric representation of the autonomic control of cardiac repolarization including mesiotemporal brain structures. These findings may explain our observation that seizure-related modulation of cardiac repolarization appears to be asymmetrically localized in people with mesial TLE. Accordingly, right- and left-sided hippocampal seizure activity may induce an asymmetric sympathetic modulation of cardiac repolarization via mesiotemporal networks  and midbrain networks (via projections to the brain stem). Interestingly, QTc intervals shortened again after the propagation of seizure activity to the contralateral hemisphere,  and returned to baseline postictally  effects on autonomic networks regulating cardiac repolarization.Whereas the above cited studies include patients or animals with a structural lesion due to ischemic stroke or in people during physiologic stress tasks (and without known brain diseases), we have assessed the effects of ongoing abnormal seizure activity in a limited neuronal network. It is tempting to speculate that, in analogy to Cardiac repolarization was assessed as QT intervals corrected with four established formulas to account for selective bias using e.g. Bazett’s formula only . QTc was not correlated with absolute ictal HR , suggesta-priori hypothesis of seizure-onset zone based on electro-clinical and MRI findings during non-invasive presurgical assessment with the ultimate goal of using as few electrodes as possible . Therefore, the number of implanted electrodes is limited with a consecutive spatial sampling bias. Ictal activity tends to spread from the hippocampus to lateral and basal parts of the temporal lobe and the insula of the same hemisphere, and later during the seizure, to the contralateral hemisphere as well This is a retrospective study capitalizing on intracranial EEG recordings. Implantation of electrodes was performed according to an per se has no measurable effect on the cardiovascular autonomic properties as compared to people with focal epilepsy without hippocampal sclerosis A further weakness of our study is the lack of information on periictal respiratory function in our patients, as seizure-related hypoxemia increases the likelihood of both QTc prolongation and shortening Figure S1QTc decreases after propagation to the contralateral hemisphere.(DOCX)Click here for additional data file.Figure S2Extent of ictal HR changes correlates with spatial spread, but not with duration of ictal activity.(DOCX)Click here for additional data file.Table S1Upper and lower normal limits of normal QTc for 4 correction formulas.(DOCX)Click here for additional data file.


Rituximab-mediated lysis of B cells in vitro increases the level of infectious HCV released from B cells. Our results, using a model where virus does not replicate in B cells, recapitulate observations seen in patients and may explain in part the rapid increase in blood HCV RNA observed after rituximab treatment.Hepatitis C virus (HCV) infected patients with vasculitis are often treated with the B-cell-depleting anti-CD20 antibody rituximab. Treatment reduces the cryoglobulins that cause vasculitis, yet it also leads to a transient increase in liver enzymes and HCV genomic RNA in the periphery. The mechanism underlying the increased viral load is unclear and both direct and indirect roles have been proposed for B cells in HCV infection. We previously reported that HCV can associate with B cells and can trans-infect hepatocytes. We established an  HCV infection predominantly affects the liver but extrahepatic manifestations have been reported The main modes of rituximab action include antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) , howeverIt has been challenging to demonstrate that HCV replicates in B cells Lake-Bakaar and colleagues studied HCV kinetics in infected patients treated with rituximab Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation from healthy volunteers who gave informed written consent for participation in the study according to the Declaration of Helsinki. Ethical approval was obtained by the South Birmingham Local Research Ethics Committee  and the University Hospital Birmingham Trust. We used the Group I Burkitt's lymphoma B cell line L3055 as a target for the ADCC assay  and Jurkat T cells as controls (ATCC). Cells were isolated and/or propagated as described PBMC were cultured in the presence of 100 IU/ml IL-2 for 24 hours to stimulate Natural Killer (NK) cells. Target L3055 B cells were incubated with HCVcc JFH-1 for 2 hours, unbound virus was removed by extensive washing and the cells were then treated with rituximab or control antibody at 10 µg/ml for 30 minutes. Activated PBMC were co-cultured with target cells at 1:10 effector:target (E:T) ratio for 3-7 hours. Rituximab activity was measured by a flow cytometric CD107a NK cell degranulation assay Data were presented as means from three or four replicates. Error bars represent standard deviations and comparisons between samples were made using nonparametric tests (Mann-Whitney for unpaired samples and Wilcoxon for paired samples) using Prism 4.0 .To mimic the effects of rituximab on CD20+ B cell depletion by blood cells, we used PBMC from healthy donors as effectors and the Burkitts lymphoma B cell line L3055 as target cells. We previously reported that B cells bound optimal levels of HCV after 2 hours We sampled the extracellular media from the ADCC assay for infectious virus released by the lysed target B cells over time . There wFor these experiments, we used HCVcc JFH-1, a genotype 2a strain that replicates in hepatocytes in vitro and in vivo but does not replicate in B cells. Despite the lack of productive infection, our study demonstrated that B cells lysed by effector blood cells could release up to four times more infectious virus following rituximab treatment.Use of primary B cells from peripheral blood of healthy donors as targets instead of L3055 cells yielded comparable results (data not shown), however this may not reflect the ability of B cells in vivo to capture virus. It is worth noting that L3055 B cells used as rituximab targets, as well as B cells from healthy donors, were not specific for HCV; indeed we previously demonstrated that primary PBMC-derived B cells and L3055 cells bind comparable amounts of HCVcc RNA as measured by quantitative RT-PCR Our in vitro ADCC assay does not take into account potential virus replication in B cells that may occur in vivo, or the role of B cells in controlling infection via immune protection or indirect clearance mechanisms; despite these limitations, we demonstrated a significant increase in the level of infectious HCV released from B cells in the presence of rituximab which may contribute to the increased viral load observed in patients. In summary, our data support a role for rituximab lysis of B cells and release of infectious HCV as a mechanism to explain the transient increase in viral load observed in treated patients.


In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization.Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection . We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window.  Atherosclerosis is the leading cause of death in the United States. Interventions for treating atherosclerosis including angioplasty, stenting and bypass frequently fail related to the development of recurrent disease (restenosis) Another key cell type integral to this process is the endothelial cell (EC). As a by-product of interventions to treat atherosclerosis, denudation of the endothelial layer of treated arteries or veins leads to deleterious consequences. First, ECs provide the vessel’s anti-thrombotic lining. Without an EC layer, platelets accumulate on the vessel surface initiating thrombus that can cause sudden death Currently, the only clinically employed method for preventing restenosis is a stent coated with rapamycin or paclitaxel used in conjunction with angioplasty e.g. lack of effect in vivo or difficulty in delivery. Moreover, the scarceness of reports identifying agents that selectively inhibit SMCs versus ECs likely reflects the fact that ECs are generally more susceptible than SMCs to anti-proliferative drugs. Thus, in order to discover candidates for selective SMC inhibition, a high throughput screening campaign is necessary to screen large libraries of compounds. To the best of our knowledge, there has been a lack of such HTS studies with this goal in mind.Thus, the optimal drug to prevent restenosis would be one that selectively inhibits SMC proliferation and intimal hyperplasia but has a minimally inhibitory effect on EC proliferation. Several such selective agents have been reported in the literature in vivo testing to be a potent inhibitor of injury-induced intimal hyperplasia. Our finding raise the possibility of repurposing idarubicin for the treatment of vascular restenosis and establish an efficient, low cost HTS that can be used through screening of large chemical libraries, to identify other candidate inhibitors of restenosis.The purpose of this study is to establish a pilot HTS system that is amenable for screening larger chemical libraries with the goal of discovering new drugs that selectively retard human SMC proliferation while leaving the growth of endothelial cells unaffected. To circumvent the shortcomings in the aforementioned studies, we have developed accurate assays for proliferation of primary human aortic SMCs and human aortic ECs, which are more relevant to human restenotic disease than animal cells. In a pilot screen we have identified that idarubicin, an FDA-approved drug currently used for treating leukemia, inhibits the proliferation of SMCs to a much greater extent than ECs through a defined concentration window. Furthermore, for the first time, this drug has been shown through The experiments involving animal use were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (Permit Number: M02273) was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.Alamar Blue was purchased from Invitrogen . Cell Titer Glo was from Promega . Primary human aortic smooth muscle cells (HuAoSMCs) and primary human aortic endothelial cells (HuAoECs) at passage 3 were purchased from Lonza; their respective optimal culture media (SmGM-2 and EGM-2) were from the same commercial source. Cells were used at passage 5 after expansion. Trypsin/EDTA solution was from Clonetics ; and DPBS was from Gibco . Microtiter tissue 96-well culture plates with transparent flat-bottoms and black-walled sides were from Costar . Resveratrol and idarubicin were products of Sigma-Aldrich . Stock solutions of these reagents were prepared in DMSO (Thermo-Fisher). The library of NIH Clinical Collection composed of 447 unique compounds of known bioactivity was available at the Small Molecule Screening and Synthesis Facility (SMSSF) of the University of Wisconsin Carbone Cancer Center (UWCCC).2. After expansion, cells at passage 5 were used for all the experiments.Cryo-protected frozen HuAoSMCs and HuAoECs  were thawed and cultured in their respective media that are optimized for cell growth by the manufacturer. HuAoSMCs were grown in the SmGM-2 medium containing 5% FBS, and HuAoECs in the EGM-2 medium containing 2% FBS in a humidified incubator at 37°C with 5% COe.g. 4 h). To verify assay consistency with a additional method, Alamar Blue dye was removed and wells were gently washed, and Cell Titer Glo reagent was then added followed by a 10 min incubation and luminescence measured using Genios Pro.Freshly collected HuAoSMCs (passage 5) were counted (>93% viability) by Cellometer AutoT4 (Nexelon Bioscience), and dispensed using Microflo Select (BioTek) to a final density of 2700 cells/200 µl/well in the SmGM-2 medium in a 96-well plate. After a 24 h incubation to allow cell attachment, 0.1 µl of DMSO (vehicle) or 0.1 µl of resveratrol (a known SMC growth inhibitor) stock in DMSO was robotically transferred using Biomek FX (Beckman) from a resveratrol stock plate into cell culture /(mc+−mc−)] where sd = standard deviation; m = mean; c+ = positive control (resveratrol); c− = negative control (DMSO). A Z′ value of 0.5 is considered the minimal robustness for an assay to perform well in HTS The HTS assay of cell proliferation was performed to screen 447 compounds included in the NIH Clinical Collection using total six 96-well plates. Each compound was tested once by the addition of 0.1 µl of 10 mM stock dissolved in DMSO to yield a final concentration of 5 µM. Each plate contained 8 wells of negative controls added with DMSO  and 8 wells of positive controls added with resveratrol . HTS assays against the same NIH Clinical Library were conducted with either SMCs or ECs. Cell growth, robotic liquid handling, and fluorescence reading were conducted as described in the preceding paragraph except that the test compounds were transferred (using Biomek FX) from preconfigured stock plates. Robustness of a HTS assay is estimated by the Z′ value In order to compare differential effects of idarubicin on the proliferation of SMCs versus ECs, dose response experiments were carried out using the two cell types on the same 96-well plate. Assays were performed as described above except that idarubicin (or resveratrol) was added at serial dilutions into triplicate wells for each concentration. Curve fitting was performed with the Prism software (GraphPad).Balloon injury of the left common carotid artery was performed in Male Sprague-Dawley rats (300–350 g) following our previously described method Immediately after re-establishment of flow, idarubicin (50 µg) or DMSO  dissolved in 300 µl of 25% F127 pluronic gel (Sigma-Aldrich) was applied around the injured segment of the carotid artery . The pluronic gel is a biodegradable polymer, which is soluble in water at 4°C but becomes a gel when in contact with tissues at 37°C Nine evenly-spaced sections through each injured carotid artery were stained using routine hematoxylin and eosin (H&E) and images were collected with light microscopy. Intimal and medial areas, and circumferences were determined by measuring the internal elastic lamina (IEL), external elastic lamina (EEL), and lumen for each section using the ImageJ software  To assess re-endothelialization, immunostaining of CD31 (an EC marker) was performed on carotid sections following our previous report. Briefly, a goat anti-CD31 primary antibody  was incubated with the sections for 1 h followed by an incubation with a biotinylated rabbit-anti-goat secondary antibody for 30 min. Immunostaining of CD31 was then visualized by using streptavidin-HRP and DAB. Re-endothelialization was quantified following previously published methods P value is <0.05.All data are presented as mean ± standard error (SEM). Statistical analysis was performed using two-tailed unpaired Student’s t-test. Data are considered statistically significant when a In order to establish a HTS system that produces consistent outcomes, we first set out to optimize assay conditions. We chose proliferation as an assay since SMC proliferation is the central event in the development of intimal hyperplasia and EC proliferation is essential to re-endothelialization of the vessel lumen and prevention of thrombosis. We chose to use primary human SMCs and ECs so that our findings would be readily translatable; human primary cells are most relevant to human diseases. For the proliferation assay we chose Alamar Blue which has been widely used for measurement of cell number as a surrogate of proliferation. This reagent is easy to use, noncytotoxic, inexpensive We then applied these optimized conditions to test the consistency of our automated HTS system for proliferation using resveratrol as a positive control. We chose resveratrol because this natural compound has been previously shown to inhibit SMC proliferation and angioplasty-induced intimal hyperplasia We then utilized this HTS system to screen 447 FDA-approved drugs in the NIH Clinical Collection. All the compounds included in this collection are clinically used drugs with diverse bioactivities. We used a compound concentration of 5 µM. Six 96-well plates were used with 8 wells of negative control  and 8 wells of positive control (resveratrol) per plate . The oveAmong the 447 tested drugs, 11 inhibited human SMC proliferation more than 50%, producing a hit rate of ∼2.5% . We assuTo identify drugs that selectively inhibit the growth of SMCs versus ECs, we performed the same HTS assay against the NIH Clinical Collection using ECs. Z′ values for the endothelial assay calculated from six 96-well plates were all >0.7, indicating an excellent consistency. We then compared percent inhibition of EC proliferation to that of SMC proliferation for the 11 hits from the SMC assay. As shown in Idarubincin is a drug used for treating leukemia, but whether it has an inhibitory effect on intimal hyperplasia has not been reported. Prompted by its favorable property of selectively inhibiting SMC proliferation, we evaluated the ability of idarubicin to suppress intimal hyperplasia using an established rat carotid angioplasty model of restenosis (which mimics the post-angioplasty pathology in humans). In order to minimize undesirable side effects that could result from systemic drug delivery, we administered idarubicin locally around the common carotid artery following injury by balloon angioplasty. The morphometric data show that on day 14 after angioplasty, an aggressive neointimal plaque develops  growth These findings suggest that it is possible to identify drugs that differentially inhibit SMCs versus ECs. To this end, we have established a 96-well HTS format through test assays using resveratrol as a control drug and pilot screens using human SMCs and ECs. We began by optimizing cell number, treatment times and liquid handling protocols to maximize the signal to background ratio and Z′ See . The AlaWhile a HTS format of one well per compound is widely used in the literature, the rationale for using this format in our HTS study is several fold. First, prior to HTS we used 40 wells for each of the positive and negative controls to specifically determine well-to-well variation. A value of Z′ (0.65) above 0.5 indicated an excellent well-to-well consistency. Second, upon HTS assays we again confirmed a low variation between wells on each plate using 8 wells for each of the positive and negative controls Z′>0.7, . Thus thetc. In the primary screen we used a relatively low drug concentration (5 µM) to minimize nonspecific drug effects. In addition, 50% inhibition of SMC proliferation measured by Alamar Blue . Since mechanisms for selective inhibition of SMC versus EC proliferation are not well understood e.g. a wider concentration window for selective inhibition of SMCs versus ECs. Ultimately, further screening studies based on our HTS format using human SMCs and ECs will allow the discovery of highly selective and potent small molecule drugs for the purpose of developing safe, efficacious treatments for vascular restenosis.In sum, using human vascular cells we have established the first HTS format that is adaptable to large-scale screening with a specific goal of discovering novel compounds that selectively inhibit SMC versus EC proliferation. We have demonstrated the validity of this HTS assay, through a screen against the NIH Clinical Library and idarubicin was identified as a selective drug that preferentially suppresses SMC versus EC growth both Figure S1Time courses of the growth of HuAoSMCs seeded at different densities. SMCs were seeded at 1000 (blue), 2000 (green), or 3000 (red) cells/well on a 96-well plate, and cultured in SmGM-2 supplemented with 5% serum. Alamar Blue dye was added at different time points (to separate wells) and after a 24 h continued incubation fluorescence was read. A background reading from cell-free wells was subtracted.(TIF)Click here for additional data file.Figure S2Re-test of the initial hits from the HTS using Cell Titer Glo assay. Following the HTS assay of HuAoSMC proliferation, Alamar Blue dye was removed and the wells were gently washed by the automated system. The plates were then subjected to Cell Titer Glo assay, and percent inhibition of SMC proliferation by some of the initial 11 hits was compared between these two different assay methods.(TIF)Click here for additional data file.


Standard methods of DNA sequence analysis assume that sequences evolve independently, yet this assumption may not be appropriate for segmental duplications that exchange variants via interlocus gene conversion (IGC). Here, we use high quality multiple sequence alignments from well-annotated segmental duplications to systematically identify IGC signals in the human reference genome. Our analysis combines two complementary methods: (i) a paralog quartet method that uses DNA sequence simulations to identify a statistical excess of sites consistent with inter-paralog exchange, and (ii) the alignment-based method implemented in the GENECONV program. One-quarter (25.4%) of the paralog families in our analysis harbor clear IGC signals by the quartet approach. Using GENECONV, we identify 1477 gene conversion tracks that cumulatively span 1.54 Mb of the genome. Our analyses confirm the previously reported high rates of IGC in subtelomeric regions and Y-chromosome palindromes, and identify multiple novel IGC hotspots, including the pregnancy specific glycoproteins and the neuroblastoma breakpoint gene families. Although the duplication history of a paralog family is described by a single tree, we show that IGC has introduced incredible site-to-site variation in the evolutionary relationships among paralogs in the human genome. Our findings indicate that IGC has left significant footprints in patterns of sequence diversity across segmental duplications in the human genome, out-pacing the contributions of single base mutation by orders of magnitude. Collectively, the IGC signals we report comprise a catalog that will provide a critical reference for interpreting observed patterns of DNA sequence variation across duplicated genomic regions, including targets of recent adaptive evolution in humans. The ancestral lineage leading to humans and great apes experienced a surge in the rate of genomic duplication and deletion A second mechanism of NAHR is non-crossover associated interlocus gene conversion (IGC). During IGC, sequence from a donor SD is copied and pasted into the paralogous position of the acceptor SD. Unlike ectopic crossovers, IGC events do not introduce changes in copy number. In addition, the converted sequence is short, usually <500 bp Despite its limited footprint, both theoretical and empirical studies suggest that IGC is an important determinant of sequence variation in duplicated regions. IGC can transfer variants that arise on the genetic background of one locus to the novel genetic context of a paralogous position, thereby enabling the spread of new variants between duplicated sequences IGC also bears on the genetic analysis of duplicated regions. Models of evolutionary divergence assume that sequences evolve independently, but duplicated sequences actively exchanging variants via IGC violate this assumption. By homogenizing paralogous sequences, IGC can slow the apparent rate of molecular evolution between paralogs and invalidate the molecular clock hypothesis. Paralogous sequence divergence times are therefore likely to be underestimated in the face of IGC. These points may deserve particular consideration in analyses of duplicated sequences in the human genome. Recently duplicated genes are enriched for signals of positive selection Focused gene family studies and genome-wide analyses have shed light on the distribution of IGC in mammalian genomes. IGC is most frequent between high-identity tandem duplicates One barrier to a more comprehensive understanding of the frequency and distribution of IGC is the difficulty of properly annotating and assembling duplicated genomic regions. High-identity SDs are often mis-placed or collapsed to a single locus in draft genomes. The active homogenization of duplicated sequences by IGC may further exacerbate this challenge. However, since the initial release of the human genome, SDs have been the overwhelming focus of targeted re-sequencing, BAC-based assembly, and manual curation genomicSuperDups table from the UCSC Table Browser to identify 1419 families of ≥4 paralogous sequences (mean number of sequences per family = 8.4) in the human reference genome (GRCh37). We limited our focus to large SDs >10 kb (mean sequence length = 34.7 kb) to ensure an adequate number of informative sites in tests of historical recombination (see below). Owing to the hierarchical, nested organization of SDs across the human genome, there is considerable sequence redundancy between SD families. Collectively, sequences within these families span 52.4 Mb of the human genome (1.7%) with each family surveying an average 29.4 kb of duplicated sequence not present in any other SD family. This initial dataset includes sequences overlapping 712 genes, with 205 genes fully contained within analyzed sequences.We used the L-INS-i algorithm implemented in mafft (v. 6.951) We constructed a high quality multiple sequence alignment for each SD family using the . We excluded an additional 34 SD families for which neighbor-joining tree topologies based on an F84 DNA distance matrix were not equivalent to the maximum likelihood topologies To infer the evolutionary relationships between paralogous sequences within each SD family, we constructed maximum likelihood unrooted trees using PhyML (v3.0) e.g., microhomology-mediated break-induced replication (MMBIR)) over time will generate complex, interwoven switches in the evolutionary history across a multiple sequence alignment Reticulate sites” (R sites). In addition to interlocus recombination, R sites can also arise from independent mutation to the same nucleotide at paralogous positions. Most double mutation events, however, are expected to result in three bases at a given site in the alignment. We refer to these sites of obligate double mutation as “Bimutational Sites” (B sites). Informative sites consistent with the dominant tree topology are classified as “Concordant sites” (C sites).Although the evolutionary history of each sequence family is summarized by a single dominant tree topology (which presumably mirrors the duplication history of the sequences), individual sites within the alignment may be discordant with the tree. As illustrated in N paralogous sequences is comprised of An SD family with Table S2).We evaluate the hypothesis of historical IGC among sequences in a given quartet by comparing the observed number of R sites to the number expected in the absence of interlocus exchange. This null expectation was derived by simulating 100 sequence datasets along the ML tree using Seq-Gen (v1.3.3) P-values for comparisons of the observed and expected numbers of R sites were computed using a strategy that accounts for uncertainty in both values. First, each draw from the bootstrap distribution of observed R sites was paired with a draw from the simulated distribution of R sites . If the simulated distribution is a good approximation to the observed distribution, the difference between these values should be zero on average. On the other hand, if more R sites are observed than expected according to our simulation, the difference between the observed and simulated R site counts should be greater than zero. P-values were calculated as the fraction of 100 paired observations for which the simulated number of R sites was greater than the corresponding value from the bootstrap distribution.Approximate pairwise output option. Tracks with a global or pairwise P-value <0.05 were considered significant and included in follow-up analyses.We used the program GENECONV to confirm the general trends documented by the quartet method and identify IGC events that may have gone undetected by the method l, we sampled one of the 1246 alignments, with sampling probabilities weighted by the total alignment length, L. Within the sampled alignment, two sequences were selected at random to undergo gene conversion. The start position of the simulated gene conversion track between these two aligned sequences was then drawn from a uniform distribution on . This procedure was repeated for each unique gene conversion track identified. A total of 1000 simulated datasets were generated, with each simulated dataset composed of a length-matched “track” for each unique gene conversion track in the actual data. We compared the observed number of tracks overlapping each tested genomic context to the distribution obtained from the 1000 simulated datasets. P-values were calculated as the quantile position of the observed value along the corresponding distribution of simulated values.We performed a series of simulations to test for an enrichment of GENECONV tracks in repetitive elements and across several genomic contexts, including: (i) transcribed, coding, and pseudogenic regions, (ii) between pericentromeric and subtelomeric duplicons, (iii) between intra- and inter-chromosomal SDs, and (iv) between paralogs with high sequence identities. Simulations were complicated by the considerable sequence redundancy and overlap among SD families. Namely, many apparent gene conversion events localizing to identical genomic coordinates were identified in distinct SD families. Inclusion of duplicate tracks in the analysis would lead us to double count any sequence features overlapping the tracks, potentially introducing erroneous signals of enrichment. We first filtered out these duplicate tracks to obtain a set of unique, non-overlapping tracks. For a given unique gene conversion track of length We extended a previously described phylogenetic quartet method P-value ≤0.05; , Table S2). Strikingly, 874 families (71.9%) had more observed R sites than expected, amounting to an average 53.2% excess relative to the simulated expectation.For each SD family, we compared the proportion of informative sites in the alignment that is reticulate to the proportion of R sites recovered in the simulated datasets. Thirty-one (2.5%) of the 1246 examined paralogous sequence families harbored no R sites and were not considered further. Of the remaining 1215 SD families, 25.4% (n = 309) had a significantly higher fraction of R sites than expected in the absence of non-allelic recombination (one-sided Table S4). This suggests that (26.1–18.2)/2 = 4% of duplicated sites in the human genome have been involved in a historical IGC event, consistent with a previous estimates from a smaller dataset In the absence of ectopic recombination and parallel mutation (see below), each site within an SD family alignment should be concordant with the duplication history of the locus. In contrast, we observe marked site-to-site fluctuations in topology across alignments Figure 3Table S2). Third, if R sites are largely due to stochastic mutational processes, they should be randomly distributed across alignments. In contrast, R sites that arise from interlocus recombination will tend to cluster within an alignment. We tested this hypothesis by applying a runs test P-value <0.05; Table S4), consistent with action of historical recombination between paralogs. Although complex mutational mechanisms can affect multiple nucleotides in a single event, these processes are overwhelmingly restricted to sites within a 10 bp window In addition to non-allelic recombination, R sites can also arise by independent mutation to the same nucleotide at paralogous positions. Although such double mutation events should be rare for the closely related sequences in our dataset, we took three measures to rule out this possible explanation. First, we replicated our analysis on alignments stripped of hypermutable CpG dinucleotides. Despite a reduction in power, a comparable number of SD families still harbor a significant excess of R sites . Because few B sites are predicted to occur by parallel mutation events in these sequence quartets, we are confident that the majority of observed R sites arose from inter-paralog genetic exchanges, especially given that our method grossly over-predicts the number of B sites . We identified 67 clusters of ≥1 consecutive R site(s) embedded within these quartet alignments . Such patterns could arise from multiple overlapping IGC events with variable track lengths, as might be predicted if events localize to discrete hotspots. Alternatively, clusters of tree switches could arise from single, complex repair events with DNA synthesis off multiple paralogs . Sub-telomeric repeats are also established hotspots of interchromosomal recombination Table S2). Numerous recombination breakpoints between subtelomeric sequences were identified in analyses of site patterns across CpG negative sequence alignments, confirming these regions as hotspots of interlocus recombination . Multiple pathogenic, NAHR-mediated rearrangements have been described in the defensin gene cluster on 8p23.1, and this locus displays considerable copy number polymorphism in human populations Our sequence quartet analysis confirms several previously reported signals of interlocus recombination in the human genome. Palindromic sequences in the male-specific region of the Y chromosome are known to undergo high rates of IGC nation ; ; S6. MulBeyond these classic examples of NAHR in the human genome, our analysis uncovers many novel signals. We highlight three compelling examples in the sections below.NBPF gene family consists of 22 genes and pseudogenes clustered in the 1p36, 1p12, and 1q21 regions. This gene family expanded during primate evolution and recent duplicates appear to be targets of adaptive evolution NBPF24, NBPF16, NBPF9, and NBPF14 are clustered in the 1q21 region and maximum likelihood phylogenetic inference suggests that NBPF24 and NBPF16 duplicated most recently (Table S3). Nine of these R sites support the alternative topology that places NBPF16 and NBPF14 as most closely related. These nine R sites are organized into five distinct clusters, including three that are complex and possibly the result of multiple IGC events or replication-based mechanisms of repair  of the informative sites across these loci may have been subject to historical interlocus exchanges . The reduced rate of molecular evolution along the GOLGA6L10 and GOLGA6L5 branches could be attributable to purifying selection, but it is difficult to reconcile the excess of R sites with this hypothesis. Alternatively, frequent IGC between these two paralogs could jointly explain the observed excess of R sites and reduced branch lengths.One consequence of IGC is a reduction in inter-paralog sequence divergence, which may manifest as a decrease in the rate of molecular evolution between duplicates. We find tentative evidence of this phenomenon in the Golgin subfamily A member 6-like gene family. Paralogs in this SD family are clustered on 15q25 with an average pairwise sequence identity of 97.5%. R sites account for 54.8% of informative sites in this alignment (95% bootstrap CI: 0.43–0.67), a ∼1.7-fold excess relative to the fraction expected the absence of interlocus recombination between paralogs . Of the 40 R sites in this alignment, 25 support a sister relationship between PSG) gene family on 19q13 show strong signals of historical interlocus recombination in our analysis. These poorly characterized genes belong to the immunoglobulin superfamily and are thought to be integral to the maintenance of pregnancy PSG sequences, each with a 1.03- to 2.83-fold excess of R sites compared with expectations derived from simulations . We used high quality SNP calls from the 1000 Genomes Project (release 20110521) PSG genes (Table S7), including 99 with minor allele frequency >0.01 in both paralogs. Average pairwise diversity between these genes is 94–95%, and incorrect read mappings may introduce spurious signals of shared polymorphism between paralogs. Nevertheless, interlocus recombination, not mutation, may account for upward of 11% of observed polymorphic variants in PSG genes (491 shared polymorphisms out of 4341 observed SNPs). Many genes involved in reproduction evolve rapidly PSG gene family. Indeed, global pairwise alignments with chimpanzee orthologs (panTro3) indicate pairwise sequence divergences between 2.2 and 6% across transcribed portions of PSG genes, considerably higher than average human-chimpanzee pairwise divergence across the genome (1.2%) PSG gene family could provide an exciting opportunity to study the interplay of IGC and diversifying selection, a prospect that motivates the analysis of high-quality clone-based sequences from this region in diverse human populations and non-human great ape species.The exchange of sequence variants between paralogous genomic regions can also increase haplotype diversity within loci Although the quartet method permits identification of SD families with an excess of R sites consistent with historical non-allelic recombination, it does not enable straightforward identification of the particular sequences driving the signal. In addition, because the method relies on the detection of sites that are discordant with the tree, interlocus exchanges between sister sequence pairs are rendered invisible. Moreover, the quartet method cannot be used to confidently resolve conversion tracks between divergent paralogs with many R sites predicted to occur by mutation. To expand our catalog of historical IGC events, we combined interlocus recombination signals from the quartet method with IGC tracks identified using a second complementary alignment-based approach that is insensitive to these limitations.P value <0.05. Six-hundred and sixty-six SD families (53.4%) have a least one significant track (Table S8), including 183 SD families that also harbor a significant excess of R sites consistent with high levels of historical interlocus recombination.Barring subsequent mutation, sequences involved in IGC events will share complete sequence identity across conversion tracks. We used the program GENECONV to find unusually long stretches of perfect sequence identity between individual sequence pairs, conditional on the overall pattern of variable sites within the alignment (P = 0.012 by 1000 randomizations of the data), many SD families are flagged by only one of the two approaches. Indeed, only 12/67 of the putative gene conversion tracks identified in the analysis of CpG minus sequence quartets above were also identified by GENECONV. To ensure that GENECONV signals are not dominated by false positives, we used simulated sequence datasets generated in the absence of interlocus exchange as input into the program. We recovered ∼80 gene conversion tracks across a full set of 1246 simulated alignments . GENECONV yields a low frequency of Type I errors in our dataset, consistent with earlier investigations on the performance of this method Although we observe a greater degree of consensus between GENECONV and the quartet methods than expected by chance (Text S1). The median conversion track length is 198 bp (mean = 385 bp) with tracks ranging in length from 3 bp to 15.0 Kb. A small percentage of tracks  are >5 kb, suggesting a low frequency of long track gene conversion. Collectively, tracks identified in the GENECONV analysis cover 1.54 Mb of sequence, or 4% of the genomic space surveyed in our analysis. Therefore, at least 0.77 Mb of sequence in the human reference genome has been directly converted by IGC.Owing to the high degree of sequence overlap and redundancy among SD families, many tracks identified by GENECONV co-occur in multiple families. We filtered out these redundant tracks to obtain a final set of 1477 unique gene conversion events, corresponding to one interlocus gene conversion event per 53 kb of analyzed sequence . IGC tracks are preferentially located in repetitive elements, and enriched for the recombinogenic PRDM9 binding motif, CCnCCnTnnCCnC Table 1 P<0.001 by comparison of mean track length across 1000 bootstrap replicates), as well as the average track length of interchromosomal exchange tracks  of These caveats aside, we can make several simplifying assumptions to derive order-of-magnitude approximations to the per generation rate of IGC in SD families. First, we assume that each paralog within a family is equally likely to be converted by any of the −7 to 1.05×10−5 per duplicated site per generation (average: 3.2×10−6), assuming μ = 1.2×10−8 mutations/site/generation (Table S2) de novo single base substitution is predicted across the duplicated sequences included in our analysis. Extrapolating these numbers to include all duplicated sites in the human genome, not just those that passed our analysis filters, yields an estimated 260 converted and two mutated sites per generation. Our calculations invoke multiple simplifying assumptions that are unlikely met in reality, and we caution against the over interpretation of these numbers. Nonetheless, it is clear that more sites in duplicated regions are converted by IGC than are targeted by de novo point mutation each generation.For the SD families studied here, IGC rates range from 6.1×10S8). In contrast to earlier studies, our analysis is not restricted to transcribed portions of the genome We applied two complementary strategies to comprehensively catalog signals of interlocus recombination between paralogous sequences in the human reference genome . Given that few IGC tracks are identified by both methods, we conclude that between 4–8% of duplicated sites in the human genome have engaged in non-allelic recombination. Notably, this fraction is consistent with other surveys of IGC in the human genome Twenty-five percent of the SD families included in our quartet analysis have a significant excess of R sites. Failure to detect an excess of R sites in the remaining 75% of SD families, however, does not exclude a role for interlocus recombination in the evolution of these paralogous sequences. Indeed, nearly all SD families have more than the predicted number of R sites, even though a minority is significant by this method Figure 2et al.A previous study used the quartet method to assess interlocus exchange patterns across a set of 30 paralog families in the human genome and identified a significant excess of R sites in 28/30 tested alignments P-values calculated from alignment-wide trends, the fine-scale distribution of R sites across an alignment  NBPF and GOLGA6L families highlighted above, as well as the LRRC37A, POM121L4, ANKRD20 and SPDYE5 gene families have been focal points for recurrent duplication and transcript innovation in human-great ape evolution e.g.Many SD families in our analysis have undergone recent expansions during primate evolution Click here for additional data file.Figure S2GOLGA6L duplicons. (A) The maximum likelihood tree relating human paralogs LOC440295, GOLGA6L9, GOLGA6L5, and GOLGA6L10 is shown. The value at the interior node of the tree is the fraction of bootstrap replicates (n = 100) supporting the topology. (B) The observed number of C sites and the number of R sites supporting the two alternate topologies are plotted, with 95% bootstrap confidence intervals (circles). Simulation derived expected values are also shown, with central 95% range of simulated values (squares).IGC and the evolution of (PDF)Click here for additional data file.Table S1SD families excluded because of low confidence tree.(XLS)Click here for additional data file.Table S2Phylogenetic quartet analysis results.(XLS)Click here for additional data file.Table S3Results from phylogenetic quartet analysis on CpG-minus alignments.(XLS)Click here for additional data file.Table S4Clustering of R sites in CpG-minus sequence quartets with <5 predicted B sites.(TXT)Click here for additional data file.Table S5Putative gene conversion tracks identified in CpG-minus quartet alignments.(XLS)Click here for additional data file.Table S6Putative non-allelic crossover events in CpG-minus quartet alignments.(XLS)Click here for additional data file.Table S7Shared SNPs in PSG genes identified from 1000 Genomes data.(XLS)Click here for additional data file.Table S8Coordinates of IGC tracks identified by GENECONV.(XLS)Click here for additional data file.Text S1Bed format custom UCSC Genome Browser track for visualizing gene conversion events detected by GENECONV.(TXT)Click here for additional data file.


Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus), in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies.We extracted peripheral blood DNA from a cohort of 540 HIV-1-positive patients  and determined the presence of XMRV and related viruses using TaqMan PCR. While we were able to amplify as few as 5 copies of positive control DNA, we did not find any positive samples in the patient cohort.In view of these negative findings in this highly susceptible group, we conclude that it is unlikely that XMRV or related viruses are circulating at a significant level, if at all, in HIV-1-positive patients in London or in the general population. Xenotropic murine leukaemia viruses (X-MLVs) are a class of endogenous gamma retroviruses. Xenotropic murine leukaemia virus-related virus (XMRV) is an X-MLV that has been detected in human samples and as such, is potentially the first gammaretrovirus to infect humans. XMRV has been detected in samples from patients with diseases such as prostate cancer and chronic fatigue syndrome, and has also been detected in 1–7% of healthy controls tested in the same studies Here we set out to assess the prevalence of X-MLVs, including XMRV, in an HIV-1-positive patient cohort in London. HIV-1-positive patients were investigated because those who have been infected by a sexual route, by intravenous drug use, by perinatal infection or iatrogenically are likely to have been at greater risk than the general population of other viral infections spread by similar routes  The University College London Research Ethics Committee has specifically exempted this study from review because it was an assay development, and waived the need for consent due to the fact the patient material used was fully anonymised.Samples were collected from consecutive patients attending Mortimer Market Centre HIV service over a period of two months and were anonymised before processing. Patients were 4.5∶1 male:female, age range 15–85 yrs, median age 42 yrs, <1% intravenous drug users, 15% were born outside the UK. 80% of patients were on highly active anti-retroviral therapy (HAART); 94% of those treated had a viral load <50 copies/ml. Approximately 8 ml blood was collected into a BD-vacutainer containing EDTA, and stored at 4°C until processing. Genomic DNA from the buffy coat fraction was extracted using the QIAamp DNA kit (Qiagen) and eluted in 60 µl.int sequence for primer set 1 or Balb/c mouse DNA (Sigma D4416) for primer set 2 (X-MLV-gag). Cycling conditions were 95°C for 15 secs and annealing/extension at 60°C for 1 min after an initial denaturation of 10 min. The HIV-1 positive samples were checked for PCR inhibitors by amplification of GAPDH as previously described TaqMan PCR of genomic DNA using primer sets 1 and 2 were performed as described , Table 1int) and 2 (X-MLV-gag)  as few as 5 copies of the XMRV-int plasmid and 0.2 pg (1/20th of a genome) of Balb/c DNA respectively (In order to assess the claim that XMRV infection is common in the human population we screened leukocyte DNA purified from anonymised blood samples of 540 HIV-1-positive patients visiting Mortimer Market HIV service. Approximately 20% of this cohort (∼108 patients) had not been treated with any anti-retroviral therapy. We performed TaqMan PCR using previously described primer sets 1 (XMRV-MLV-gag) , Table 1ectively .To assess the sensitivity of the PCRs when detecting XMRV integrated into genomic DNA, we extracted DNA from 22Rv1 cells. This cell line contains around 10 copies of the XMRV provirus 3 genomes). We did not detect positive PCR signals from any of the patient DNA samples using either primer sets 1 or 2, indicating that neither XMRV nor any other X-MLV amplifiable with these primers were detectable in these samples. All samples were positive for GAPDH by TaqMan PCR at expected levels, showing that no PCR inhibitors were present.In the HIV patient screen, the quantity of leukocyte DNA per patient per PCR reaction averaged 362 ng (IQR 188–468 ng, 54–135×10In order to establish whether XMRV infects the human population and whether it is associated with human disease it is extremely important to be able to detect and quantitate XMRV specifically and sensitively. The majority of screens carried out so far have used highly sensitive nested-PCR protocols gag or env sequences. 101 of the patients tested were not receiving antiretroviral drugs. Similarly, Kunstman et al We could not detect XMRV or any other X-MLV sequences in HIV-1-positive patients using TaqMan PCR, suggesting that XMRV and related viruses are either entirely absent or at least extremely uncommon in this population cohort. There are several possible explanations for the discrepancy between this and some previous studies, including that XMRV does not in fact establish infection in human peripheral blood at detectable levels, or that the geographic differences between the cohort studied here and elsewhere are critical. Alternatively, the possibility that the positive findings reported by others were due to occult laboratory contamination should be seriously considered gag sequences, allowed us to detect viruses closely related to XMRV in addition to XMRV itself. By assaying DNA rather than RNA we sought proviral DNA rather than evidence for active replication. Nucleic acid testing remains the gold-standard for monitoring blood-borne viral diseases such as HIV and HCV, and is more specific than serological tests. Sequencing positive PCR products provides confirmatory evidence, and can illuminate when contamination has occurred At the current time there is no consensus on definitive strategies for testing for XMRV, or X-MLVs, and it remains unclear why the results of published studies differ so widely in their reported prevalence of XMRV in patient populations and healthy controls, although several independent studies have demonstrated how PCR contamination from mouse DNA or DNA from human cell lines infected with xenotropic MLVs might explain XMRV detection In conclusion, this study failed to find any evidence of XMRV or X-MLV infection in a cohort of HIV-1-positive patients. In view of these negative findings in this highly susceptible group, we conclude that it is unlikely that XMRV or related viruses are circulating at a significant level, if at all, in HIV-1-positive patients in London or the healthy population.