Microarray data normalization is an important step for obtaining data that are reliable and usable for subsequent analysis. One of the most commonly utilized normalization techniques is the locally weighted scatterplot smoothing (LOWESS) algorithm. However, a much overlooked concern with the LOWESS normalization strategy deals with choosing the appropriate parameters. Parameters are usually chosen arbitrarily, which may reduce the efficiency of the normalization and result in non-optimally normalized data. Thus, there is a need to explore LOWESS parameter selection in greater detail.In this work, we discuss how to choose parameters for the LOWESS method. Moreover, we present an optimization approach for obtaining the fraction of data points utilized in the local regression and analyze results for local print-tip normalization. The optimization procedure determines the bandwidth parameter for the local regression by minimizing a cost function that represents the mean-squared difference between the LOWESS estimates and the normalization reference level. We demonstrate the utility of the systematic parameter selection using two publicly available data sets. The first data set consists of three self versus self hybridizations, which allow for a quantitative study of the optimization method. The second data set contains a collection of DNA microarray data from a breast cancer study utilizing four breast cancer cell lines. Our results show that different parameter choices for the bandwidth window yield dramatically different calibration results in both studies.Results derived from the self versus self experiment indicate that the proposed optimization approach is a plausible solution for estimating the LOWESS parameters, while results from the breast cancer experiment show that the optimization procedure is readily applicable to real-life microarray data normalization. In summary, the systematic approach to obtain critical parameters in the LOWESS technique is likely to produce data that optimally meets assumptions made in the data preprocessing step and thereby makes studies utilizing the LOWESS method unambiguous and easier to repeat. DNA microarray technology has become a standard tool in biomedical research for large-scale transcriptional monitoring . A growif of neighboring samples to be included in the weighted polynomial fit is particularly sensitive and its variation greatly affects the normalization results. This parameter should be carefully chosen through a systematic procedure where experimental assumptions are clearly specified. Benefits in the normalization process may be considered to be small in their own right, but these improvements are extremely meaningful in the context of searching for subtle biological differences in gene expression.Microarray data preprocessing contains three phases: quality control, within-slide normalization, and between-slide normalization. Within-slide normalization aims to correct dye incorporation differences which affects all the genes similarly, or genes with the same intensity similarly . One scaf in print-tip LOWESS normalization. Results are compared to common, arbitrary selections of f. The proposed procedure first examines a case study where we have utilized three quality filtered, self versus self hybridization experiments. With self versus self experiments, we are able to clearly detect normalization differences. Such analysis also verifies that the optimized method produces properly calibrated ratios. Our proposed technique is also demonstrated on a typical set of quality filtered microarray data. We utilize a set of breast cancer data that has replicated measurements for four different tumor cell lines [f. Our self versus self data, including the original TIFF images, are available online [We outline an optimization-based procedure for obtaining a systematic value for ll lines . In addie online  and the e online . denote the background corrected selection for the intensity of the jth gene of the Cy3 (green) colored sample. Similarly, let  denote the jth gene of the Cy5 (red) colored sample. One key issue for the dyes is that they are consistently imbalanced [Within-slide normalization is used to correct the dye intensity errors introduced across one microarray slide. The result of this step provides the normalized, calibrated ratios. Let balanced ,13. Diffbalanced ,24. Dye n total gene expression ratios and we denote the observed vector of ratios for a single experiment as r ∈ ℝn × 1. The calibrated ratio of expression for each gene is obtained by dividing the test by the reference sample intensities with the proper normalization factor in the denominator,For a single microarray experiment, there are i = 1, 2..., n, where n is the total number of spots on a microarray. The normalization factor, denoted by Φ(·), is a function of data-dependent variables. If the dyes are linearly dependent, it can be assumed that the normalization function is a constant, namely Φ(·) = φ. Many studies have looked at linear dependencies [for ndencies , as wellMany studies perform within-slide normalization in a global manner by assuming the error effects are stationary across an entire slide. This is currently true for the cases of Affymetrix GeneChip or Agilent oligonucleotide microarrays. For cDNA microarrays, however, the sources of variation typically originate in a localized or spatial manner , mainly et al. [Ig, Ir)-scatterplot in log2-space for determining the value of Φ(·). It has been suggested in separate works by Dudoit et al. [et al. [2-based scatterplot of the average fluorescence intensity A versus the transformed ratio M should be used instead of a simple, log2-based intensity scatterplot. This type of scatterplot is commonly known as a Bland-Altman plot in the statistics literature. The values for A and M are given as,One of the most widely used nonlinear correction techniques is the LOWESS method, which was first applied to microarray data by Yang et al. . The maiet al. . Subsequt et al.  and Yang [et al.  that a li = 1, 2,..., n. Equations (2) and (3) are preferred over the original intensities because the (A, M)-scatterplot may reveal artifacts that are not clearly visible in the ordinary intensity scatterplot. Such a transformation represents a scaled, 45° rotation of the (Ig, Ir)-coordinate system [for e system .Mj = g(Aj) + εj, where the jth transformed ratio Mj is a function of the corresponding overall intensity Aj and a zero mean random variable εj. The smoothed point at Aj using LOWESS with a degree d polynomial is (Aj, ), where  is the fitted value of the regression. The LOWESS estimate, , is a weighted linear combination of the MiThe smoothing procedure has been designed to accommodate measured scatterplot data obeying the form hi(Aj) depend on Ai, ∀i, but not on the Mi. The LOWESS algorithm contains four data-specific parameters, namely the polynomial order d, the number of LOWESS algorithmic iterations t, the weight function w(·), and the fraction of the data points used in the local regression f. Consequently, these parameters all affect the values of the weights hi(Aj) in Eq. (4). For a complete outline of the LOWESS algorithm, consult [d = 0, 1, or 2, depending on the choice of (Ig, Ir)- or (A, M)-coordinate systems, the tri-cube weight function is quite standardized for all types of data [t = 3. The final parameter must be chosen where f ∈ . The value of each ψk,i(·) is a function of experiment-specific parameters such as temperature or other environment settings which may differ from sample to sample in a single experiment. Accordingly, the cost function to be minimized for the kth print-tip group across all transformed ratios isFor a microarray experiment, there are a total of ℓ print-tips used on a single slide. In order to reliably determine the value of fk ∈  is unknown, reliable estimates that reflect experiment-specific assumptions may be used. Usually there are tens of thousands of genes in a microarray study and a plausible assumption is that the mean of the log2-transformed ratios after normalization is zero. Also, in a variety of experiments, platform-dependent control transcripts that are known to have certain expression at a constant level may be utilized in the optimized approach. Furthermore, in our breast cancer case study we show how to obtain statistically reliable estimates of ψk,i(·) from replicate slides. We also show how our approach may be used if replicates are not available for typical microarray studies. Ultimately, the optimized approach requires experimenters to explicitly state their assumptions behind the study, which is systematically better than arbitrarily choosing parameter values. In addition, determining an experiment-specific fk by trial and error may be time consuming and will oftentimes lead to non-optimal results. The chosen optimization algorithm for minimizing the corresponding cost function is based on a combination of golden-section search and successive parabolic interpolation as outlined by Forsythe et al. [fk for minimizing δk(fk) for each print-tip, k = 1,..., ℓ within a tolerance of ±0.01. Each print-tip, resultingly, may have a different, optimal bandwidth parameter.with the constraint that e et al. . This ap have been obtained, calibrating the intensities for all the Ak,i is given asAfter the estimates i = 1,..., nk, and k = 1,..., ℓ. For the local LOWESS normalization within each print-tip group, the issue of how the total intensities are spread about the sample mean for the group becomes a factor to consider when normalizing the data [2-ratios from the different print-tip groups are usually centered around zero. Some print-tips may have larger variances compared to others and an appropriate scale adjustment is needed to account for such differences. One proposed approach is to find the maximum likelihood estimate for the scale of the variance for each print-tip group [2-ratios from the kth print-tip group follow a normal distribution with mean zero and variance σ2, where σ2 is the variance of the true log2-ratios and  is the estimated scale factor for the kth print-tip group. However, this is only valid for certain types of data that reasonably follow a normal distribution and in our work we observe that this assumption may often times lead to undesirable results. Refer to [for the data . After nip group . This meRefer to  for furtAnother approach proposed here that is able to deal with the variance scaling issue is to introduce a weighting factor in the calibration function that is of the formi = 1,..., nk, k = 1,..., ℓ, and where the weight is given as . The bias-corrected sample variance for the kth print-tip is denoted by  and is given asfor  denotes the sample mean for print-tip k. Furthermore, the minimum sample variance is given aswhere λk takes values over , we have obtained nearly identical sample means, but less total variance for the resulting data compared to previously published techniques. The computation of λk is straightforward and easy to calculate but our novel variance stabilization procedure does not take into account any heteroscedasticity in the data, namely observed increasing ratio variance with decreasing measurement intensity A. A rigorous comparison of print-tip scaling is beyond the scope of this contribution, but it is noted that the different scaling procedures affect the overall calibration scheme.Compared to the maximum likelihood method outlined by , this meet al. [et al. [et al. [fk was performed and print-tip LOWESS normalization results are compared to the results using arbitrary choices of the parameter fk. The implementation took a few minutes to run on a standard desktop PC running MATLAB.To demonstrate the utility of our optimized LOWESS normalization procedure, we first utilized a set of three self versus self experiments , BT-474,et al. . For eac [et al.  and the  [et al. . Optimiz2 - log2 space should be distributed along a straight line that intersects zero with a slope of unity. In the (A, M)-coordinate system, all values of M should lie on a straight line at M = 0 for all values of A; this means that the calibrated ratios should ideally be unity for all variables. Correspondingly, the cost measure is given when ψk,i(·) = 0, (∀k, i), in Eq. (5) for the (A, M)-coordinate systems. Separate trials were conducted using weighted, zeroth-order (d = 0), first-order (d = 1), and quadratic (d = 2) polynomial fits. For all trials, the number of print-tip LOWESS iterations was fixed at t = 3. The weight function used is given by Cleveland [fk. As a comparison to arbitrary selections of fk, the print-tip normalization was also carried out using fk = 0.2, 0.4, 0.6, and 0.8 in separate trials. Figure M(Arb), M(Opt))-scatterplot comparison between the calibration results with d = 1 using optimal fk and arbitrary fk for the BT-474 self versus self experiment. The points that deviate from the blue line are the genes that are most affected by the choice of fk. The MArb)  surely will not be equal to zero for all genes. Accordingly, implementing the cost function in Eq. (5) becomes an immediate challenge since the normalization reference level of all the genes for a typical microarray experiment may be diffcult to determine with complete accuracy. We note that our cost function still may be used with the assumption that the sample mean for each print tip before log2-transformation is unity. In most microarray experiments, many genes may be assumed to have constant RNA concentrations while smaller numbers of genes may be over or under expressed, namely their sample mean over all the genes is zero, . Using this assumption in Eq. (5), our experiments show that by minimizing the cost function in this context, like in the self versus self case study, we are able to systematically choose fk and the only consequence is that the minimum of the cost will not be as low as in the self versus self scenario where all genes should be constantly expressed. The main benefit of utilizing LOWESS for microarray normalization is that it is robust to extreme outliers and the cost function implemented in this fashion further restricts the effects of such extreme points in the regression. Ultimately, this implementation results in reliably calibrated ratios compared to the arbitrary formulation where different choices of fk affect the resulting data.One immediate concern for typical experimental microarray data is that many genes may be over- or under-expressed and the true, transformed gene expression ratio fk. After an initial round of optimized LOWESS normalization for each replicate slide with ψk,i(·) = 0 in Eq. (5), the sample mean for each gene may then be calculated using the replicates. The normalization reference levels ψk,i(·) were reassigned these average gene expression values in Eq. (5). Each experiment was then separately calibrated a second and final time using the optimization approach and the final results were noticeably different compared to the normalized data using f = 0.2 that Järvinen et al. posted on their website [M = 0 in the (A, M)-scatterplot if the cost function approach is used. Consequently, the calibrated data reach a stable domain with a small dynamic range. Empirically, we found that performing optimized normalization in an iterative manner will not propagate regression effects through to disrupt the underlying structure of the data.Since a single microarray experiment represents an observation, multiple observations would be needed to compute a reliable estimate of the true transformed ratio values. The use of only a small number of replicate slides may be satisfactorily used to determine reliable estimates of true gene expression and one study showed that three replicates suffce for significantly reducing experimental variability . With th website . A notewfk for the first replicate BT-474 hybridization. Some genes in this plot report 4-fold differences and ultimately these differences affect data analysis. Consequently, the errant choice of this parameter fk may have deleterious effects on different biological studies. To illustrate the differences for one representative print-tip in this breast cancer study for the first replicate of the BT-474 cell line, Figure k = 16 are highlighted in black. In this plot, we show that the regression obtained by the optimized choice of f16 differs from the one obtained by arbitrarily selecting f16 = 0.2 and the calibration results are thus affected. Figure fk.Figure kth print-tip groupAs further illustration of the calibration differences between the optimized and arbitrary calibration results, we employ a goodness-of-fit test . We wishM(Arb) and M(Opt) are the arbitrary and optimized calibration results, and the denominator within the summation is simply the variance of the difference between M(Arb) and M(Opt). The null hypothesis is defined to be H0: the normalized ratios using arbitrary f are comparable to ones using optimized f. We tested against p < 0.05 for the  distribution and reported the alternative hypothesis for a few print-tip groups on almost all the slides. In this analysis, we compared optimized choices of f for each print-tip to the arbitrary choices f = 0.2, 0.4, 0.6, and 0.8. By looking across each replicate of the calibrated data for all four breast cancer cell lines, almost all slides in this study reported at least one print-tip to have statistically different calibration results based on the choice of fk. Often times a single slide would report two or three print-tip groups that had statistically different calibration results.where et al. [homeo box B7, which was validated with RT-PCR [v-erb-b2, which is known to be over-expressed in the BT-474 cell line [et al. [homeo box B7 gene shows that it falls within the top 18% of overall gene expression, but by using the optimized approach we report it to be within the top 13%. For the v-erb-b2 gene, both calibration techniques report that this gene falls within the top 1% of the genes in terms of expression. As a result, for the homeo box B7 gene, the calibration factor fk is responsible for about 5% change in the reported gene expression. This is a dramatic result that may influence how the expression for this gene may be interpreted in comparison to the accepted biological knowledge of a certain experiment. As public data from microarray experiments continues to become available, the knowledge of certain genes will undoubtedly be uncovered for well-studied cell lines and this information will help further assess normalization and microarray quality control tasks.In addition to statistical analysis, genes that exhibit known over-expression in the BT-474 cell line data  were selet al. , our reset al. . Two genh RT-PCR , and v-eell line . The res [et al.  for caliet al. [f was chosen arbitrarily and its value was not reported in the study. Consequently, reproduction and verification of these results may be diffcult. For instance, some of the important biological findings, such as start and end points of amplifications and deletions, may be adversely affected by different choices of f.The LOWESS method has recently been applied in other applications for the biological sciences. Comparative genomic hybridization (CGH) is a molecular cytogenetic method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and they reveal a characteristic pattern that includes mutations at chromosomal and subchromosomal levels. Our proposed optimized scheme is directly applicable to the application of calibrating CGH microarray experiments, as well as for data analysis aspects. For example, the work of Clark et al.  utilizedet al. [f result in noticeably different correction effects and the optimization method proposed here may be suitable for enhancing such a study. Adaptations to the cost function may be utilized to handle this type of data. In addition, analysis of other types of scatterplot data by utilizing the LOWESS method with an arbitrary choice for the bandwidth parameter is undoubtedly susceptible to varied interpretations or errant conclusions [In addition to CGH analysis, LOWESS has found application in case-control studies where logistic regression has been used to model the relationship between binary responses and continuous predictor variables . In theset al.  clearly clusions ,39.d in the weighted polynomial fit. A higher-order (d > 2), weighted polynomial is rarely needed based on the argument that such an assumption is, to a certain extent, over-fitting the data. From the findings of our study, we find that it is better to use a linear estimate based on minimizing the estimate errors across (A, M)-scatterplots. Consequently, different choices of d resulted in different optimized values for f. The reason is that for the higher-order polynomial, it is beneficial in general to retain a larger fraction of the values of A for the weight function in computing the polynomial coeffcients. It is very important to carefully select f since ultimately, the bandwidth is a function of the polynomial order.Another result of this optimized calibration study is that we uncovered a better understanding of choosing the parameter f . For one, print-tips are physically different and they are considered to have different types of errors introduced based on these properties. In the formulation of normalization, it is imperative to address such subtle issues when choosing and implementing any algorithm.Here, we also reaffirmed the idea that the quality filtering of ratios and spots is a necessary step that should precede all experimental microarray data handling procedures, whether it is scatterplot-based normalization or any other normalization method, since errant ratios would surely have a deleterious affect on the calibration. For instance, in the BT-474 data, the first replicate slide had poor ratio quality for a handful of genes. Calibration without considering or removing these errant spots resulted in less reliable results. This study addresses the issue of locating sources of experimental error for print-tips that have high sensitivity for the parameter f for the LOWESS algorithm results in noticeably different normalization results. This proposed method requires the calibration step to clearly state the assumptions used for within-slide normalization. Our optimization algorithm is more systematic than simply choosing an arbitrary parameter value or through trial and error techniques since the optimized approach relies on the actual underlying structure of the data. We also stress that such an optimization algorithm may also be utilized for other studies in addition to DNA microarray normalization treatments. Proper changes need to be made to Eq. (5) to reflect the ideal model for the data captured in the function ψk,i(·), but in some studies, such a function may be satisfactorily determined or estimated from the data.The systematic choice of the parameters in the LOWESS algorithm has not been previously addressed in the microarray literature and the method proposed here may be utilized in different microarray platforms. Such a treatment is also important for a wide variety of applications that employ scatterplot-based regression. The findings of this study illustrate that by choosing different values of et al. [et al. [et al. [et al. [et al. [For the self versus self hybridizations, custom cDNA microarray experiments proceed as follows. Altogether, three microarray hybridizations were performed using custom printed cDNA microarray slides from the same print batch. Labelling, hybridization and washing were done as described previously by Monni et al.  and Järv [et al. . Briefly [et al. . Microar [et al. . For the [et al.  and deta [et al. .et al. [et al. [All microarray experiments contained in this work were conducted and spotted using groups of ℓ = 32 print-tips, with each tip being responsible for either 384 or 420 spots in their respective subarray. In order to reduce the effects of spots whose intensities are not reliable due to experimental or printing errors, we used two separate quality filtering methods and normalized the intensities after discarding values that were detected unreliable. The assessment of ratio quality was performed using the method proposed by Chen et al.  and rati [et al. . In this [et al. . The traJAB developed the mathematical formulation of the problem, implemented the optimized normalization algorithm in MATLAB, developed the statistical analysis, and wrote the manuscript. SH developed the LOWESS normalization software in MATLAB, coordinated spot quality filtering, and assisted in drafting the manuscript. AKJ conducted the self versus self microarray experiments and performed ratio quality filtering for data analysis. HE assisted in data preparation and in drafting the manuscript. SKM participated in the design and coordination of the study and assisted in drafting the manuscript. JA reviewed the statistical analysis and participated in the design and coordination of the study. All authors read and approved the final manuscript.


Although serotonin reuptake inhibitors are effective in the treatment of OCD, many patients fail to respond to these agents. Growing evidence from open-label and placebo-controlled trials suggests a role for augmentation of SRIs with atypical antipsychotics in OCD. Quetiapine is generally well tolerated and previous open-label data has produced mixed results in OCD and additional controlled data is needed.We undertook a double-blind, randomised, parallel-group, flexible-dose, placebo-controlled study of quetiapine augmentation in subjects who had responded inadequately to open-label treatment with an SRI for 12 weeks. Following informed consent and screening, forty-two subjects were randomised to either placebo or quetiapine for six weeks.There was significant improvement from baseline to endpoint on the Yale-Brown Obsessive-Compulsive Scale in both the quetiapine and placebo groups  with 40% (n = 8) of quetiapine and 47.6% (n = 10) of placebo treated subjects being classified as responders. Quetiapine did not demonstrate a significant benefit over placebo at the end of the six-week treatment period (p = .636). Similarly quetiapine failed to separate from placebo in the subgroup of subjects (n = 10) with co-morbid tics. Quetiapine was generally well tolerated.In this study, quetiapine augmentation was no more effective than placebo augmentation of SRIs. A number of limitations in study design make comparisons with previous studies in this area difficult and probably contributed to our negative findings. Future work in this important clinical area should address these limitations. Obsessive-compulsive disorder (OCD) is a prevalent, chronic and disabling disorder . ControlFrom this it is clear that a need exists to pursue more effective treatments for those with OCD who fail to respond or respond inadequately to SRI's. To this end, preliminary evidence supports a role for the addition of atypical antipsychotics to SRIs in OCD. These agents combine serotonin-dopamine antagonism with the advantage of being well tolerated including a low potential for inducing motor side-effects.To date a number of open-label studies have suggested that augmenting SRI's with atypical antipsychotics is an effective strategy for treatment-refractory OCD. These include support for risperidone -7, olanzThe outcome of the first controlled study in this area with the antipsychotic haloperidol demonstrated preferential benefit for refractory OCD subjects with co-morbid tic disorder . In two Despite some mixed evidence in this area, in general the available literature appears to support the use of relatively short trials with low doses of antipsychotic agents as augmentation to SRIs. Quetiapine has a particularly interesting profile in that it is the only available antipsychotic with significant 5-HT1D effects and this serotonin receptor subtype has been implicated in OCD ,27.Our objective was to examine the effects of quetiapine augmentation in subjects with OCD who had failed to respond adequately to a 12 week trial of an SRI, employing a double-blind, placebo-controlled, six week study design.Forty two subjects aged 18–65 years inclusive were recruited in our multi-centre study comprising five sites in South Africa and one in Canada. Recruitment took place between May 2002 and November 2003. Prior to commencement, all sites in the study received approval from their relevant Research Ethics committees/Institutional review boards and regulatory authorities. All subjects provided written informed consent prior to the commencement of any study-related procedures. Diagnosis was confirmed using the MINI Neuropsychiatric Interview (Version 5.00, 1998)  to ensurAll subjects were treated and monitored by investigators for the minimum twelve week duration of SRI-alone treatment phase before inclusion into this study. This was to ensure that patients met criteria for duration of SRI treatment which included at least 6 weeks on the maximum tolerated dose of the relevant SRI. Sample size calculations were based conservatively on similar work in this area ,21. AccoA double-blind, randomised, parallel-group six-week augmentation with quetiapine or matching placebo of the SRI to which participants had not responded adequately, was undertaken. Specific SRI's, mean doses and dose range are provided in Table At baseline participants were randomly allocated to receive treatment with either quetiapine or placebo using a computer generated schedule and numbered dispensing wallets. A flexible dosing schedule was initiated at 25 mg per day for one week and then doubled weekly to the start of week 4. Based on Clinical Global Impression of Improvement (CGI-I) scores of minimally improved or worse, clinicians were permitted to increase the dose to a maximum of 300 mg per day for the final two weeks of the study. In addition to clinical measures of improvement, clinicians also considered patient tolerability in their decision to adjust doses. Following completion of the treatment phase, subjects were withdrawn from study medication while continuing their SRIs. All subjects were then followed up for any adverse effects.Patients were assessed by clinicians at baseline and on completion of weeks 2, 4 and 6. Telephonic assessments were performed on completion of weeks 1 and 3. Symptoms of obsessive-compulsive disorder were measured by the same clinician where possible at all study visits using the Yale Brown Obsessive Compulsive Scale YBOCS) ,31. A glOur primary outcome measures for OCD symptoms were (1) the change in YBOCS score from baseline to endpoint and (2) the clinical global impression of improvement (CGI-I) at endpoint. In the final analysis, treatment response was defined as a 25% or greater reduction in YBOCS score and a CGI-I of 1 (very much improved) or 2 (much improved) from baseline to endpoint. Secondary outcome measures included the MADRS, SDS and YGTSS (in subjects with co-morbid tics).Thirty-nine of the forty-two randomised subjects successfully completed the six-week treatment phase. Two subjects withdrew from the study prematurely (Week 1 and Week 4) due to severe levels of sedation. In both of these cases at least one week of study medication had been taken and at least one post-baseline clinical assessment was completed. Both of these subjects were included in the final analysis using data from the last observation carried forward (LOCF). The single subject not included in the efficacy analysis completed the study, but was found not to have correctly fulfilled the study definition of treatment refractoriness and was excluded. Twenty subjects were allocated to the quetiapine arm and twenty-one to the placebo arm. Student's t-tests were used to determine any baseline differences in the groups for age, gender, number of previous trials of SSRI's, severity of symptoms in relation to OCD, depressive symptoms, and CGI-S. Analysis of variance was undertaken with group and tics as factors. All tests were two-tailed with p-values of less than 0.05 considered significant.For the final analysis, our sample comprised 19 men and 22 women. Baseline characteristics of two treatment groups did not differ with respect to age (years) (quetiapine group 33.8(SD 9.66), placebo group 31.81 (SD 12.14); p = 0.57), gender (p = 0.29), number of previous adequate SRI trials (quetiapine 1.55 (SD 1), placebo 1.62 (SD 1.02) (p = 0.83), baseline severity of OCD , depressive symptoms (p = 0.91), patient-rated disability (p = 0.28) or the presence (n = 11, p = 0.66 and severity (p = 0.87) of co-morbid tics.For the primary outcome measure of severity (YBOCS), quetiapine p < 0.0001) and placebo (p = 0.001) augmentation of an SRI significantly improved symptoms of OCD. However quetiapine did not demonstrate significant benefit over placebo at the end of the six-week treatment period  ; placebo n = 1(20%)) were classified as YBOCS responders. The reduction in the YGTSS did not differ significantly between treatment groups with tics .Severity ratings for depressive symptoms (MADRS) were low at baseline (mean 10.6, SD 4.8), showed little change over the study period, and at week 6 remained similar for both groups .The mean daily dose at week 6 for the quetiapine group was 168.75 mg (SD 120.82) compared to 228.57 mg (SD 99.46) per day for those on placebo. Quetiapine responders (187.5 mg, SD 124.6) did not differ significantly from quetiapine non-responders (156.25 mg, SD 122.1) in their mean daily dose at Week 6 (p = .585). Furthermore, within the quetiapine group, participants receiving ≥ 200 mg/day (10/20 at week 6 demonstrated non-significant differences (F = 6.837, p = .988) and a marginally lower percentage reduction in YBOCS at endpoint (26.7%, SD 20.34) compared to those receiving a dose ≤ 200 mg/day (26.9%, SD 36.24) at endpoint.Quetiapine was generally well tolerated and no serious adverse events (SAE's) were reported through the course of the study period. Two patients on quetiapine withdrew from the study due to severe sedation (Week 1 and Week 4) that was judged to be drug related. Otherwise adverse events were in the mild to moderate range and were mostly self-limiting. No subjects on placebo withdrew from the study. Table Our findings indicate that both quetiapine and placebo significantly reduced symptoms in subjects with OCD who had failed to respond adequately to 12 weeks of an SSRI and, that the difference between groups was not significant. Similarly in the subgroup with co-morbid tics, no preferential benefit was noted for quetiapine. Interestingly, the high placebo response was similar to that seen in a recent failed controlled trial of olanzapine , but staFirst, the duration of a therapeutic trial of an SRI prior to augmentation with an antipsychotic should be of adequate dose and duration. In our study the majority of participants had failed only the single trial of an SRI on which they continued during the study (63.4% mean 1.59). Notably only six weeks of this treatment was required at the maximum tolerated dose. Despite the notion that an optimum trial of pharmacotherapy in OCD is 12 weeks, it may be argued that higher and ultimately effective doses of an SRI had not been maintained for an optimum duration prior to randomization. Given that therapeutic doses of SRIs in OCD are usually on the upper end of the dose range, it seems feasible that the high placebo response rate may reflect a response to SRI's once they had been administered at these higher doses for the additional six weeks of the study. It seems possible that the recent study by Shapira et al  may haveIn a second and related point; the number of previous SRI trials in the subgroup receiving quetiapine did not predict a poorer response to treatment. This effect is probably related to the lack of statistical power to detect these differences in a group in which the low number of previous SRI trials was a distinguishing characteristic. Certainly, previous positive studies in this area have used relatively more refractory groups based on the number of previously failed SRI trials. Taken together with the first point above, we suggest that future work in this area should consider longer periods at maximum tolerated doses of SRI's prior to categorisation of subjects as treatment refractory.Third, the use of a slow up-titration resulted in a relatively low mean daily dose being administered for the majority of the study. For instance, a mean daily (week 6) dose of 168 mg/day in the quetiapine group (median 175 mg) had only been achieved for the final two weeks of the study. These doses are comparably low to those used in the negative single-blind study using low dose quetiapine by Sevincok et al . In contFourth, the impact of repeated clinical assessments and rating of relatively small changes in clinical severity combined with regular dose increases, may conceivably have increased the placebo response rates resulting from increased optimism, a tendency to over-report improvements and belief that higher doses are more likely to be more effective than lower doses. This may be particularly true for the placebo-treated group that were considerably less likely to report sedation as an adverse event and as such were more likely to have their treatment dose increased at each visit. Our results differ, with respect to placebo response, from a considerable literature that suggests a consistently lower placebo response rate in treatment trials in OCD than in other mood and anxiety disorders. While we believe that the reasons (1–3) discussed above probably provide the main reasons for our finding, the impact of repeated assessments and the potential effect thereof cannot be entirely discounted.Despite significant improvement in each of the study groups, response to quetiapine augmentation in SRI non-responders, failed to separate from placebo treated subjects at the end of the six week treatment phase. A number of limitations in study design make comparisons with previous studies in this area difficult and probably contributed to our negative findings. Future work in this important clinical area should address these limitations.Dr. Stein has received research grants and/or consultancy honoraria from Astrazeneca, Eli-Lilly, GlaxoSmithKline, Lundbeck, Orion, Pfizer, Pharmacia, Roche, Servier, Solvay, Sumitomo, and Wyeth.PDC drafted manuscript and analysed data, BV protocol design, SS protocol design, JEM drafting of manuscript, statistical design, data acquisition, MVM protocol design, DJS principal investigator, protocol design, drafting of manuscript.The pre-publication history for this paper can be accessed here:


We have previously proposed a theoretical model for studying physical disability and other outcomes in rheumatoid arthritis (RA). The purpose of this paper is to test a model of impairment and functional limitation in (RA), using empirical data from a sample of RA patients. We based the model on the disablement process framework.We posited two distinct types of impairment in RA: 1) Joint inflammation, measured by the tender, painful and swollen joint counts; and 2) Joint deformity, measured by the deformed joint count. We hypothesized direct paths from the two impairments to functional limitation, measured by the shirt-button speed, grip strength and walking velocity. We used structural equation modeling to test the hypothetical relationships, using empirical data from a sample of RA patients recruited from six rheumatology clinics.The RA sample was comprised of 779 RA patients. In the structural equation model, the joint inflammation impairment displayed a strong significant path toward the measured variables of joint pain, tenderness and swelling (standardized regression coefficients 0.758, 0.872 and 0.512, P ≤ 0.001 for each). The joint deformity impairment likewise displayed significant paths toward the measured upper limb, lower limb, and other deformed joint counts (standardized regression coefficients 0.849, 0.785, 0.308, P ≤ 0.001 for each). Both the joint inflammation and joint deformity impairments displayed strong direct paths toward functional limitation (standardized regression coefficients of -0.576 and -0.564, respectively, P ≤ 0.001 for each), and explained 65% of its variance. Model fit to data was fair to good, as evidenced by a comparative fit index of 0.975, and the root mean square error of approximation = 0.058.This evidence supports the occurrence of two distinct impairments in RA, joint inflammation and joint deformity, that together, contribute strongly to functional limitations in this disease. These findings may have implications for investigators aiming to measure outcome in RA. Physical disability is an important outcome of rheumatoid arthritis (RA) ,2. The Apathology → impairment → functional limitation → physical disability [pathology occurs at the level of molecules, cells, or tissues, and is measured using tests such as the erythrocyte sedimentation rate, the C-reactive protein concentration, cytokine expression patterns, or images of the joints obtained with X-ray or MRI. Impairments are dysfunctions or structural abnormalities that occur at the level of organs or organ systems. They include signs and symptoms of disease such as pain, morning stiffness, joint tenderness, swelling and deformity. Functional limitations are restrictions in basic physical or mental actions, and they involve the whole person. Although they can be measured in a number of different ways, we have chosen to use performance-based functional tests such as the grip strength, walking velocity and the timed shirt-button test to measure functional limitations [Disability involves difficulty with a physical or mental activity, within a social context. The measurement level therefore should include the person and the societal environment. We have used self report measures of physical disability such as the Health Assessment Questionnaire, or the physical function scale of the SF-36, to measure physical disability [Inquiries into physical disability in RA, needing to weigh the influence of numerous variables interacting over time in complex ways, benefit from a conceptual framework, or model . A modelsability . A usefuitations . Disabilsability .functional limitation and physical disability [As noted above, we based our initial model and its proposed measurement strategies on purely theoretical grounds . Since isability -14. In tFrom 1996 to 2000, we enrolled consecutive patients meeting the 1987 RA criteria , into a We recruited patients from six outpatient rheumatology clinics in San Antonio, Texas: 1) An Army Medical Center, 2) An Air Force Medical Center, 3) A private, university-based clinic, 4) A community-based, seven-rheumatologist private practice, 5) A county-funded clinic and, 6) A Veterans Administration clinic. All evaluations were done on location in these facilities.Our study was approved by the Institutional Review Board of each of the clinical facilities were we went to recruit patients, and all patients gave written, informed consent. A physician or a research nurse, assisted by a trained research associate, conducted evaluations at the clinic where the patient was recruited. The evaluation lasted approximately 90 minutes, and consisted of a comprehensive interview, physical examination, review of medical records, and laboratory and X-ray tests. Interviews were conducted in either English or Spanish, as preferred by patients.We measured impairments using self-report and physical examination. We used a validated, one-page joint mannequin for patients to mark the joints that were painful or swollen . This va® Dynamometer (Sammons Preston, Bolingbrook, Illinois). In a sitting position, with the elbow held at 90 degrees, and the forearm supported on a flat horizontal surface, patients were asked to squeeze the handle with as much as strength as possible. Three repetitions from each hand were recorded, in kilograms. The mean value of all repetitions for both hands is shown; 2. Walking velocity. Starting from a standing position, patients were asked to walk at their usual pace, for a distance of 50 feet, or 25 feet if they had difficulty covering the full distance. No effort was made to conceal the stopwatch used to time the patients. Results are expressed in feet per second. Patients unable to walk were assigned a velocity of 0 feet per minute; 3. Timed button test. Patients were asked to don and fasten the front buttons of a standard eight-button, men or women's extra-large, denim shirt . A stop watch was activated when the patient took the shirt as it was offered by the examiner, and stopped when the last button was fastened. This test quantifies the performance and large and small upper extremity joints. Results are expressed as buttons per minute. Patient unable to don the shirt were assigned a value of 0 buttons per minute.We measured functional limitations using the following performance-based rheumatology function tests: 1. Grip strength. We measured grip strength with a hand held JAMARWe began the analysis by inspecting summary statistics and histograms of all study variables. Skewed variables were square root transformed toward normality. We specified a structural equation model (SEM) of impairment and functional limitation in RA . We hypoWe recruited 779 patients from 1996 to 2000. We have described the clinical characteristics of the study sample in earlier publications ,14. BrieA graphical display of the proposed model is shown in Figure The latent variable of joint deformity is measured by the deformed joint count, which we disaggregated into counts for the joints in the upper and lower extremities, and other joints. The path coefficients for upper and lower extremity deformities were stronger than that of the other joints (i.e. temporo-mandibular, acromio-clavicular and sterno-clavicular joints), but all three remained significant Table To obtain a better understanding of the properties of the latent variables, we used the path coefficients from the two impairment latent variables to compute their estimated values, generating a new variable for each one. Figures In the structural equation model, both of the impairment latent variables displayed strong direct paths toward functional limitations. The standardized coefficients were -0.5 or less, representing a change of more than half a standard deviation in the functional limitations for every standard deviation change in the impairments Table . The squpost hoc, we tested a direct path between joint inflammation and joint deformity, but the resulting coefficient was small and did not reach statistical significance. We therefore omitted an inflammation → deformity path from the final model.The initial CFI and NFI of the model were both 0.95, suggesting a close fit of the model to the data. The initial RMSEA was 0.07, suggesting reasonable fit . Modificbody structures and functions in the ICF are analogous to the stages of pathology and impairment, while activities and participation correspond to functional limitations and disability in the disablement process [Several models have been proposed to study disability in the general population ,9,20, an process .We chose the disablement process over other disability models, among other reasons, because it considers the stages in disablement as an explicit sequence of linked events, one leading to the next. Rather than suggesting specific functions or activities to measure, it offers broad definitions of each stage, leaving it up to investigators to find ways to test them. Several of the variables we include in the present analysis are represented in the ICF classification, including joint pain and deformity, and walking. Variables not represented in the ICF, but which we did obtain, include joint tenderness and swelling, and grip strength. Our measurement of the stage of disability, described elsewhere, has considerable homology with that of the ICF . The mosimpairment to be theoretical construct that cannot be directly quantified. We studied it as a latent variable, the articular signs and symptoms listed above serving as the tools we used to tap into the impairment construct.We used the disablement process framework to build a model of impairment and functional limitation in RA . ImpairmThe concept of impairment as a stage in the disablement process is not intended to oversimplify the anatomical or physiological derangements that occur within that stage. In fact, the derangements can be quite complex, depending on the nature of the initial pathology, and the organ system under study. In the case of rheumatoid joints, the initial pathology can be broadly classified into two discrete, but related groups: inflammation and damage . It shouBoth impairment latent variables displayed strong, statistically significant path coefficients toward the measured variables, providing evidence that the measures we chose adequately tap into the proposed impairments. In the diagram of our model, these paths are shown as arrows from the latent to the measured variables, to indicate that it is the joint inflammation or damage (both of them latent variables), that are "causing" the joint signs and symptoms that we are able to measure. Not included in our final model because it did not reach statistical significance, was a path from joint inflammation to deformity. This is likely because we restricted the present analysis to the stages of impairment and functional limitation. Although there is considerable evidence that inflammation leads to damage in RA joints, the link between the two processes occurs during the stage of pathology, not impairment. We expect to find a strong link between inflammation and damage when we extend our analyses to include pathology measures such as the erythrocyte sedimentation rate, C-reactive protein, joint erosions and joint space narrowing.According to disablement theory, impairments lead to functional limitations ,10. We eThe disablement process was proposed as a framework to aid investigators in their efforts to understand the development of disability in aging, and in specific disease states . The fraOne of our goals with this and earlier efforts to model RA's stages -12, is tThe success with which the ACR and similar response measurement systems have been used in clinical trials, does not preclude the possibility that they could be improved . Mixing Certain constraints apply to the interpretation of our findings. The maximum likelihood estimator that we used for SEM assumes multivariate normality, a requirement that is not strictly met by some of the variables we used in this analysis. Non-normality may affect standard errors, and thus significance testing, about the parameter estimates, albeit not the value of the parameters themselves. The overall structure of the model we propose, i.e. two distinct impairments linked to functional limitations, is thus likely to be unaffected by this deviation from assumptions. The patient sample we studied is sufficiently large that the potential deleterious effect of non-normality on the significance of the path coefficients may be offset. It should also be noted that data we used here to test the impairment → functional limitation relationship are cross-sectional. The sequential link between the two stages we propose has face validity in that joint tenderness, swelling and deformity are causes, not consequences, of diminished grip strength, walking velocity and shirt button speed. Nevertheless, confirmation of our findings in a longitudinal dataset would strengthen the evidence for the model.We conclude that two distinct impairments occur in RA, one characterized by signs and symptoms of joint inflammation, the other by joint deformity. Both of these contribute substantially to the functional limitations that occur in this disease.RA = Rheumatoid arthritis;ÓRALE = Outcome of rheumatoid arthritis longitudinal evaluation;CFI = Comparative fit index;NFI = Normed fit index;RMSEA: Root mean square error of approximation;SEM: Structural equation model;ACR = American College of RheumatologyThe author(s) declare that they have no competing interests.AE designed and obtained funding for the study, directed the statistical analysis, and drafted initial and final versions of the manuscript; RWH performed the statistical analysis and edited the manuscript; IDR designed and obtained funding for the study, supervised its implementation, and edited the manuscript.The pre-publication history for this paper can be accessed here:


Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed.We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them. Cell cycle checkpoints preserve genome integrity by monitoring the fidelity of DNA replication and segregation. In mammalian somatic cells, the best-characterized checkpoints are the DNA damage/replication checkpoints and the mitotic spindle checkpoint. The DNA damage/replication checkpoints result in cell cycle arrest if DNA is not fully replicated, or is damaged . The mitThere is also evidence that defects in events relating to cell division itself can result in cell cycle arrest. Lanni and Jacks  and CaseOther evidence suggests that mammalian cells are not sensitive to tetraploidy. Rao and Johnson used cell fusion to study the regulation of DNA synthesis and mitosis by fusing cells at different cell cycle stages ,8. BinucHere we investigate whether tetraploidy or other cellular defects in binucleate cells lead to cell cycle arrest. We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause DNA damage that is the likely cause of G1 arrest.Immortalized cell lines often have altered checkpoints, therefore we used early passage primary cells to investigate the tetraploidy checkpoint. All experiments were performed with human diploid fibroblasts (HDF) from infant foreskin and used prior to passage 10. We had previously developed methods for synchronizing these cells , and tesWe first tested HDF cells for the previously described G1 arrest following cytochalasin-induced failure of cytokinesis . Cells wAt 6 h after the removal of cytochalasin, 50% of the untreated control cells had entered S phase, whereas only about 10% of either the mononucleate or binucleate cytochalasin-treated cells had entered S phase Figure ; these nA potential problem with cytochalasin treatment is that depolymerization of the actin cytoskeleton is likely to have effects other than blocking cytokinesis. Indeed, we found that even at 2 μM, cytochalasin had a strong cytotoxic effect, delaying cell cycle progression significantly, with slow recovery after release (data not shown and ). To detHDF cells were synchronized in mitosis by double thymidine block followed by nocodazole treatment, then released into medium containing BrdU, +/- 12.5 μM blebbistatin. By 10 h after the addition of blebbistatin, most cells had exited mitosis; in the presence of blebbistatin approximately 30% of the cells were binucleate and the remaining cells were mononucleate, presumably completing cytokinesis successfully. Blebbistatin was removed, and cells were assayed for S phase entry over time.At 6 h after the removal of blebbistatin, 44% of the untreated control cells and 53% of the mononucleate blebbistatin-treated cells had entered S phase, whereas only 18% of the binucleate blebbistatin-treated cells had entered S phase Figure . By 24 hCells that have failed to divide after mitosis differ from normal cells in that they have two nuclei, two centrosomes, and a decreased surface area to volume ratio. We tested each of these defects individually for an effect on G1 arrest. To test the role of centrosome number, serum-starved G0 cells were fused with enucleated G0 cytoplasts to create cells with two centrosomes, but only one diploid nucleus Figure . The celTo test the role of tetraploidy, serum-starved HDF cells were fused to create binucleate cells. Creating binucleate cells by fusion avoided disruption of the actin cytoskeleton, allowing us to examine the effect of ploidy alone. The binucleate cells resulting from fusion were both tetraploid and had two centrosomes; we showed above that centrosome number was not a factor in the G1 arrest. As above, unfused cells in the population served as an internal control. At 24 h after fusion, 72+/-2% of the unfused cells and 75+/-1% of the fused, binucleate, cells had entered S phase. Therefore, tetraploidy does not cause the observed G1 arrest resulting from cytochalasin-induced failure of cytokinesis.Cells that fail to divide at cytokinesis are larger than normal cells. Larger cells have a decreased surface area to volume ratio, which might affect the response to perturbation of the cytoskeleton. Thus, the apparent sensitivity to cytokinesis failure might derive directly from a difference in size. To test this, we created large binucleate cells by fusing serum-starved G0 cells to each other. The fusion products were released into growth medium for 3 h to allow for reattachment to the culture substrate. We then added 25 μM blebbistatin, 5 μM cytochalasin, or 5 μM AKI-1 to cells for 10 h, followed by release into BrdU-containing medium. Figure If cells were sensitive to failure of cytokinesis, one might expect that the sensitivity would be expressed as a delay in exit from mitosis, a time when a cytokinetic defect could be corrected. This would be similar to the known DNA damage, DNA replication and spindle assembly checkpoints . We testSince we had ruled out most of the cellular defects associated with division failure as being the cause of the G1 arrest, we attempted to further characterize the arrest. Andreassen et al.  reportedp53-p21-dependent G1 arrest often results in either apoptosis or senescence ,19. To dWe have shown that failure of division of synchronized cells results in a p53-dependent arrest, but that the arrest is not due to ploidy, centrosome number, or cell size, and that the arrest is not preceded by a delay in mitotic exit, suggesting that it is not a classical checkpoint. The characteristics of the arrest are similar to those of the G1 arrest caused by the DNA damage checkpoint in HDF cells, which respond to irreparable DNA damage by entering senescence, instead of apoptosis . These sIn the untreated control cells, only 3.4% of cells contained γ-H2AX foci, whereas in the hydrogen peroxide treated cells, 33% of cells contained γ-H2AX foci Figure . RemarkaThe binucleate cells that persisted in culture were arrested in G1, as they did not incorporate BrdU after the previous round of mitosis, and they did not proceed to the next round of mitosis, as evidenced by the preservation of the binucleate phenotype. The presence and persistence of nuclear γ-H2AX foci in the G1-arrested binucleate cells suggested that DNA damage might be the cause of the arrest. However, not all the arrested binuclear cells contained visible γ-H2AX foci, indicating that γ-H2AX-associated DNA damage might not be the only cause of the arrest. The percentage of binucleate cells with nuclear γ-H2AX decreased from 52% to 29% over 8 days of culturing, possibly indicating that some cells were able to correct the DNA damage after being arrested in G1 for several days. In contrast, the percentage of mononucleate cells that displayed γ-H2AX foci decreased dramatically over 8 days of culturing, however this was likely due to proliferation of normal mononucleate cells in the culture rather than a difference in response of the mononucleate and binucleate cells to the treatment.The presence of γ-H2AX foci in the mononucleate cells that successfully completed cytokinesis after blebbistatin treatment suggested that the DNA damage might have been the result of the synchronization treatments, prior to the addition of blebbistatin. Therefore, we tested whether any of the cell synchronization treatments alone had an effect on cell cycle progression. Asynchronous cells were treated with double thymidine block, nocodazole, or blebbistatin individually, following the same protocols used above in multiple treatments. Cells were released from drug and S phase entry was assayed at time points. Figure Since none of the single drug treatments resulted in a cell cycle arrest, we reasoned that some combination of the treatments must be responsible. To determine which combination of treatments caused a G1 arrest, cells were subjected to the treatments in the order double thymidine, nocodazole, blebbistatin. Samples of cells were taken after release from each drug, and S phase entry was assayed Figure . AlthougTo determine whether the thymidine – nocodazole combination caused the DNA damage we observed, cells were treated with both drugs as above and stained for γ-H2AX. At 3 h after release from the drug treatments, about 33% of cells had γ-H2AX foci Figure . Thus thWe have shown that tetraploidy, aberrant centrosome number, increased cell size, and failure of cytokinesis do not lead to G1 arrest in primary human diploid fibroblasts. Rather, we found that the observed G1 arrest in cells that have failed to divide is likely due to cellular damage caused by standard synchronization treatments. We note that all published observations of a G1 arrest in response to division failure involved extensive manipulation of mammalian cells in culture. It seems likely that these manipulations resulted in DNA damage, or in other damage, that resulted in a G1 arrest, but was not directly associated with division failure. For example, Uetake and Sluder  found thHuman diploid fibroblasts (HDFs) were from infant foreskin. Wt, p53 -/- and p21 -/- mouse embryo fibroblasts (MEFs) were the kind gift of Laura Attardi (Stanford, CA). HDFs and MEFs were cultured in DMEM (Gibco) with 10% fetal bovine serum. HDFs were used prior to passage 10 and MEFs were used prior to passage 5. HDFs were synchronized in G0 by serum starvation  and S phCells were incubated with 20 μM BrdU (Sigma) for indicated times and fixed in -20°C methanol for at least 10 min. Fixed cells were treated with DNase I (Boehringer Mannheim) and exonuclease III (New England Biolabs) to expose the BrdU epitope prior to incubation with anti-BrdU antibodies, as described . Nuclei Cells were fixed with 2% paraformaldehyde at room temperature for 10 min, washed 3× with PBS, then permeabilized with-20°C methanol for 5 min and stained with 27 ng/ml γ-H2AX antibody (Trevigen, MD). Only cells with multiple, clearly labeled foci were counted as being γ-H2AX positive.Cells synchronized in S phase by double thymidine block were released from the block for 6 h to allow completion of S phase. Nocodazole (100 ng/ml)  was then added for 6 h to arrest the cells in mitosis. Cells were released from the mitotic arrest for 30 min, during which time most cells formed a bipolar mitotic spindle Fig . At 30 mCW participated in the design of the study and carried out the experiments. TS conceived of the study and participated in its design.


Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment.Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella using a freely available oligo design software package. 21 ± 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65°C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure.Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65°C, 28 ± 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Secondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly. Target molecules  hybridize transiently to the probe oligomers until they form stable double helices with their specific probes. At some point, the rate of on and off reactions reach equilibrium, and the concentration of the target in the sample solution can be calculated. Transcript abundance is assessed by the relative intensity of signal from each spot on the array. This interpretation of array data relies on the assumption that each hybridization reaction goes to completion within the timeframe of the experiment and that the behavior of all pairs of intended reaction partners in the experiment is somewhat uniform.Sequence-specific hybridization of a long single-stranded labeled DNA or RNA target molecule to shorter oligonucleotide probes is the basis of the gene expression microarray experiment. In this type of microarray experiment, gene specific There are three major types of DNA microarrays, which differ in the approach used for probe design: Affymetrix type microarrays , which aA number of oligonucleotide design software packages have been published in recent years, each having design strengths in one of a number of criteria -14. SeveFew of the available array design packages explicitly consider the possible structures of the transcript-derived molecules in the sample solution and their impact on whether the microarray will provide an effective assay, although the OligoDesign web server  does comBrucella suis. We have assessed the stability of structures formed in the transcript and the accessibility of the binding sites of optimal probes generated using commonly applied design criteria. Because random shearing of the full-length target molecule is used in some protocols, we have also modeled the effects of shearing to an average length on the prevalence of secondary structure in selected targets.In order to estimate the prevalence of stable secondary structure in long target molecules, and thus the impact such structures might have on the analysis of microarray data, we have modeled secondary structure formation in mRNA transcripts of the intracellular pathogen Brucella suis  is near 70% of the average for Brucella, while the average ΔG for transcripts from the GC-poor genome (L. lactis) are even lower (30% at 52°C). However, even in the most GC-poor genome, stable target secondary structure in the single-stranded target is widespread.Our modeling results obtained for the genome-wide set of intact single-stranded DNA or RNA targets demonstrate that stable secondary structures are widespread in target mixtures from s Figure  and in rOur results demonstrate that a significant fraction of nucleotide sites in the average target mixture, whether single stranded DNA or RNA, will be found in stable secondary structure under the hybridization conditions used in oligonucleotide microarray experiments, and will be relatively inaccessible for intermolecular interactions. Figure Figure Figure in silico shearing, revealed the likely presence of stable secondary structures in both full-length target and sheared target mixtures. These structures do not convert completely to random coil with either increasing hybridization temperature, more extensive shearing, or both. These secondary structures may therefore compete with the intended target for effective probe annealing in a microarray experiment, resulting in a misinterpretation of the amount of target present in the sample.Lack of bioinformatics tools that incorporate experimentally validated biophysical properties of nucleic acids as a criterion for synthetic oligomer probe design is a major challenge for do-it-yourself microarray designers. One biophysical characteristic, which we predict will reduce the binding efficiency of microarray probes to their targets, is the propensity of long single-stranded DNA or RNA molecules to form stable secondary structure. 3-D structures such as hairpins and stacked regions have the potential to pre-empt target nucleotides, thus blocking regions of the target molecules from hybridizing to their intended probes. Prediction and thermodynamic analysis of secondary structure at a range of temperatures in full length target sequences, as well as in subsequences formed by in silico experiment, secondary structure prediction in the target is being used to develop a new criterion for oligonucleotide probe design. Our results from this modeling experiment demonstrate that the implicit assumption used until now – that eliminating probe secondary structure by avoiding self-complementarity eliminates target secondary structure as well – is valid only when the target and probe are of the same length. Use of target secondary structure as an explicit criterion will allow for masking or preferentially avoiding the regions of the target sequence in which base pairs are directly involved in secondary structure formation, to eliminate these regions from the sequence for the purpose of the search for the optimal probe.Based on the results of this In this study we have assigned accessibility scores to sites in the target sequence based only on the fraction of predicted structures within 5% of the energy optimum, in which a residue is found in a single-stranded conformation. While this measure is not too computationally intensive to compute, and can be applied to genome-scale problems using readily available software (Mfold), it is not the most physically rigorous definition of accessibility. By equally weighting each possible structure in the ensemble of optimal and suboptimal structures that a molecule can form, it is possible that secondary structure at some positions in the molecule is overcounted; bonds which form only in rare conformations are considered equal to bonds which are present in the lowest-energy structure. The program Sfold -18 assigWhen we compared MFold-based accessibility predictions for an individual transcript to those generated by SFold and RNAFold, we found that the difference in average predicted accessibility over an entire transcript is small. We computed accessibility for the transcript of human 1CAM-1, which has been mapped experimentally to determine its accessibility . The aveIn this study, we focused specifically on the DNA/RNA base pairs that are actively involved in hydrogen bond formation. We realize that other accessibility considerations will have to be added to the scoring scheme in practice. The structure of a long single stranded DNA or RNA molecule can contain many nucleotides that, while not part of a double-helical stem, remain inaccessible to hybridization due to their location inside small loops within the target secondary structure. A loop is a somewhat constrained structure as well, and the length at which it presents accessible sequence that favors hybridization has been shown to be on the order of 10 nucleotides and longer , while nB. suis example, use of a low annealing temperature, e.g. 42°C which is the temperature used in some published 70-mer array experiments [Development of a target secondary structure criterion for oligonucleotide array design is expected to impose restrictions on the probe selection beyond the sequence similarity and melting temperature criteria that are currently used, especially in cases where short probe length restricts the annealing temperature used in the hybridization protocol to 22–37°. In the eriments , would rWe have shown here that while shearing reduces overall ΔG of secondary structure formation for individual molecules in the target solution, shearing does not in itself eliminate formation of secondary structure in single-stranded DNA or RNA. The question of whether shearing should be used for long oligomer arrays is still an open one. While some signal may be gained by reducing the stability of secondary structure in the target molecule, random shearing by its nature creates a mixture of targets that may have substantially different affinities. For instance, in a 300 nt transcript that is targeted by a 70mer oligonucleotide, there is nearly a one in four chance that a random break in the sequence will occur within the target site for which the probe is designed. Short fragments may present a substantially different binding site, and therefore have a different binding affinity, than the full-length transcript that is considered when the probe is designed. This is illustrated in Figure 8d, where binding of a 50mer sheared fragment to a 70mer probe leaves a dangling end in the probe. A break very close to one end or the other of the target site may create a target that still binds to the probe, though with reduced affinity; a break closer to the middle of the target site may produce fragments that bind partially to the probe, competing for binding with perfect matches.In other experimental contexts where hybridization is critical to success, the impact of secondary structure in single stranded polynucleotides on results has been recognized and is now being systematically studied (18–21). Intramolecular folding of mRNAs is so extensive that only 5–10% of most transcripts is accessible to binding of complementary nucleic acids; however the modeling of long molecules has not proven to give very accurate binding predictions -25. In fBrucella had binding sites in the target which were predicted to contain a stretch of unpaired bases of at least 14 nt in length; at 65°C, that fraction increased to 93%. Based on these findings we would expect that at 52°C only 57% of our probes would encounter optimal conditions for hybridization and therefore would demonstrate the expected behavior in the experiment, where intensity is expected to scale with target concentration. We predict that the remaining probes, which have shorter, or no, accessible sequences, will exhibit modified binding behavior, and we plan to conduct experiments to characterize this behavior. We have shown conclusively that avoiding self-complementarity in the probe when designing an oligonucleotide array is insufficient to eliminate secondary structure from the binding site in the target. By combining the procedure for systematic computational assessment of transcript accessibility described in this study with selective experimental validation of the impact of predicted accessibility on hybridization, we will develop a useful criterion for avoiding troublesome secondary structure when designing microarray targets.The results of the current study suggest a significant role for target secondary structure in hybridization to oligonucleotide arrays, which will warrant further investigation. Oligonucleotide probe binding sites in a significant fraction of transcripts are found in double-stranded conformations even in cases where self-complementarity was avoided during the probe design process. We find that at 52°C, for example, approximately 57% of probes designed for Brucella suis 1330 genome. Brucella suis has a relatively high (57%) genomic GC content. Brucella suis was chosen for this experiment because our collaborators have previously acquired a custom synthetic oligomer microarray for this organism, developed using standard oligo array design software, and we have access to both target sequences and to a set of unique probe sequences that define the interaction sites for which expression results have been obtained by the laboratory.Prediction and thermodynamic analysis of secondary structure was performed for all protein-coding gene transcripts predicted from 3264 CDSs in the Escherichia coli), and 50 from the GC-poor genome of the nonpathogenic AT-rich gram-positive bacterium Lactococcus lactis (35% genomic GC content). The Brucella suis genes ranged in length from 90 to 4,803 bp, with an average transcript length of 851 bp. The E. coli genes ranged in length from 140 to 2,660 bp, with an average transcript length of 792 bp. The range of GC content in the genes chosen was 37% to 57% with an average value of 50%, which is reasonably representative of the E. coli genome. The L. lactis genes chosen ranged in length from 140 to 2,730 bp, with an average transcript length of 765 bp., and ranged in GC content range from 30% to 42% with an average value of 35%.In order to determine whether Brucella sequences form atypical structures we randomly picked and analyzed 50 gene coding sequences from a compositionally balanced genome  using ArrayOligoSelector (pick70) [70-mer probes for each (pick70) . ArrayOlProbe and transcript secondary structure were predicted using the Mfold 3.1 software package ,29. MfolAccessibility in folded single-stranded DNA or RNA has recently begun to be addressed in a few experimental studies, mainly with the goal of targeting appropriate sites for RNAi. Because the structure of single-stranded nucleotide molecules is much more dynamic than that of proteins, with each molecule likely to exist in an ensemble of structures, and because the 3D structure of these molecules is rarely known, there is not yet a consensus representational standard of per-residue accessibility for single-stranded nucleic acids. Ding et al. ,18 impleIn this study, we chose to use the less physically rigorous approximation of probability of single strandedness as a simple fraction of predicted optimal and suboptimal structures in which a residue is found to be part of a single stranded structure, as computed by Mfold. Accessibility scores derived from MFold predictions have been used in limited studies of RNA structure focused on hammerhead ribozymes, antisenB. suis. Secondary structure and thermodynamics were computed for each of these fragments individually.Random shearing of the target mixture is an approach that is often offered as a solution for the problem of target secondary structure. The actual content of a sheared mixture of DNA or RNA fragments is complex. Shearing breaks the molecule not in predictable locations, but in random locations that give rise to a distribution of fragments around an average fragment length. In order to simulate the effects of different degrees of shearing on structure formation and stability in a transcript, we picked fragments of 200, 100, or 50 bases in length, choosing the start position via a sliding window of 10 bases. Secondary structure prediction for all fragments derived from every transcript in the B. suis genome is computationally intensive and produces an extremely large amount of output. Since our initial goal was to determine how much the method would affect the number and type of secondary structures probes would be expected to bind the shearing simulation was performed for fragments derived from the 300 bp Ure-1A gene of VGR participated in the design of the study, carried out the simulations and analysis, and drafted the manuscript. JWW participated in the design of the study and helped to draft the manuscript. CJG conceived of the study, participated in its design, coordinated the research and analysis, and drafted the manuscript.


The aim of this experimental study was to determine the effect of mild hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model with a human melanoma xenograft.A human melanoma xenograft was implanted into the hindlimbs of 30 athymic nude rats. The animals were randomized into five groups: group I: control, group II: sham group, group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP (tissue temperature 37°C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5°C for 30 minutes). Tumor response was evaluated by tumor size determination and immunohistochemical analysis 6 weeks postoperatively. Tissue sections were investigated for expression of CD34 and basic fibroblast growth factor (bFGF).3 to 1388 mm3. In the sham operated group (II) tumor volumes were significantly larger than in group I. Tumor volumes in group IV were significantly smaller than in group I and lowest in group V. There were no significant differences in size between group I and group III after six weeks. In group III and IV each, 5 animals showed tumor progression and one had a partial tumor response. In group V only 2 animals showed tumor progression. Immunhistochemical analysis of the tissue sections demonstrated that angioneogenesis was more pronounced in group II than in group I and less pronounced in group IV and V compared with group I.Average tumor volumes of the controls (I) increased from 105 mmOur results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF.External hyperthermia of 41.5°C tissue temperature for 30 minutes only has no impact on tumor growth and angioneogenesis in vivo. Hyperthermic isolated limb perfusion is an established treatment for multiple, locoregional intransit metastases of malignant melanoma and soft tissue sarcomas of the extremities. It was introduced 1957 by Creech and Krementz . Isolatein vivo [in vitro [Angioneogenesis has been recognized for many years to play a central role in the growth of primary tumors and the formation of metastases in general ,6. Melanin vivo  and in vin vitro . Antisenin vitro .Hyperthermia inhibits angioneogenesis. Some of the antineoplastic effects of hyperthermia are caused by ischemia due to obstruction or destruction of the tumor vessels. Eikesdal et al. described by hyperthermic temperatures disrupture of 25–50 % of the vasculature in malignant tumors. The heated tumors (44°C) had a blood flow reduction of 40–60 % after 24 hours .In vitro data indicate that capillary endothelials cells of malignant neoplasms are more thermosensitive than endothelial cells of normal tissues. The vascularization of tumors can be significantly damaged at temperatures which may alter but do not damage the vasculature of normal tissue [l tissue . The extl tissue . The cril tissue .Hyperthermia increases the effects of angioneogenesis inhibitors on tumor growth and tumor angioneogenesis . The aimad libidum. Maintenance and care of all experimental animals were carried out according to the guidelines of the local responsible Animal Protection Commission and carried out in compliance with national guidelines .Thirty inbred male nude athymic rats (Rowett rnu/rnu) weighting 200 to 280 g were used in this study. Animals were kept separately during the experiment with 12 hours of light per day. They were fed a standard laboratory diet and tap water A solid sample (2 × 2 × 2 mm) of a human melanoma xenograft (SK-MEL-3), derived from a lymph node melanoma metastasis, was implanted into the hindlimbs of 30 nude rats. The largest width and the maximum tumor diameter perpendicular to the width were measured with a micrometer. Only tumors with a diameter greater than > 12 mm in the largest diameter were included in the study.2; 0.85% CO2) and warmed in the heat exchanger. The warmed arterialized perfusate is driven by a roller pump with two synchronously running pump-heads on a single axis for the arterial and venous lines (Masterflex®). The outer diameters of the tubes corresponded with the diameter of the femoral vessels of nude rats weighting about 250 g .The details of the perfusion system have been described previously . The equRat hindlimbs were perfused with ringer's solution and sodium heparin  at 37 or 41.5 degrees C for 30 minutes with a flow rate of 4 ml/min. External hyperthermia was applied by an infrared lamp positioned in a fixed distance to the tumor.For temperature measurement during limb perfusion, a nickel-chrom-nickel thermocouple of 0.6 mm in diameter (Standard Integrated Thermocouple Thermocoax, Phillips, Hamburg, Germany) was placed at the macroscopic tumor margin. The thermocouple was calibrated before use in a high-precision water bath. Baseline temperature was recorded for 5 minutes before treatment. Temperature was continuously measured during application. In group I (control) and group II (sham operated) temperature measurement was not performed.The animals were randomized into a control and four study groups of six animals each: group I: control (no therapy), group II: sham group (skin incision without ILP), group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP (Ringer's solution as perfusate, tissue temperature 37°C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5°C for 30 minutes). ILP was subsequently performed with the above-mentioned parameters. Antineoplastic agents were not applied.®, 80 mg/kg i.m.; Pearl-Davis, Berlin, Germany) and Xylazin , and fixed in a supine position. For perfusion, the animals underwent a 3-cm incision of the groin. The left femoral artery and vein were isolated using an operation microscope. A vascular clip was placed across the artery and a silicon catheter (outer diameter 0.7 mm) was introduced via a transverse arteriotomy. The femoral vein was cannulated with a silicon catheter of 1 mm outer diameter . The other part was fixed in 0.9 % saline solution, frozen and stored at -80°C until use.Vascular density and tumor morphology were evaluated by immunohistochemistry. Tumor tissue was tested for expression of basic fibroblast growth factor (bFGF) and CD34.20 and counterstained with hemalaun. Negative controls without primary antibody were run for each sample.Tissue specimens were fixed in 10 % formaldehyde and embedded in paraffin according to routine protocols. Sections 4 μm thick were deparaffinized and rehydrated using graded ethanols following routine protocols. For antigen retrieval all sections were microwave treated for 20 minutes in target retrieval solution  or 10 mM citrate buffer (for CD34) at 700 W. The sections were incubated overnight with primary mouse anti-bFGF  and anti-CD34 antibody . After rinsing in Tris buffer detection was carried out with a biotinylated secondary rabbit anti-mouse antibody diluted 1:50 (DAKO, Glostrup, Denmark) for 30 minutes at room temperature. The sections were washed again as above in Tris buffer and then incubated with streptavidin-biotin-alkaline-phosphatase (10 μl each solution A+B (Sigma, Deisenhofen, Germany) in 10 ml Tris). Immunohistochemical staining was visualized with Fast Red (2 mg Naphtol-As-Mx-phosphate, 10 μl 1 M Levamisol and 10 mg Fast-Red (Sigma) in 0.2 ml N, N-dimethylformamide, (Merck, Darmstadt, Germany) and 9.8 ml 0.1 M Tris-HCl buffer at pH8.6). The sections were rinsed in HTumor response was graded according to the system described by de Wilt et al. . ProgresIn areas of intense neovascularized spots microvessels were counted in a 100× field. A microvessel was defined as a lumen surrounded by a rim of endothelial cells highlighted by immunostaining with anti-CD34 antibodys or anti-bFGF antibodys. Five separate intense neovascularized areas were assessed, and the mean was calculated as microvessel density of each tumor. The score was assessed by two independent observers.Data were analyzed using SPSS/PC+ statistical software. The mean and range of tumor and lesion volumes were calculated for each group. For comparison of tumor volume and microvessel density between the diffent groups a non-parametric test was used . A p value < 0.05 was considered to be significant.During intervention, the required limb tissue temperature was reached within 8–12 minutes in group IV and 10–15 minutes in group III. Stable temperatures were then maintained for a further 30 minutes with a mean of 41.4°C ± 0.5. To reach limb tissue temperature the perfusion fluid maintained 43°C ± 0.4.3 (4.3), 98 mm3 (5.7), 108 mm3 (3.4), 95 mm3 (5.0) and 107 (4.1) mm3 respectively, with no significant difference between the groups. Average tumor volume of the controls (I) increased to 1388 mm3 (101) during six weeks. In the sham operated group (II) tumor volume was significantly larger than in group I (2350 mm3 (198.6), P = 0.021). Tumor volume in group IV was significantly smaller (1009 mm3 (122.5)) than in group I and lowest in group V 405 mm3 (103.6) (P= 0.036 and P= 0.021, respectively.). There were no significant differences in size between group I and group III (1135 mm3 (99)) after six weeks (P > 0.05) volume of the treated tumors in groups I, II, III, IV and V was 105 mmAfter the perfusion, body weight decreased in all groups during the first 5 days with a maximum of 8 %. In the following weeks, body weight mounted up to 130 % of the preoperative value. In the last week of the experiment, the weight almost reached a plateau. No significant differences between the five experimental groups were found.In the H&E staining the untreated melanoma (group I) presented as a subcutanous unilocular nodule of moderate differentiation. The tumor showed mainly tubular structures with vacuole like lumen formation and was infiltrated with fibrous septa. The spontaneous rate of necrosis was estimated to be 20 %. Numerous mitoses were detected. Tumor morphology did not change with ageing or tumor size. Histology was similar in group II.In group V the tumors showed signs of irreversible cell damage after treatment. Tumor cells displayed clear shrinkage and partial loss of cell contact. Thromboses of the larger adjacent vessels were found on the tumor-skin border. Macrophage infiltration was present in all groups, but was more numerous pronounced in group V. In group III and IV there were less signs of irreversible cell damage after treatment than in group V.Immunhistochemical analysis of the histological sections for bFGF demonstrated that microvessel density of group I Fig  ranged fImmunhistochemical analysis of the histological sections for CD34 showed that microvessel density of group I ranged from 17 to 23 with a mean value of 19. Angioneogenesis was more pronounced in group II than in group I (p = 0,001) and less pronounced in group IV and V compared with group I (p = 0.023, p = 0.017). There were no significant differences in the expression of angiogenetic markers between group I and group III (p > 0.05).Strong expression of bFGF and CD34 was found in the cytoplasm of intimal endothelial and medial smooth muscle cells. Expression of bFGF and CD34 was stronger at the invasion front than in the centre of tumors.Summarized data of the expression intensity of bFGF and CD34 in the tumors are given in table In group I and II tumor progression was observed in all animals macro- and microscopically. In group III and IV 5 animals showed tumor progression and one had a partial tumor response. In group V, 2 animals showed tumor progression, 2 had a complete remission. One had a partial tumor response and one had no change in tumor response  in which tumors grew more rapidly than in the control group (group I). This implies that resection is associated with an accelerated growth in residual tumors. It is assumed that the surgical manipulation induces liberation of growth factors that in addition to their effect on healing also have a stimulating effect on tumor proliferation.This hypothesis is supported by the results of our immunohistochemical investigations, which revealed an enhanced expression of fibroblast growth factor (bFGF) in group II. In an intraperitoneal tumor model, EGGERMONT et al. showed that the surgical intervention of laparotomy induces an increase of carcinomatosis in the entire peritoneal cavity. At the same time, the immunotherapeutic effect of interleukin 2 and natural killer cells is significantly reduced. It must therefore be speculated that surgical trauma induces a temporary immunosuppressive effect that influences tumor growth ,18,19.In our experiments, group V (hyperthermic perfusion) showed a significant slowing of tumor growth in comparison with the control group. This was also confirmed by the immunohistochemical investigations that showed a reduction in the expression of relevant markers of the vascular endothelium. Indeed, two of the six animals occurred a complete remission of their tumor. The observation that two animals in this group experienced tumor progression can probably be explained by the duration of the intervention, since factors affecting the kinetics of cell death during hyperthermia are not only the maximum temperature induced, but also the duration of the elevated temperature.In an overview report BHUYAN noted that, as a function of various tumor cell types, a temperature of 43°C must be applied between 30 and 150 minutes to irreversibly damage the tumor cells. At a temperature of 45°C, the application time required varied between 13 and 85 minutes . In our Although the temperature in group III  was also 41.5°C applied for 30 minutes, no significant changes in the growth pattern of the tumor could be observed. Furthermore, the histological and immunohistochemical evaluations showed a mild hyperthermic damage at best. A comparison of treatment groups III and V revealed no differences in terms of duration of treatment and induced tissue temperatures.In contrast to group III, however, hyperthermia in group V has been mediated directly by the vascular system. In normal tissues subjected to warming, blood perfusion increases reactively via dilatation of the vessels together with an increase in vessel wall permeability. A decisive factor is that the tumor cells themselves, as well as the endothelial cells of tumor vessels are more thermosensitive than normal tissue ,21,22. OHeat transfer between the vascular system and the tissue is determined in particular by the nature of the blood vessels. Important factors are the number, length, and diameter of the vessels, and also the velocity of blood flow . CREZEE On the other hand, tumor vasculature carries away the externally applied heat (group III) which is then no longer available to destroy the tumor cells. In this case, the temperature gradient decreases from the tumor to the vascular system.Furthermore, in the presence of hyperthermia, microcirculation in the tumor tissue is more affected than in normal tissue . In addiOur results have also confirmed that surgical manipulation of the vascular system and perfusion have an influence on the tumor. Overall, the effect of the intravascular application of hyperthermia appears to be mediated by a combination of hypoxia and heat.Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF. External hyperthermia of 41.5°C tissue temperature for 30 minutes has no impact on tumor growth and angioneogenesis in vivo. Hyperthermic isolated limb perfusion effectively suppresses tumor angioneogenesis respectively tumor growth.None declared.JP carried out the treatment and drafted the manuscript.MM carried out the treatment.CS carried out the immunohistochemical studies.JG participated in the design of the study.AD carried out the histological studies.WH participiated in the design of the study.TM participiated in the design and coordination of the study.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: