In acute renal failure, a pronounced net protein catabolism occurs that has long been associated with corticoid action. By competitively blocking the glucocorticoid receptor with the potent antiglucocorticoid RU 38486, the present study addressed the question to what extent does corticoid action specific to uremia cause the observed muscle degradation, and does inhibition of glucocorticoid action reduce the protein wasting?RU 38486 was administered in a dose of 50 mg/kg/24 h for 48 h after operation to fasted bilaterally nephrectomized (BNX) male adult Wistar rats and sham operated (SHAM) controls. Protein turnover was evaluated by high performance liquid chromatography (HPLC) of amino acid efflux in sera from isolated perfused hindquarters of animals treated with RU 38486 versus untreated controls.Administration of RU 38486 reduces the total amino acid efflux (TAAE) by 18.6% in SHAM and 15.6% in BNX and efflux of the indicator of net protein turnover, phenylalanine (Phe) by 33.3% in SHAM and 13% in BNX animals as compared to the equally operated, but untreated animals. However, the significantly higher protein degradation observed in BNX (0.6 ± 0.2 nmol/min/g muscle) versus SHAM (0.2 ± 0.1 nmol/min/g muscle) rats, as demonstrated by the marker of myofribrillar proteolytic rate, 3-Methylhistidine (3 MH) remains unaffected by administration of RU 38486 (0.5 ± 0.1 v. 0.2 ± 0.1 nmol/min/g muscle in BNX v. SHAM).RU 38486 does not act on changes of muscular protein turnover specific to uremia but reduces the effect of stress- stimulated elevated corticosterone secretion arising from surgery and fasting. A potentially beneficial effect against stress- induced catabolism in severe illness can be postulated that merits further study. Persike and Addis[Bondy et al. [Schäfer et al. [Schäfer et. al used the experimental approach of inhibiting activation of the glucocorticoid receptor by enteral application of the potent glucocorticoid antagonist RU 38486 in acutely uremic rats [As part of the complex uremic metabolic syndrome, pronounced disturbances of carbohydrate and lipid metabolism are commonly observed, as are pathologic changes of amino acid and protein turnover . An incrand Addis, and in y et al.  showed tr et al. ,5 who pomic rats ,8, so famic rats , in whicWith this experimental design and by comparing sham-operated and bilaterally nephrectomized animals, the present study looked at the question to what extent does corticoid action specific to uremia cause the observed muscle degradation, and does inhibition of glucocorticoid action reduce the protein wasting?Animal experimentation was carried out on male Wistar rats, aged 11–15 weeks, weighing 217–225 g, from the animal experimentation facilities of Heinrich Heine University, Düsseldorf. Permission to use animals for experimentation was given by Regierungspräsident Düsseldorf, file nr. 26.4203.1-217/87 according to German federal law. Surgery for nephrectomy, sham nephrectomy and preparation for perfusion was carried out under narcosis with hexobarbital (EVIPAN- Na: 15–20 mg/ 100 g BW).Bilateral nephrectomy was performed using a dorsal access, ligation of renal vessel string, and excision of the kidney, leaving the adrenal glands in place. Sham operated animals underwent the same manipulations except for the ligation and excision. After surgery, animals were fasted for 48 h until perfusion. Nephrectomized animals had access to drinking water on the day of surgery for 8 hours, then were deprived of liquid to avoid lung edema. Sham animals had free access to drinking water throughout.Animals were randomly assigned to one of four groups: bilaterally nephrectomized (BNX) and sham operated (SHAM) treated with RU38486 and untreated BNX and SHAM animals. For treatment, RU38486 was dissolved in phenylmethanol, then mixed with sesame oil to form a milky suspension which was injected into subcutaneously into the lateral abdomen in three subdoses within 24 h, adding up to a total dose of 5 mg/100 g/BW.48 h after initial surgery, animals were narcotized and prepared for perfusion as previously described.The hindquarter was linked to the recirculation system after full passage of 70 ml of pre- perfusion medium, as shown in illustration 1. The pre- perfusion medium was discarded and not used for the recirculation experiment.-6 mmol/l phentolamine were added to avoid vessel contractions.The perfusion was carried out with a half- synthetic medium on the basis of Krebs- Ringer- bicarbonate buffer (KRB), pH 7.38 . Oxygen During perfusion, the arterial pH and perfusate oxygenation were monitored using a pH- meter and a total oxygen content analyzer . At the beginning and end of perfusion, plasma samples were frozen for amino acid analysis. Amino acid analysis by HPLC was carried out using 25 μl of deproteinized perfusate sample, mixed with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid to form OPA- adducts that were separated on a reversed phase column and measured by fluometry. Quantification was done by comparison with a standard amino acid mix including 3-methylhistidine. Of the 20 proteinogenic amino acids, cysteine, proline, and asparic acid were not included in HPLC analysis.Statistical analysis was done using the "Student" t- test for ungrouped, non- paired data with f = n1 + n2-2 and a significance level of p < 0.05.During the 48 h fasting period between operation and perfusion, animals had a pronounced loss of BW table . In SHAMOxygen utilization in the perfused muscle tissues is roughly the same in all four groups (data not shown) and equal to in vivo data previously reported  from ratTotal amount and spectrum of amino acids released during reperfusion: During perfusion, amino acids are released in varying amounts as shown in ill. 2. Nephrectomized animals (BNX) showed a general increase in amino acid release. This, however, is significant only in a few individual amino acids. The total amino acid efflux increases by 10,4 % (p = 0.05) without qualitative change. Notable exception is glycine, which is released to a lesser amount in nephrectomized animals. Amino acid release after treatment with RU 38486: Nephrectomy equally increases the amino acid efflux in animals treated with RU 38486 by 14.4% (p < 0.05) without change in spectrum. The total efflux of amino acids, however, is significantly reduced in the comparison SHAM/ SHAM+RU  and BNX/ BNX+RU . Release of 3-methylhistidine: 3-methylhistidine, a derivate of histidin mainly from actin and myosin in sceletal muscle and intestinal mucosa [l mucosa , is not Acute renal failure is a catabolic state, and unfortunately the inherent acceleration of protein breakdown cannot be suppressed effectively by provision of exogenous nutritional substrates . The sitOne factor that has for long been associated with the disturbances of protein metabolism is glucocorticoid action. The first observations date back to the nineteen-forties, when Persike and Addis  reportedIn order to evaluate the relative importance of glucocorticoid action on protein metabolism in acute renal failure, an experimental setting was chosen that allowed to study glucocorticoid action indirectly by selective blockade of the glucocorticoid receptor with the potent antiglucocorticoid RU38486, a substance that binds to the receptor without activating the further process of transcription . ParenteThe isolated perfusion procedure introduced by Ruderman is well In the given experimental setting, it is difficult to account for the in detail contribution of protein degradation, amino acid intermediate metabolism, and protein synthesis. Factors that modify the efflux are transport systems in the cell membrane -28, whicin ultima causa remain still unclear[Sham- operated animals are catabolic at the time of perfusion, having lost about 36 g BW (see results), which is approximately 16% of initial BW. Rats of this age are still growing, with an increase of approx. 5 g/ day (2–3% BW). The weil unclear. Nephrecl unclear, of whicl unclear. At firsBy contrast, in the comparison of the two groups treated with RU38486, the amino acid release remains increased in nephrectomized animals, albeit to a lesser extent, stressing the very point that glucocorticoids are only one of several factors that contribute to the net protein wasting. Chronically uremic rats with increased corticosterone- secretion , and a pLowell et al. found no reduction of the efflux of 3-methylhistidin after adrenalectomy in the perfused hindquarter of fasted animals [Teschner [This presumption is supported by the finding that RU 38486 had no effect on the efflux of 3-methylhistidin. While this result is in contrast to Schäfer et al. [ animals , and in Teschner . As respTeschner  and actiTeschner  comparedTeschner , it seemThe presented data fail to show that RU38486 inhibits glucocorticoid action in the specific uremic setting while it clearly reduces the elevated net protein catabolism compared to non- operated animals. This suggests that glucocorticoid mediated protein wasting in acute uremia is rather a by- product of the overall stress, in the present experimental setting caused by surgery and fasting, than due to an independent action specific to uremia.While this finding abolishes hopes to counteract muscle wasting in uremia by administration of an anticorticoid drug and indirectly rather stresses the well described clinicalBoth sham- operated and nephrectomized animals show an increase in net protein catabolism. RU38486 clearly reduces the net protein wasting in both groups, but the increase in net protein catabolism observed over sham- operated animals remains unchanged in nephrectomized rats. The effect of antiglucocorticoid RU38486 may be attributed to an inhibition of fasting and operative stress- induced cortisol action which, even when within the physiological range, promotes increased protein turnover , and to The author(s) declare that they have no competing interests.A.M. carried out animal experimentation, sample analysis, statistical analysis and wrote the manuscript.The pre-publication history for this paper can be accessed here:


The incidence of reported adolescent and adult pertussis continues to rise in the United States. Acellular pertussis vaccines for adolescents and adults have been developed and may be available soon for use in the U.S. Our objectives were: (1) to describe patient valuations of pertussis disease and vaccination; and (2) to compare valuations for short-term and long-term health states associated with pertussis.We conducted telephone surveys with 515 adult patients and parents of adolescent patients with pertussis in Massachusetts to determine valuations of pertussis-related health states for disease and vaccination using time trade-off (TTO) and contingent valuation (CV) techniques. Respondents were randomized to complete either a short-term or long-term TTO exercise. Discrimination between health states for each valuation technique was assessed using Tukey's method, and valuations for short-term vs. long-term health states were compared using the Wilcoxon rank-sum test.Three hundred three (59%) and 309 (60%) respondents completed and understood the TTO and CV exercises, respectively. Overall, respondents gave lower valuations  to avoid a given state for adolescent/adult disease compared to vaccine adverse events. Infant complications due to pertussis were considered worse than adolescent/adult disease, regardless of the method of valuation. The short-term TTO resulted in lower mean valuations and larger mean differences between health states than the long-term TTO exercise.Pertussis was considered worse than adverse events due to vaccination. Short-term health-state valuation is better able to discriminate among health states, which is useful for cost-utility analysis. The incidence of reported pertussis continues to rise in the United States despite high levels of childhood vaccination ,2. WaninRecently completed clinical trials suggest that the booster may prevent cough illness related to pertussis among adolescents and adults ,9. ThougMethods commonly used for measuring health-state valuations include contingent valuation (CV) and time trade-off (TTO) . ContingAnother common approach to measuring the benefits and harms of health interventions relies on health-state utilities. Utilities measure a person's preferences for specific outcomes on a scale of 0 to 1, on which 0 typically represents a state equivalent to death while 1 represents the best imaginable health. The time trade-off method is one of several approaches used to assess health utilities. Using the TTO method, respondents are asked how much longevity they would be willing to give up, if any, to avoid living with a particular health outcome. Traditionally, TTO questions have been framed as giving up time to avoid a long-term or chronic health state . HoweverWe conducted a survey using TTO and CV methods to determine the health-state valuations of adult patients and parents of adolescent patients diagnosed with pertussis. We compared two alternative approaches to framing TTO questions, based on either short-term or long-term health states. We hypothesized that framing questions as short-term rather than as long-term health outcomes would allow for better discrimination between states.Structured telephone interviews were conducted with adult patients (≥ 18 years) and parents of adolescent patients 11–17 years) diagnosed with confirmed pertussis in Massachusetts from December 1, 2001 to January 31, 2003 [–17 yearsDescriptions of the health states were derived with input from 3 pertussis experts Table . Adults For TTO questions, respondents were asked the maximum amount of time they would be willing to trade from the end of their lives to avoid a particular health outcome now. Adults were asked how much time they would give up from the end of their lives to avoid living in a particular health state themselves, while parents were asked how much time they would give up from the end of their own lives to avert the health state in the adolescent ,20. ThisAlthough TTO questions traditionally have been framed using permanent health states for valuations of chronic disease , the heaCV questions elicited the amount of money that a respondent would be willing to pay to avoid living in a particular health state for 8 weeks. We chose to frame the CV questions as short-term health states for both versions of the survey based on prior work ,22. RespTelephone interviews were conducted in English or Spanish using standardized forms. Some respondents either were unable to complete or refused to answer the entire set of TTO questions or CV questions. If any answers were missing within a set of questions, respondents were excluded from analyses (of that set) in order to assess population means. If the respondent completed either set of questions, trained interviewers judged how well the respondent understood the TTO or CV questions separately based on a 3-point scale . Respondents were excluded from further analyses if they were thought to have either some or limited understanding of the tasks presented .We calculated utilities based on the TTO exercise, under alternative assumptions about discounting of future health outcomes. For long-term health states, the utility was based on the proportion of time that the respondent would be willing to give up to avoid a lifetime health state for themselves, for their adolescent child, or for a hypothetical infant Figure . Life exIt is most straightforward computationally to start with the disutility, rather than the utility, of a particular health state, computed as the ratio of the duration of life that would be given up  to the expected duration of time lived in the health state. In the absence of discounting, the disutility was calculated simply by dividing the amount of time traded from the end of the respondent's life by the LE of the beneficiary. The utility was then calculated by subtracting this result from one. With discounting, we assumed that individuals compared the present values of the two different streams of life in the trade-off, in a way that reflects declining relative weight for future consequences, and we computed utilities based on a discount rate (r) of 3% per year . As empiUsing the formula for discounting a continuous stream of life , we obta-r (LE of respondent)) - (1/r) * (1 - e-r (LE of respondent - years of life traded))(1/r) *  was-r (LE of beneficiary))(1/r) * ) - (1/r) * (1 - e-r (LE of respondent - years of life traded))(1/r) * )(1/r) *  and medians (with interquartile ranges). We assumed that the maximum amount of discounted time traded from the end of the respondent's life could not exceed the duration of the present health state; thus, any utilities that would be negative based on the computations described above were instead set to 0. For parent respondents who were asked questions about how much time they would trade to avoid long-term health states in their adolescents, we used interval regression with left censoring to calculate mean utilities . In inteTo compare demographic characteristics of respondents for the short-term vs. long-term TTO surveys, we used the chi-squared test for categorical variables and the t-test for continuous variables. To determine if mean health state utilities and WTP values were significantly different from one another, we used Tukey's method, which is a non-parametric test that allows for multiple pairwise comparisons assuming all sample sizes are equal  ComparisFive hundred fifteen adult pertussis patients and parents of adolescent pertussis patients were eligible and participated in the survey Figure . CharactWe compared demographic characteristics of respondents who completed and understood the survey with those who did not. Parents of adolescents with higher household incomes (p = 0.022) and higher educational levels (p= 0.017) were more likely to complete and understand the CV survey. Also, parents who were white (p = 0.011) with higher educational levels (p = 0.010) were more likely to complete and understand the TTO survey. Adult respondents who completed and understood the CV and TTO survey were significantly younger  and had higher educational levels  than those who did not.CV and TTO responses for short-term and long-term health states for adolescents are described in Table Short-term and long-term TTO responses for adult respondents are described in Table TTO and CV responses for infant health states are described in Table Overall, mean utilities were higher for long-term health states than for short-term health states. These differences were significant for adolescents with mild cough (p = 0.045), severe cough (p = 0.001), and pneumonia (p = 0.001). No significant differences were found between short-term and long-term health states for adults. For infants, we also found significant differences with higher mean utilities reported for long-term health states compared to short-term health states .We evaluated associations between demographic characteristics such as age, race/ethnicity, education, and household income and estimates provided by respondents for the TTO or CV exercise. Older age was associated with lower utilities for short-term health states for adolescent/adult disease  and infant disease (i.e. respiratory and neurologic complications) (p < 0.05). Older age was also associated with lower utilities for long-term health states such as systemic reaction, adolescent/adult disease, and infant disease (p < 0.05). Higher income was associated with lower utilities for long-term mild cough and short-term respiratory complications, although older respondents were more likely to report higher household incomes (p < 0.001).Higher income was significantly associated with higher WTP values for the following health states: systemic reaction, adolescent/adult disease, and infant disease (p < 0.05). We also found an association between higher respondent education and higher WTP estimates for pneumonia, although we note that education and income were themselves positively correlated .Tables Vaccination programs in the US have traditionally been life-saving and cost-saving ,32. Howeet al. is an alternative approach for assessing process utility [We explored differences in utilities for short-term and long-term health states using open-ended TTO questions. Other methods for short-term health state valuation described in the literature include chained TTO, sleep tradeoff (STO), and waiting tradeoff (WTO). The chained TTO has been shown to have good consistency and reliability ,33,34. H utility . While tWe found that the rankings of health states based on mean utilities were essentially the same using either short-term or long-term health states. However, the short-term TTO resulted in lower mean utility estimates and larger mean differences between health states than the long-term TTO exercise, thus allowing for better discrimination of health states in cost-utility analyses. These results suggest that responses may not fulfill the constant proportional trade-off assumption, which requires that the TTO utility be independent of the duration of the specified health state. While previous studies using TTO and other valuation methods have also found that the constant proportional trade-off assumption does not always hold, the direction of the discrepancy has been mixed -40.The short-term health state approach may have violated the constant proportional trade-off assumption because respondents were less averse to giving up small amounts of time from the ends of their lives (days or weeks) compared to large amounts of time (months or years) in the long-term approach. In other words, asking respondents to give up a few days or weeks from the ends of their lives may not be considered a significant loss, even to avoid a short-duration health state lasting only 8 weeks. However, giving up months or years of life is considerably more difficult for individuals, even to avoid an intermediate- or long-term health state. It may be that a threshold exists whereby individuals are more willing to give up a very small portion of their lives for perfect health, but as the duration of health states increase, they are less willing to give up time per health unit gained, resulting in failure to behave according to the constant proportional trade-off assumption. This aversion to giving up larger amounts of time may play an important role in measuring utilities, particularly since the short duration of these health states over the lifetime of individuals would otherwise lead to nearly imperceptible, but arguably important differences in terms of quality-adjusted life years.The impact of discounting is important to consider since assuming no discounting can lead to a downward bias, while a high discount rate can lead to an upward bias, which may strongly devalue the benefits of any preventive intervention ,41-43. WThe CV exercise resulted in similar mean health state rankings to our TTO exercise, and these estimates were positively correlated with income, which is not surprising since individuals were asked to respond in consideration of their actual household income . For theAs always, there are limitations to our study. First, our respondents were either adult pertussis patients or parents of adolescents with pertussis. We elicited patient and caregiver valuations as part of a larger study to determine societal costs of pertussis in adolescents and adults. While the U.S. Panel on Cost-effectiveness in Health and Medicine suggests that community preferences be used where possible, there iSecond, selection bias might arise from our survey completion rate of around 60%, although this is comparable to other published valuation studies ,23. In oBecause of the complexity of the task required, it is not surprising that respondents with higher educational levels were more likely to complete and understand the preferences exercise. In addition, most respondents were white, well educated, and had relatively high household incomes. Household income was associated with utilities as well as willingness-to-pay to avoid pertussis. To address this limitation, economic analyses of pertussis should vary willingness-to-pay and utilities over wide ranges that would reflect the preferences of a general population. Further research in more socioeconomically diverse populations should also be considered.et al. found that a mother's WTP to prevent a cold is approximately twice as large for the child as for the mother[Another issue that should be explored more thoroughly is the impact of parents as surrogate respondents for children and the method of preference elicitation. We asked parents of adolescents to serve as proxy respondents for their child. Interestingly, parents of adolescents were less likely to provide answers to the long-term TTO exercise than adult respondents, which may suggest that parents had difficulty answering the TTO question for long-term illnesses in their children. Also, trading time from the parent's life to avoid illness in a child may result in preferences that incorporate other aspects of their relationship such as altruism. Previous work in the CV literature has suggested that altruism may significantly affect valuations. For example, Liu he mother. While pIn this study, we estimated health-state valuations regarding pertussis disease and vaccination among adult patients and parents of adolescent patients. Patient preferences in conjunction with health outcomes will be key factors in deciding whether or not to implement a universal vaccination policy for adolescents or adults. The results from our study suggest that short-term health-state valuation may provide a reasonable approach to assessing preferences given its superior ability to discriminate between states, which may be particularly useful for cost-utility analyses for future vaccination programs.Dr. Lee had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Lee, Salomon, LieuAcquisition of data: Lee, LieuAnalysis and interpretation of data: Lee, Salomon, LeBaron, LieuDrafting of the manuscript: LeeCritical revision of the manuscript for important intellectual content: Lee, Salomon, LeBaron, LieuStatistical analysis: Lee, SalomonObtained funding: LieuAdministrative, technical, or material support: LieuStudy supervision: Salomon, LieuThis study, part of the Joint Initiative on Vaccine Economics Project, was supported by the National Immunization Program, Centers for Disease Control and Prevention, via cooperative agreement with the Association of Teachers of Preventive Medicine, Task order #TS-0675. Dr. Lee's work was also supported in part by the grants T32 HS00063 and K08 HS013908-01A1 from the Agency for Healthcare Research and Quality, U.S. Department of Health and Human Services. Dr. Salomon was supported by the National Institute on Aging (Grant P01 AG17625).


MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis.One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a  A transgenic mouse model demonstrates that developmental context may be the reason why the spectrum of tumors differs in children and adults The frequency of cancer development varies depending on the age of the host. In humans, the most common childhood cancers include tumors of the hematopoietic system, nervous system, and skeletal muscle system. In contrast, in the adult population, solid tumors of the lung, colon, breast, and prostate are more common. Differences in types of cancers in hosts of different ages may reflect the abundance of cells in a differentiative state susceptible to tumorigenesis . Indeed,C-MYC oncogene to induce tumorigenesis in mice of different ages using a novel conditional transgenic model system for C-MYC–induced hepatocellular carcinoma (HCC).We recently reported that upon oncogene inactivation tumor cells can differentiate into mature cells, and in this new differentiative context the reactivation of an oncogene fails to restore tumorigenesis . Based oC-MYC (now referred to as MYC) is a member of a family of proto-oncogenes comprising C-MYC, N-MYC, and L-MYC. MYC encodes a transcription factor that, as part of a heterodimeric complex with MAX, regulates the expression of a multitude of genes involved in regulating cellular proliferation and growth  and other oncogenes  in murine hepatocytes results in HCC  . We founMYC in murine hepatocytes  transgenic for both TRE-r masses B. The nor masses C. Tumorsr masses A–2C. Thescid mice. Tumors formed in the inoculated mice after an 8–10-week latency  succumbed to neoplasia within 8 weeks. Mice in which MYC was induced at 4 weeks or 6–12 weeks of age developed tumors after a mean latency of 15 and 35 weeks, respectively . We did not observe any histological changes in the liver after 10 d of MYC activation . Thus, there may be an absolute limit to the ability of MYC to induce liver growth. MYC activation in adult hepatocytes was not associated with a change in overall liver weight . However, this latency of tumorigenesis in adult mice after partial hepatectomy was still up to two magnitudes longer than what was observed when MYC was activated in embryonic and neonatal mice    and 3A. i of HCC B and 11CMYC's ability to induce cellular growth versus proliferation and tumorigenesis depends on the differentiative context of a cellular lineage. Our results have general implications for the mechanisms by which MYC and other oncogenes induce and are restrained from causing tumorigenesis.Here we demonstrate that MYC during embryonic development and at birth caused gross liver enlargement and rapid emergence of neoplasia. This increased liver size resulted from increased cell number or cellular hyperplasia. In contrast, in adult murine hepatocytes, MYC activation induced cell growth, resulting in cellular hypertrophy and endoreduplication. MYC overexpression caused HCC in adult mice less frequently than in neonates, and only after a prolonged latency. We conclude that the ability of MYC to induce cellular proliferation and tumorigenesis appears to be determined by the relative developmental maturation of a cellular lineage.We found that the initial consequences of MYC overexpression, as well as its ability to induce tumorigenesis in murine hepatocytes, depend on the host age. The activation of MYC induction. We were able to address this possibility directly by examining the consequences of MYC overexpression in adult hepatocytes after partial hepatectomy, which resulted in the immediate induction of adult hepatocyte proliferation associated with complete liver regeneration within 2 weeks. Adult mice only exhibited a modest increased susceptibility to MYC-induced tumorigenesis observed when MYC was induced in embryonic mice. This is despite the fact that after partial hepatectomy the total number of proliferating hepatocytes would be at least two magnitudes greater than in a neonatal or embryonic liver, whose total mass is much smaller. We conclude that developmentally specific parameters other than proliferation are more likely to play a causative role in the differential susceptibility of embryonic and neonatal hepatocytes to MYC-induced tumorigenesis.We recognized that an alternative explanation for our results is that only hepatocytes that are actively proliferating are susceptible to neoplastic transformation upon Several different development-related factors could account for our observations. The relative ability of MYC to induce tumorigenesis in developmentally immature hosts may reflect the relative increased abundance of immature hepatocytes. Homeostatic mechanisms that regulate liver size and hepatocyte proliferation may permit MYC-induced proliferative expansion in the developing livers of young hosts and restrain proliferation in the mature liver of adult hosts. Differences in rates of tumorigenesis may be related to telomerase function that monitors the distance from senescence, as recently described . An unliMYC, from inducing cancer in adult somatic cells. MYC activation alone is capable of enforcing DNA replication, but not cell division  from the drinking water of mice transgenic for both TRE-MYC and LAP-tTA.The TRE-eviously . The LT-eviously . The p53MYC was activated in the liver by removing doxycycline treatment from the water. MYC was activated during embryonic development by removing doxycycline treatment from the mating cage before conception. MYC was activated at birth by removing doxycycline from the mating cage immediately after the birth of the litter. Mice were monitored daily and were sacrificed when moribund. During necropsy, liver tissues were saved by fixation in 10% buffered formalin or by being snap frozen in liquid nitrogen. For transplantation, liver tumor specimens were sliced into small pieces, incubated first in calcium-free Hank's Buffered Saline Solution (HBSS) on a stirring plate at 37 °C for 20 min, then washed and resuspended in 1× digestion buffer with 1.5 mg/ml collagenase, and incubated on a stirring plate at 37 °C for an additional 40 min. The solution was then filtered through a 100-μm filter, washed, and resuspended in PBS twice. After the first wash, the solution was resuspended in 10 ml of PBS. After the second filtering and wash, the solution was resuspended in 500 μl of PBS. A quantity of 106 cells was injected subcutaneously into scid mice (250 μl per injection) using a 1-ml syringe and 27-gauge needle. Mice showed signs of tumorigenesis within 2–3 weeks of inoculation. Mice were sacrificed when tumors reached 2 cm in size. To prepare 5× digestion buffer, 1.12 g of KCl, 37.94 g of NaCl, 0.69 g of NaH2PO4*H2O, and 9.9 g of dextrose monohydrate were dissolved in 1 liter of H2O.The median and left lateral lobes comprise about 70% of the liver and their removal is recognized classically as a partial hepatectomy. We performed a one-third partial hepatectomy, removing only the median lobe of the liver. Mice were anesthetized with 16 mg/kg ketamine/xylazine. An incision was made through the midline ventral abdominal skin and abdominal muscles, extending from just above the xiphoid cartilage to about halfway towards the base of the tail. A small bolster was placed under the thorax, causing the liver to fall forwards away from the diaphragm. The liver was pushed out and the suspensory ligaments were cut with blunt-end scissors. The median lobe was raised and a ligature was tied around it with the blood vessels at the base. The liver lobe was removed by cutting close to the ligature with sharp scissors. The bolster was removed and the muscle and skin incisions were closed. The mice were monitored hourly for pain and dehydration after the procedure.Liver tissues were fixed in 10% buffered formalin for 24 h and then transferred to 70% ethanol until embedding in paraffin. Tissue sections 4 μm thick were cut from paraffin-embedded blocks and placed on glass slides. Hematoxylin and eosin (H&E) staining was performed using standard procedures. The Stanford Histology Core laboratory prepared paraffin sections and performed H&E staining. We measured Ki67 expression by immunofluorescence using a mouse anti-human Ki67 monoclonal antibody . We used the Vector M.O.M. Basic Kit . Sections were deparaffinized with xylene and rehydrated through graded alcohol washes, followed by antigen retrieval in a microwave for 15 min in Vector Antigen Unmasking solution (H-3300). The slides were then incubated in 100 mM glycine for 2 × 8 min to reduce fluorescent background. Slides were blocked by incubation in avidin for 10 min followed by biotin for 10 min using the Dako biotin blocking system  and subsequently incubated for 1 h in M.O.M. IgG-blocking reagent diluted 1:4 in PBS. Slides were then incubated for 1 h in mouse anti-human Ki67 monoclonal antibody diluted 1:100 in M.O.M. diluent. Slides were washed in TBST for 3 × 5 min to reduce background and were then treated with M.O.M. biotin-labeled anti-mouse IgG, diluted 1:250 in M.O.M. diluent. Following another 3 × 5 min of TBST washes, slides were incubated for exactly 45 min in Cy3-conjugated streptavidin diluted 1:800 in PBS  in the dark. To visualize nuclei, slides were counterstained with 0.2 μg/ml DAPI. Ki67-positive cells were visualized by fluorescence microscopy.Western analysis was performed using conventional techniques. Liver tissues were disrupted and protein was isolated using a pestle and tube homogenizer in NP-40 lysis buffer. Equal protein was loaded in each lane, as quantitated by the Bicinchoninic Acid (BCA) Protein Assay . Proteins were electrophoresed on 10% Tris-HCl polyacrylamide gels at 100 V for 60 min and transferred on PVDF membranes at 100 V for 60 min. The membrane was blocked in 5% nonfat dry milk solution in TBS at 4 °C overnight. MYC protein expression was detected using the C-19 rabbit polyclonal antibody that recognizes mouse and human MYC . p53 protein expression was detected using the NCL-p53-CM5p rabbit polyclonal antibody (Vector Laboratories). As a positive control, we used a hematopoietic tumor previously shown to overexpress p53 that was generously provided by Dr. Kevin Smith.Images of H&E-stained liver sections were made with a Nikon Eclipse E800 microscope utilizing a Spot RT Slider digital camera  and Spot Advanced Software . To estimate cellular and nuclear volume, the radii of at least five hepatocytes per field were measured in at least three fields.Hepatocytes were collected by a two-step in situ perfusion technique . First, Cell size was determined by FACS forward versus light scatter of isolated hepatocytes utilizing a Becton Dickinson FACSCaliber (BD Biosciences). Data were analyzed using Cellquest v3.3 Software (BD Biosciences).Nuclei were prepared for staining by touching a cut piece of liver to superfrost slides. The smears were then air dried, fixed in formalin for 5 min, and stored in 70% ethanol. The nuclei were stained for DNA content analysis according to the Feulgen technique .Apoptosis was detected using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, using the In Situ Death Detection Kit . In order to visualize the nuclei, cells were counterstained with DAPI (0.2 μg/ml). TUNEL-positive cells were visualized by fluorescence microscopy.p21, MDM2, and glyceraldehye-3-phosphate dehydrogenase (GAPDH) were used as probes  product specifications. cDNAs corresponding to s probes . The proTo evaluate loss of heterozygosity of liver tumors derived from mice heterozygous for p53 deletion, PCR analysis was performed as previously described .260 measurements. The quality of RNA was measured on a formaldehyde, 1% agarose gel.Total cellular RNA was isolated from snap-frozen liver tissue using the Invitrogen Micro-to-Midi Total RNA Purification System according to the manufacturer's instructions. The amount of total RNA isolated from tissues was quantified using spectrophotometric ODMYC and rodent GAPDH were measured by real-time quantitative RT-PCR using the 5′ nuclease technology on an ABI PRISM 7900HT Sequence Detection System . The probe sequences for human c-MYC were forward primer 5′-CCCCTGGTGCTCCATGAG-3′ and reverse primer 5′-GCCTGCCTCTTTCCACAGA-3′. The human c-MYC probe, 5′-TCCTCCTCAGAGTCGC-3′, was labeled with FAM dye-MGB. A VIC TaqMan rodent GAPDH control reagents kit (Applied Biosystems) was used to measure mouse GAPDH. The RNA was reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's protocol with a minor modification, the addition of RNase inhibitor (Applied Biosystems) at a final concentration of 1 U/μl. Samples were incubated at 25 °C for 10 min and 37 °C for 180 min. PCR reactions were prepared in a final volume of 20 μl, with final concentrations of 1× TaqMan Universal PCR Master Mix (Applied Biosystems) and cDNA derived from 20 ng of input RNA as determined by spectrophotometric OD260 measurements. Thermal cycling conditions comprised an initial UNG incubation at 50 °C for 2 min, AmpliTaqGold DNA polymerase activation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. Each measurement was performed in triplicate and the threshold cycle t), and cDNA prepared from Universal Mouse Reference RNA (Stratagene). Universal Human Reference RNA was used for human c-MYC and Universal Mouse Reference RNA was used for rodent GAPDH.The RNA of human c-http://us.expasy.org/sprot/) for the loci discussed in this paper are the following: Cdkn1a (mouse), P39689; Mdm2 (mouse), P23804; Myc (human), P01106; Myc (mouse), P01108; and Tp53 (mouse), P02340.Swiss-Prot accession numbers (


Many studies associated nosocomial infections with increased hospital costs due to extra days in hospital, staff time, extra investigations and drug treatment. The cost of antibiotic treatment for these infections represents a significant part of hospital expenditure. This prospective observational study was designed to determine the daily antibiotic cost of nosocomial infections per infected adult patient in Akdeniz University Hospital.All adult patients admitted to the ICUs between January 1, 2000, and June 30, 2003 who had only one nosocomial infection during their stay were included in the study. Infection sites and pathogens, antimicrobial treatment of patient and it's cost were recorded. Daily antibiotic costs were calculated per infected patient.Pseudomonas aeruginosa infections was the most expensive infection treated. Piperacillin-tazobactam and amikacin were the most prescribed antibiotics, and meropenem was the most expensive drug for treatment of the nosocomial infections in the ICU.Among the 8460 study patients, 817 (16.6%) developed 1407 episodes of nosocomial infection. Two hundred thirty three (2.7%) presented with only one nosocomial infection. Mean daily antibiotic cost was $89.64. Daily antibiotic cost was $99.02 for pneumonia, $94.32 for bloodstream infection, $94.31 for surgical site infection, $52.37 for urinary tract infection, and $162.35 for the other infections per patient. The treatment of Daily antibiotic cost of nosocomial infections is an important part of extra costs that should be reduced providing rational antibiotic usage in hospitals. Nosocomial infections are frequent complications of hospitalization and also an important public health problem in developing countries, as well as in developed ones. The socioeconomic impact, ie, prolongation of hospitalization, mortality, and cost of these infections adversely effects patients and nations' economic well-being . The cosThe aim of our study was to determine daily antibiotic cost of nosocomial infection per infected patient in a university hospital.Akdeniz University Hospital is a 600-bed tertiary referral centre in Antalya, Turkey, treating 27 000 patients per year. The study was conducted in six adult medical and surgical intensive care units (ICUs) with a total of 51 ICU beds. Neonatal ICU was not included in the study. Since 1993, the institutional policies of hospital infection control have been implemented by infection control team.In our hospital, routine prospective, active surveillance of nosocomial infections in all ICUs is performed by one infection control nurse, supervised by an infection control physician. Nosocomial infections are defined using the Centers for Disease Control and Prevention criteria ,6. We doBetween January 1, 2000 and June 30, 2003, all inpatients hospitalized in one of the adult ICUs were included in this study. Data on antimicrobial treatment were recorded for patients aged 15 or above presenting with only one nosocomial infection. For all patients included in the study, the following were recorded: age, sex, infection site, microbiologic data, antimicrobial therapy and antibiotic cost.In our ICUs, all antimicrobial prescriptions are recommended by an infectious disease consultant. Antimicrobial agents prescribed only for therapeutic indications were recorded. The daily antibiotic cost was calculated in US dollars based on June 2003 prices of antimicrobial agents provided by the hospital pharmacy. The daily antibiotic cost per infected patient was calculated by the multiplication of box price and number of daily doses that was used for that infection. Two costs were calculated for each antimicrobial; the minimal cost (min) was based on the lowest recommended parenteral daily dose and the maximal cost (max) was based on the highest recommended parenteral daily dose.Between January 1, 2000, and June 30, 2003, a total of 8460 patients were admitted to the adult ICUs. Overall, 817 patients developed 1407 episodes of nosocomial infections, accounting for an infection rate of 16,6%. Among them, 233 patients  had only one nosocomial infection. Mean daily antibiotic cost was found $89,64 per infected patient .Among the sites of nosocomial infections, urinary tract infections had the lowest daily antibiotic cost per infected patient Table . The meaPseudomonas aeruginosa was the most prevalent bacteria followed by Klebsiella spp. and Acinetobacter spp.. P. aeruginosa infections had the highest overall daily antibiotic cost per infected patient than other pathogens. Among 21 Staphylococcus aureus strains 15 (72%) were resistant to methicillin. The overall daily antibiotic cost of methicillin resistant S. aureus (MRSA) infections was two to three times higher than infections with susceptible strains. However, median daily cost per infected patient for MRSA infections were lower than susceptible strain infections.Out of 233 patients, 206 patients had microbiologically documented infections, 177 patients were infected by a single pathogen while 29 patients were diagnosed as having polymicrobial infections  declare that they have no competing interests.DI collated and analyzed the data, participated in the study design and was principal writer of manuscript. ANY conceived the study. GO carried out the laboratory studies. RS, FG, OT and LM participated in the patient management. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


PLoS Biology, biochemist Jeremy Brockes and his colleagues at University College London tie this muscle cell de-differentiation to expression of a single gene, Msx1, a player in limb development and regeneration across species.Cell and molecular biologists have a good start at understanding the adult salamander's enviable ability to completely regrow a lost limb or jaw.  Happily, mammals share many of the required cellular skills—though in an untapped form. In response to injury, fully differentiated salamander muscle cells can produce less specialized cells that are capable of multiplying and recreating lost tissue. In this month's Msx1 appears at the borders of maturing tissues in salamanders, chickens, and mice, and represses differentiation of muscle cells  during development. Msx1 promotes amphibian limb and tail regeneration, zebrafish fin repair, and regrowth of the tips of digits in mice. Here, Anoop Kumar et al. clarify the gene's role in muscle repair by demonstrating that Msx1 expression is required for de-differentiation of salamander myofibers—mature muscle tissue without the ability to produce new cells—into cells with single nuclei (mononucleate cells) that are able to multiply.The protein encoded by Large, elongated cells, myofibers are distinguished by a striped pattern of actin and myosin, the proteins that produce muscle contractions. They develop from precursor cells, which proliferate and fuse together into multinucleate intermediates called myotubes. As myotubes bulk up on actin and myosin, pushing their many nuclei to the cell periphery, they mature into myofibers. Once differentiated, myofibers are committed to the life of a muscle workhorse. They cannot divide; mature muscle in mammals adds strength and repairs injury by accumulating more actin and myosin, and by fusing single-nucleus “satellite” cells into new or existing myofibers.Msx1. A fraction of mouse myotubes made to express Msx1 break off mononucleate cells that can be induced to express markers for bone, cartilage, fat, or muscle.Unlike their mammalian counterparts, salamander and newt myofibers respond to injury by splitting into several multinucleated fragments, or by budding off several of their nuclei to create individual cells. These mononucleate progeny multiply and develop to replace lost tissue. Kumar et al. found that more than half of salamander myofibers spontaneously fragment and bud when dissected and placed in cell culture. Prior study has shown that mouse myofibers don't normally behave this way, although cultured mouse myotubes can imitate amphibian myofibers if given a push, in the form of Msx1 RNA and protein appeared in actively fragmenting and budding myofibers, especially in and around their nuclei. When Kumar et al. blocked the translation of Msx1 mRNA, the myofibers did not generate new cells.In the salamander cells, Msx1 as a pivotal regulator of muscle cell re-entry into the cycle of cell division and tissue growth. In this context, it doesn't drive cell division—on the contrary, the authors showed that Msx1 induced cell splitting without DNA replication. The gene drove committed muscle cells to donate nuclei, creating a pool of new cells to divide, differentiate, and repair damage. Given what it can do for mouse cells in culture, the question remains whether Msx1 might help awaken a latent capacity for regeneration in living mammals.The results confirm


S. cerevisiae as a model system. Through the use of arrayed yeast strains harboring ordered deletions of open reading frames, we determined that 54 out of the 4,507 yeast genes examined significantly influence AHR signal transduction. In an effort to describe the relationship between these modifying genes, we constructed a network map based upon their known protein and genetic interactions. Monte Carlo simulations demonstrated that this network represented a description of AHR signaling that was distinct from those generated by random chance. The network map was then explored with a number of computational and experimental annotations. These analyses revealed that the AHR signaling pathway is defined by at least five distinct signaling steps that are regulated by functional modules of interacting modifiers. These modules can be described as mediating receptor folding, nuclear translocation, transcriptional activation, receptor level, and a previously undescribed nuclear step related to the receptor's Per–Arnt–Sim domain.The aryl hydrocarbon receptor (AHR) is a vertebrate protein that mediates the toxic and adaptive responses to dioxins and related environmental pollutants. In an effort to better understand the details of this signal transduction pathway, we employed the yeast  4500 yeast deletion mutants, transformed with a reporter construct that is sensitive to dioxin, were used to create a protein-interactive map that outlines five distinct steps required for dioxin-mediated cell signaling The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor found in a variety of vertebrate species. The AHR is a prototype member of the Per–Arnt–Sim (PAS) superfamily of signaling molecules. Members of this superfamily regulate cellular responses to a variety of environmental stimuli, including pollutants, hypoxia, and external light cues . Our iniCYP1A1, CYP1A2, and CYP1B1  strategy to provide a framework to describe AHR signaling. By coupling both computational and experimental annotations, we were able to deduce the minimum number of genetic loci and signaling events required for AHR signaling.Saccharomyces cerevisiae is a valuable model system for the study of signaling by mammalian nuclear receptors  and large-scale databases describing protein and genetic interactions  . This chSaccharomyces Genome Deletion Project (wt) BY4742 strain (p < 10–6). To minimize false positives, we selected clones that influenced signaling at no less than two of the six concentrations of agonist tested. In addition, we retested each positive strain in a secondary screen with another AHR system containing the same LacZ reporter and a high-copy AHR–LexA chimera (pAHR)  . By thest strain .To eliminate those deletions that influenced the AHR pathway in a nonspecific manner, each of the 92 deletion strains was examined with a control plasmid pGal4TAD see . This coOf these “AHR-specific” factors, Hsc82p and Cpr7p were previously described AHR modifiers, and the other 52 were novel  Table S. The anaRecent experiments from a number of laboratories have provided data to support the idea that protein interaction network (PIN) can be used to portray the workings of complex biological systems . To invemax). One common feature of AHR–PINs with Dmax values greater than 1 was that the majority of M-nodes were interconnected in a single large network with no breaks (max was set at low stringency (Dmax ≥ 3), the representation of M-nodes in AHR–PIN was high. For example, at Dmax = 3, 46 of 54 M-nodes were included. However, AHR–PINs resulting from these inclusive, yet low-stringency conditions exhibited high complexity, which made it impossible to assess the interactions visually (max was set at higher stringency (Dmax = 2), the resultant AHR–PIN now comprised 34 closely interconnected M-nodes and was much easier to visualize  are interconnected, while in corresponding random PINs with mock M-nodes, this number drops to 18.5% (10/54). Although the AHR–PINs at Dmax = 3 and Dmax = 4 also exhibited statistically significant differences from random PINs, these AHR–PINs were not considered further for two reasons. First, these networks were visually complex and could not be simply annotated in two dimensions. Second, the ratios of interconnected M-nodes in these AHR–PINs to those of random PINs were quite low . This observation suggests a much greater potential for displaying false positive interactions at these settings as compared to the AHR–PIN at Dmax = 2, where this ratio was 3.4 (34/10).For further exploration, we chose to focus on the network with the greatest statistical significance, i.e., the PIN generated at DOur next objective was to use the PIN to enumerate and define steps in AHR signaling. It has been suggested that PINs exhibit a modular nature, with each module comprising highly interconnected proteins of related cellular functions . Our hypmax = 2). Each distance (d) in the matrix refers to the length of the shortest path between a pair of nodes in the full network space of yeast genomic PIN and was transformed into an “association” value (21/d). The resultant pairwise association matrix was used to identify network clusters in the AHR–PIN by a hierarchical average-linkage clustering algorithm  at various agonist concentrations, times, and temperatures, as well as after exposure to two distinct AHR agonists, α-naphthoflavone (αNF) and β-naphthoflavone (βNF). The relationship between each modifier and signaling was then examined using a hierarchical average-linkage clustering algorithm  Figure A. It waswt strain was transformed with the plasmid pAHRGFP, it was found that the fusion protein was evenly distributed in the cell in the absence of AHR agonist. In the presence of the agonist βNF, the AHR–GFP protein translocated to the nucleus (wt strain (group I). Approximately 30% of the strains were found to contain a marked reduction in the level of AHR protein in the cell (group II). Approximately 10% of the deletion strains displayed receptor aggregates in the cell (group III). The final 10% of the deletion strains displayed a normal level of AHR protein, but the receptor failed to translocate into the nucleus in the presence of agonist (group IV). When overlaid with the previously determined experimental layers, group I was found to overlap with the modules of C and D, and groups II, III, and IV corresponded to modules B, A, and E, respectively . When the  nucleus A. To exa nucleus B. About ectively C. Accordectively C.Our initial objective was to identify the number of loci that are required for AHR signal transduction. In this regard, our high-throughput deletion screen identified 52 novel and two known AHR modifiers. Although this is a surprisingly large number of modifiers for the function of a single protein, it is probably an underestimate since the deletion screen cannot identify modifiers that are encoded by essential genes. Moreover, our criteria of including only strong modifiers (influence of 4-fold compared to control) may have caused us to miss some important modifiers of this pathway. Nevertheless, the number of AHR modifier loci reported here is approximately 10-fold greater than what has been reported using mammalian cell culture and animal models .max was set at 2, 3, or 4, the resultant AHR–PIN was of a complexity that could not have resulted from random chance. Furthermore, the comparison of various simulations at different Dmax settings guided us to select the linking parameter at Dmax = 2. This setting of intervening links resulted in the highest level of statistical significance, displayed the lowest potential for false positive interactions, and decreased the map's visual complexity to a level that was readily understood in a two-dimensional map.Once we identified these AHR modifiers in yeast, we sought a way to position and characterize them in the context of the AHR pathway. Given the idea that PINs can be used to portray the cellular workings, we attempted to use our deletion data to generate and annotate an AHR–PIN . To consOur analysis of the AHR–PIN revealed an underlying modular structure. That is, there are areas in the AHR–PIN that display high interconnectedness of nodes, and these regions represent functionally related modifiers. The modularity of AHR–PIN was revealed by both computational and functional tests. In our initial computational approach, a total of ten clusters were identified, and the functional enrichment of each cluster was calculated by hypergeometric distribution .Although the computational approaches of module identification and annotation were useful in hypothesis generation, they did not provide a direct description of AHR signaling. Therefore, we set out to annotate the AHR–PIN with a number of functional tests. In our first annotation experiment (“domain influence”), we found that the AHR–PIN could be divided into three discrete functional modules . Additionally, each of these modules was found to overlap with one to several network clusters see . This tiA module that regulates AHR folding was identified by the known activities of its constituents, as well as the appearance of receptor aggregates when these modifiers were absent see . In addiThe AHR folding module can also be used to explain the existence of I-nodes within a functional module. Given their “linker” position and the observation that they often share similar annotated function with their neighboring M-nodes (data not shown), it is a logical prediction that I-nodes play a role in AHR signaling that is functionally similar to their modifier neighbors. We propose that I-nodes most commonly arise as the result of their essential gene nature  or because they represent a redundant gene product (white nodes in the figures). We offer two examples that support this idea. First, one essential gene I-node in the folding module, Cns1p, has recently been reported to be involved in AHR signaling . Second,The composition of the transcriptional activation module suggests that the AHR activates target genes via the coordination of histone acetylation, ATP-dependent chromatin remodeling, and direct recruitment of basal RNA polymerase II transcriptional apparatus see . We baseOur network analysis has also identified a functional module that regulates the ligand-dependent translocation of the AHR see . This nuA module that regulates the amount of receptor protein was also identified in our AHR–PIN see . This moA novel PASB-dependent step in AHR signaling appears to have been revealed by this network analysis see , group IWe began this study with the objective of defining the AHR signal transduction pathway in a manner that would allow us to quantify the number of loci and enumerate the steps involved in signaling. By integrating our deletion screen with the PIN framework and through subsequent computational and experimental annotations, we were able to identify modifier modules that regulate five distinct AHR signaling steps. In this regard, we found that the integration of multiple annotation approaches is vital for the reconstruction of the final picture by connecting and cross-validating individual information pieces. As interaction datasets become more fully developed and annotated, such a map will steadily improve and provide more accurate description of AHR signaling. Lastly, the systematic strategy that we developed in this work should be readily applicable to the study of most mammalian proteins to reconstruct corresponding modifier networks that regulate their signaling.S. cerevisiae strain BY4742  was used in this study. This deletion set was obtained from Research Genetics  in a 96-well arrayed format. The plasmid pCEN-AHR (PL1605) was constructed by replacing the TRP1 autotrophic marker of PL883 (HIS3 marker using a “marker swap” method (ADH1) promoter. LexA-AHRNΔ166 is a chimeric AHR, with its amino acid residues 1–166 replaced by residues 1–202 of bacterial repressor LexA, and is referred to in the Results section simply as “AHR” for convenience. The reporter plasmid pSH18–34 (PL623)  is a 2μ-based, URA3-selectable vector that contains the bacterial LacZ gene, as a reporter, under the control of eight LexA-binding sites. The plasmid pEG202  is a 2μ-based, HIS3-selectable plasmid containing the LexA1–202 sequence under the control of the ADH1 promoter. The plasmid pAHR (PL700) has been described previously (GAL4 inserted into the EcoRI site of pEG202. The control plasmid pAHRΔPASB (PL1799) is the same as pAHR except for the removal of the C-terminal half of the PAS domain. This pAHRΔPASB plasmid was constructed by subcloning the EcoRI fragment of PL248 . The OD600 was measured using a SpectraMax 250 microplate reader . Cells were then subjected to centrifugation at 3,500 rpm for 8 min, and the supernatant was decanted. The 96-well plates were placed upside-down on a stack of paper towels for 10 min to drain residual medium. For transformation, each plate was vortexed at maximal speed for 15 s before dispensing 22 μl of DNA in “OneStep” buffer  into each well. To make the DNA in “OneStep” buffer, one volume of DNA  was mixed vigorously by vortexing with ten volumes of “OneStep” buffer. After DNA was dispensed, the plate was quickly vortexed again at maximal speed for 10 s to resuspend the cells, followed by incubation at 45°C for 40 min. After this “heat shock” step, 5 μl of the transformation mix from each well was inoculated into a fresh 96-well flat-bottomed plate containing 96 μl per well of dropout medium without Trp, Ura, and His (dropout minus TUH medium) plus G418. The inoculum was gently mixed by vortexing and incubated at 30°C for about 4 d until transformants grew out.A high-throughput protocol was developed for 96-well transformation based on work previously described . Unless LacZ reporter assay, transformants from the 96-well plates were rearrayed into 384-well stock plates containing 30 μl per well of dropout minus TUH medium. The inoculum was incubated at 30°C for 2–3 d to allow cell growth. For the LacZ reporter assay at each agonist concentration, 0.5 μl of cell culture was transferred from the 384-well stock plate (30°C) into a clear-bottomed/black-walled 384-well assay plate  using a disposable 384-pin replicator (GenomeSystems/Incyte Genomics). In the 384-well assay plate, each well contained 18 μl of dropout minus TUH medium (diluted 1:4 in water) plus agonist at the tested concentration. The plates were then incubated at 30°C for 48 h to allow all strains to reach stationary phase. Cell growth was monitored by measuring the OD600 of each well using a SpectraMax Plus384 microplate reader (Molecular Devices). To initiate the fluorescence assay, 18 μl of lysis/assay buffer was added to each well. Lysis/assay buffer contained a mixture of CUG substrate , 10% SDS, 1 M NaPO4, and 25× TAE in the ratio of 1:1.4:350:17.5. For assays with pCEN-AHR transformants, no TAE was required. Plates were vortexed at medium speed for 1 min and left at room temperature for 20 min. The reaction was stopped by dispensing 6.5 μl of 25× TAE to each well and vortexing at medium speed for 1 min. The fluorescence emission of each well was detected using a Wallac “VICTOR V” microplate reader . The fluorescence reading was normalized to the corresponding OD600 value to obtain the LacZ reporter activity of each deletion strain.To perform the Selected deletion strains were transformed with the plasmid pAHRGFP. Transformants were incubated in a 96-well microtiter plate containing 100 μl per well of dropout minus TH medium at room temperature. Given that we have observed that small temperature shifts can affect AHR's localization, we found it more convenient to both grow and examine cells at the same temperature. For some samples, assays were repeated at 30°C using a heating chamber attached to the microscope. Such results were found to be comparable to those obtained at room temperature. For strains that reached early log phase, 0.5 μl of culture was mounted on a glass slide, and the AHR–GFP subcellular localization was examined using a Zeiss  Axiovert 200M microscope (α Plan-FLUAR 100× objective). Images were captured using an AxioCam HR digital microscope camera (Zeiss). To stain the nucleus in living cells, 4,6-diamidino-2-phenylindole (DAPI) was added to the dropout minus TH medium to a final concentration of 5 μg/ml.LacZ reporter activity of each deletion strain was compared to the average of wt BY4742 strain controls included in the same plate, and the fold change was obtained and log2 transformed. These data-processing steps, as well as subsequent modifier selection, were performed automatically using Perl scripts written “in house.” In brief, for the primary screen involving 4,507 deletion strains with low-copy pCEN-AHR system, a stringent cutoff of 4-fold change over wt control was chosen for selecting a pool of most significant AHR signaling mutants. This cutoff corresponds to a p value of less than 10–6 at all six assessed concentrations (null distribution: wt control). The initial positives were subject to validation and characterization in secondary screens with high-copy pAHR and control systems. The cutoffs for control pathways pGal4TAD and pAHRΔPASB in the secondary screens were chosen at 2-fold change over wt control, which corresponds to p values of 3.3 × 10–2 and 5.6 × 10–4 (null distribution: wt control), respectively.To identify deletions that modify AHR signaling, the http://dip.doe-mbi.ucla.edu) and the genetic interaction table from the MIPS database (http://mips.gsf.de/proj/yeast/). Perl scripts, written “in house,” were used to search the combined physical and genetic interaction database and identify all valid paths  that linked each pair of modifiers. The network graph was rendered using the Graphviz tool kit (http://www.research.att.com/sw/tools/graphviz/)  .Within experimental annotation layers of the AHR–PIN, the region corresponding to each functional module was outlined by a closed line (boundary) drawn manually on the network map. This boundary was delineated to include the maximal number of modifier nodes that are members of the corresponding functional module and the minimal number of modifier nodes that are nonmembers. This boundary was also defined in such a way that all enclosed modifier nodes were interconnected via paths within the enclosed region or through at most one modifier node outside. When defining functional modules in the summary AHR–PIN, the highest weight was given to the results from the localization influence experiments because these results provided the most direct indication of a modifier's effect on AHR signaling, and the lowest weight was given to the pharmacology clustering result because this result was highly sensitive to the choice of clustering algorithm.Table S1wt BY4742 strain. Also shown are their known gene names, products, gene descriptions, and Gene Ontology (GO) annotations .Click here for additional data file.Table S2This table contains all of the ORFs that were observed to influence the signaling of the AHR but not the pGal4TAD control. Also shown are their known gene names, products, gene descriptions, and GO annotations .(27 KB XLS).Click here for additional data file.Table S3This table summarizes the annotation on cellular functions of AHR modifiers. The annotation was derived from the YPD database, as of May 2002  .(23 KB XLS).Click here for additional data file.Table S4max = 2). Also shown are their known gene names, products, gene descriptions, and GO annotations .Click here for additional data file.Table S5p value is shown .This table summarizes the functional enrichment of each network cluster as calculated by the hypergeometric distribution of MIPS and GO annotations. For each cluster, the functional enrichment is determined by using M-nodes alone and both M- and I-nodes, respectively. In each case, the annotation that corresponds to the largest number of nodes in the cluster and the smallest (22 KB XLS).Click here for additional data file.


Vertebrates come in a dazzling array of shapes and sizes, from blue whales to pygmy bats, their overt morphology determined largely by the skeleton. The head skeleton in particular has undergone remarkable diversification, as is beautifully illustrated in Darwin's examination of beak morphology in Galapagos finches. It is now appreciated that a large part of the facial skeleton is derived from a newly identified, vertebrate-specific population of cells, called the cranial neural crest, that has its origins at the border of the dorsal neural plate (the future brain).Vertebrates develop from three germ layers—the endoderm, mesoderm, and ectoderm—which each give rise to distinct elements in the emerging body plan, and interactions between these layers are a common feature of embryogenesis. For example, early in development, cranial neural crest cells migrate to positions along the bottom  of the future head, where they form a series of developmental intermediate structures called pharyngeal arches. The arches facilitate interactions between crest cells (derived from ectoderm) and neighboring tissues (such as endoderm and surface ectoderm), which induce specific bone and cartilage patterns in the face. Recent chick studies showed that head endoderm, which contributes to the lining of the pharynx and gills, can pattern the facial skeleton. But the question remained, by what mechanism does endodermal signaling induce specific patterns of cartilage and bone?PLoS Biology, Justin Crump, Mary Swartz, and Charles Kimmel study the patterning of a jaw-support cartilage called the hyosymplectic in the larval zebrafish and find a “hierarchy of tissue interactions” at work. In zebrafish mutated for a gene called integrinα5, the authors report, a specific region of the hyosymplectic cartilage fails to develop. The loss of this cartilage region correlates with the loss of the first endodermal pouch. Pouches are outpocketings of the head endoderm that fuse with the skin to form the gill slits later in development. By labeling individual crest cells with fluorescent dye and making time-lapse recordings of these cells in transgenic fish, Crump et al. show that the hyosymplectic cartilage regions lost in the integrinα5 mutant are normally derived from crest cells directly adjacent to the first pouch.In this issue of Integrins are transmembrane receptors that promote cell adhesion and signaling. Although integrins function in crest cell migration, Crump et al. show that the Integrinα5 receptor is required in endoderm for hyosymplectic cartilage development and appears to promote development of the first pouch. The first pouch in turn acts as a template, by promoting both the survival and local clustering of crest cells, to pattern a specific region of the hyosymplectic cartilage.integrinα5 mutants also have region-specific defects in cranial muscles and nerves, the first pouch may serve to organize an entire functional unit in a region of the head. As the hyosymplectic element has undergone considerable change during evolution—from a jaw-support element in fish to a tiny, sound-conducting bone called the stapes in mammals—Crump et al. speculate that such a local, interconnected strategy of development would facilitate evolution of the vertebrate head. Changes in endodermal signaling would allow a particular skeletal element to vary in shape or size, in coordination with the muscles and nerves that move the skeletal element and independent of other regions of the head.But the pouch may have more far-reaching effects. Since It will be interesting to determine, the authors note, whether this hierarchical organization applies to other skeletal elements in the head. But for now, these results will inform efforts to understand the specificity of interrelated defects seen in human craniofacial syndromes such as DiGeorge Syndrome, whose underlying causes lie in the development of the endoderm.


Many trials of nebulized therapy have used nebulized saline as a "placebo". However, nebulized isotonic saline is sometimes used to assist sputum expectoration and relieve breathlessness in COPD patients. We designed this study to establish if nebulized saline had a placebo effect or a clinical effect.40 patients were studied following hospital admission for exacerbated COPD (mean FEV1 30% predicted). Patients were randomised to single-blind administration of either 4 mls of nebulized isotonic saline using an efficient nebulizer (active group n = 20) or an inefficient nebulizer (placebo group n = 20). Spirometry and subjective breathlessness scores  were measured before nebulized treatment and 10 minutes after treatment.There was no significant change in FEV1 after active or placebo nebulized saline treatment. Patients reported a 4% improvement in mean breathlessness score following placebo  compared with 23% improvement following active nebulized saline (p = 0.0001). 65% of patients given active nebulized saline but only 5% of the placebo group reported that mucus expectoration was easier after the treatment.This study lends support to the current use of nebulized saline to relieve breathlessness (possibly by facilitating sputum clearance) in COPD patients. Lung function was not affected. Nebulized saline can therefore be used as a placebo in bronchodilator studies involving COPD patients but it cannot be used as a placebo in trials assessing symptom relief. Nebulized saline is used by some doctors and physiotherapists to assist mucus clearance and to relieve breathlessness in patients with COPD, bronchiectasis and Cystic Fibrosis. This practice is justified by a small number of studies which have demonstrated enhanced sputum expectoration or improved breathlessness after nebulized saline or humidified oxygen -4. NebulHowever, nebulized saline has also been used as a placebo in several trials involving nebulized bronchodilator therapy ,11. For 40 patients were studied during a hospital admission for an exacerbation of COPD. Patients were recruited at a time when their condition had stabilized prior to their planned discharge from hospital. Clinical details of the patients are summarised in table Patients were randomised to receive 4 mls of 0.9% saline using an efficient nebulizer system (active group) or an inefficient nebulizer system (placebo group). The active nebulizer was a System 22 Acorn nebulizer  driven by the hospital's piped oxygen supply at a flow rate of 9 l/min for 10 minutes. This nebulizer system was found to deliver 95% of particles in the size range 2.5 to 2.8 microns using a Malvern laser system. . This small particle size was selected to achieve effective delivery to the airways. The placebo nebulizer was an old model (1980s) Bard Inspiron nebulizer (no longer manufactured) driven by oxygen at a flow rate of 3 l/min. This nebulizer system delivered 95% of particles in the size range 9.5 to 9.9 microns. This particle size was selected to achieve a placebo effect with deposition in the tubing of the system and in the pharynx but little penetration to the airways . Both neThe trial was conducted in a single-blind manner. 40 slips of paper were labelled "Treatment A" or "Treatment B" and placed in opaque brown envelopes. These were shuffled in random order and each patient was asked to select one envelope. This was then opened by the investigator and the appropriate treatment was administered . For the six patients who took part in the study twice, the second treatment consisted of whichever treatment they had not received previously.Patients were told that we wished "to observe the effects of a nebulized treatment which is not a new or experimental drug". They were not informed of the exact nature of the nebulized treatments as this might have led patients to try to guess if the treatment which they received was a "placebo". The Ethics Committee agreed that it would not have been possible to measure a true placebo effect if patients were made aware that both treatments were saline (not a bronchodilator) and one of the nebulizers was deliberately made to run inefficiently.Patients were recruited on the Respiratory Wards of a University hospital. We recruited patients who had diagnosis of COPD confirmed by a respiratory consultant (patients with asthma or bronchiectasis were excluded from the study). Patients were approached by one of the investigators whilst in a relatively stable phase prior to discharge from hospital following an admission for exacerbated COPD. All testing was undertaken between 12.00 and 16.00, at least four hours after bronchodilator treatment.Prior to participating in the study, patients gave informed consent and undertook baseline measurement of FEV1 and FVC using the best of 3 blows on a Microlab 3300 Spirometer  was measured using a Wright's Peak Flow meter. Each patient also recorded an assessment of their perceived level of breathlessness using a seven point modified Likert scale .Ten minutes after completion of nebulized therapy, FEV1, FVC and PEF measurements and subjective breathlessness scores were repeated. Patients also recorded a subjective assessment of benefit using the following modified Likert scale. .Patients then received 4 puffs of salbutamol (400 mcg) using a Metered Dose Inhaler and 750 ml Volumatic spacer (Glaxo Smith Kleine UK).Fifteen minutes later, FEV1, FVC and PFR measurements and subjective breathlessness scores and symptom relief scores were repeated.All data was entered on a SPSS version 9 statistical package. Mann Whitney U-test was used to compare lung function tests and symptom relief scores. Wilcoxon Signed Rank test was used to compare the change in breathlessness scores for matched pairs before and after nebulized saline.The study was approved by Salford and Trafford Research Ethics Committee. All patients gave written informed consent to partake in the study and to receive a single dose of nebulized treatment .34 patients completed the study; patient details are summarised in table The baseline FEV1 of the two treatment groups was well matched. Both groups had a non-significant fall in FEV1 after nebulized saline therapy and a small rise in FEV1 after 400 mcg salbutamol from MDI-spacer Table . FVC, anThe placebo group had a 4% improvement in breathlessness after treatment (Wilcoxon p = 0.37) compared with a 23% improvement after active nebulized saline (Wilcoxon p = 0.0001). This corresponded to a reduction from 4/7 (moderate breathlessness) before treatment to 3/7 (mild breathlessness) after treatment in the active treatment group.The mean symptom relief score (patient's assessment of benefit) for the active treatment was 3/7, (slight benefit) almost identical to the response to 400 mcg salbutamol from MDI. The placebo group had a score of 2/7(very slight benefit) after nebulized placebo and 3/7(Slight benefit) after 400 mcg salbutamol from MDI.15 patients in the active group felt better after nebulized saline, 5 felt the same and no patient felt worse. Six patients in the placebo group felt better after nebulized treatment, 12 felt the same and 2 felt worse (Chi Squared test p = 0.013).Patients were asked if the nebulized treatments had any effect other than relief of breathlessness. 13 patients in the active group (65%) said that the nebulized treatment assisted sputum expectoration. Only 1 patient in the placebo group reported this effect . No patient reported any adverse effects from either nebulized treatment.This is the first study which has compared active nebulized saline with placebo nebulized saline. The results suggest that nebulized saline has non-bronchodilator therapeutic effects that are possibly explained by airway-moistening and sputum-inducing effects of nebulized saline. Sputum volume was not measured in the present study but two thirds of patients who were given nebulized saline through an efficient nebulizer system reported that it helped them to expectorate sputum. This finding is consistent with the results of previous studies which have shown improved sputum clearance and decreased breathlessness following the open administration of nebulized saline ,3 The rePrevious studies have shown that nebulized saline can have a bronchoconstrictor effect in some patients which is greater with hypertonic saline than with isotonic saline and greater in asthma patients than COPD patients ,6,8,9 NeThe main strength of the present study is inclusion of a placebo limb using an inefficient nebulizer system. Patients in the placebo group believed that they were receiving a nebulized treatment because a placebo effect could have been abolished if patients were told that both treatments involved no active medication and one of the patients involved an inefficient nebulizer system. This issue was discussed fully with the ethics committee and found to be acceptable because the patients did not miss any of their regular medication and they did not receive any pharmacological treatment. The use of a mouthpiece ensured that patients could not see or feel that the output from the experimental system was different to the nebulized bronchodilator therapy which they had received during their hospital admission . Furthermore, only 6 patients took part in both limbs of the study so most patients could not have tried to guess which treatment was more effective.The 23% improvement in breathlessness in the active group was equivalent to the subjective benefit following 400 mcg of salbutamol from MDI-spacer. This improvement in breathlessness occurred without bronchodilation, mirroring the findings of Poole et al . PatientThis study is in agreement with previous studies which have shown no bronchodilator effect  when nebulized saline is given to patients with COPD . This juHowever, as nebulized saline has non-bronchodilator therapeutic effects, it cannot be used as an inert placebo treatment in clinical studies where breathlessness or quality of life are to be measured. For example, Jenkins et al concluded that nebulized treatment had a strong placebo effect because patients expressed a preference for nebulized treatment even though the same bronchodilator effect could be achieved for most of their patients when nebulized saline was given with active bronchodilator therapy from a MDI device . It is lBritish and European nebulizer guidelines state that most patients with airflow obstruction should be treated with hand-held devices unless they have demonstrated clear additional benefit from the use of nebulized treatment in carefully monitored domiciliary studies ,14. The FEV1 Forced Expiratory Volume in 1 secondFVC Forced Vital CapacityPEF Peak Expiratory FlowCOPD Chronic Obstructive Pulmonary DiseaseMDI Metered dose inhalerThe authors declare that they have no competing interests.BROD developed the concept for the study and both authors designed the study protocol.SYK recruited patients and performed all study measurements.Both authors assisted in analysis of the data and preparation of the manuscript.The pre-publication history for this paper can be accessed here:


Swedish patient associations for breast cancer patients (PABCPs) offer patients with breast cancer unlimited meetings with a breast cancer survivor, a contact person (CP). We applied the voluntary action perspective in this interview study with members of Swedish PABCPs in order to explore how women with breast cancer experienced their contact with a CP from a PABCP.Audio-taped narratives from 8 women were analysed using Reissman's monitoring and Gee's analysis structure.Three themes appeared: 1. Shared experiences give new perspectives on having cancer, 2. Feelings of isolation are a part of the identity of the illness and 3. Relations with others enable self-help. However, the relationship with the CP is sensitive to timing, correct information and understanding.CPs act as sounding boards and should optimally have capacity for listening, gives support and act as partner in this conversation. On the other hand, CPs should be aware that their presence and limited general medical knowledge could at times disturb the patient's psychological recovery and strengthen feelings of isolation. Thus, PABCPs must be careful in selecting CPs and offer relevant educational activities related to the themes identified herein. Reach to Recovery Program (RtoRP), a world-wide rehabilitation program initiated in the U.S. in 1952 and was later established also in Europe. RtoRP is based on the opportunity to meet with individuals with similar experiences and thereby learn different ways of dealing with disease-related feelings and problems  understandswhat I mean, like she understandsthat I can float above itand thought like it wasn't so serious (informant 8)yes, I think thatshe took me seriouslywhen I felt like it was hardnot to be able to identify myself in the group [the PACP group] (informant 8)One woman described being one among others like oneself by talking about the special feeling that came over her when she was welcomed into the association. It was not necessary to say that she had had a breast cancer operation since everyone there knew; this was obvious in the association. Another woman described the importance of meeting people in the same age and situation in life, which took place at regular theme meetings for younger women. A younger woman exemplified the importance of being the same age by saying that women of the same age had common questions to discuss – questions that could not be talked about with health professionals. The following two stanzas illustrate how the women used the CP meetings to talk with women with similar problems:it could for example behow I could make up my eyes nowwhen I don't haveany eyelashes (informant 6)if there was anyonewho had childrenbecause the ones I'd met were only older peoplewho didn't have children living at home (informant 6)At each contact meeting, the women felt that they could gain knowledge through the different experiences that were described about different forms of treatment and their effects on well-being. One woman describes how she could balance her worry in this environment by talking about what she thought about, which is illustrated in the stanzas below:how will I go through this psychologicallyin the futurewill I trust this answeror how much will I worry (informant 2)alot of questions like thatthat I've thought aboutand that I couldtalk about with other people (informant 2)Another woman's reactions to some of the meetings were feelings of sadness, while other meetings could give her hope for the future. However, while the meetings made her react differently, she felt it was important to come to the CP meetings regularly to see with her own eyes that the other women were alive and led good lives. It came out in another narrative how it is gradually possible to talk with any of the members of the association that have experience even though it is the CP that is the person one initially talks with. Thus in the long run the experiences are felt as being most important, where the CP acts to open the door to them.Time, in the sense of a particular phase in the process, is important when the women want to make contact with others with experience of having breast cancer. One woman felt that she was not ready to contact the association and meet others with similar experience despite having many questions. At first, she said, she wanted most just to crawl into herself and manage on her own without involving other people. Nevertheless, this woman was content having received a card from the CP early in the process, telling her about the possibility of establishing a contact. The possibility of getting a contact is thus given higher priority than hearing others' experiences at an early stage.One woman evaluated the meeting with the CP positively in part because the CP was a trained CP and in part because the CP knew what she needed to hear. This is illustrated in the following stanza:it was most of all goodto hear [name of CP]maybe because she had trainingknew what you needed to hear (informant 8)Another woman described how she had used her CP as a "sounding board" in her choice of surgical method. It comes out in this narrative that the sounding board function and not the counselling function made the woman finally decide in favour of a surgical method that the CP had not recommended on the basis of her own experience. The fact that the CP had given an opinion helped the woman to form an opinion herself. Thus it is not necessary to share the exact same experience; at times it is enough to share the experience of cancer.One of the women said that she would have appreciated there being someone with experience to contact when she was forced to wait for a long period before having her operation. Access to a CP for one's own personal problems is illustrated in the following stanza:I know she's the contact personwhich makes me thinkthat I can turn to herand talk about my special problems (informant 8)Women describe not only the benefit of the CP meetings but also say that there can be a feeling of more or less indifference in the contact with the CP. In one example the CP acted as a guide to the association's premises but had otherwise not spoken of anything in particular. Another woman had come into contact with the association's CP at an educational class but had never contacted a CP herself. For these women it was sufficient to contact with other women in educational classes organised by professionals.Since the CP activity is central within the PABCPs, we aimed to investigate how women with breast cancer described their experiences from having contact with a CP. The narratives allowed the women to reflect and formulate themselves through this opportunity to tell their own stories with the aim to analyse their experiences . The strThe narratives in our study illustrate the importance of meeting women of the same age and in a similar life situation and of being in an environment (CP meeting) that is free of need to explain. Meeting others and experiencing their' reactions help these women feel normal . "CatchiEven though all patients do not wish to be confronted with the experiences of others via personal contact with a CP, they may be ready to be confronted with the disease in other ways. A confrontation with women who have undergone partial or complete mastectomies can take place in water therapy sessions and in the sauna. The results show that this meeting also gave a feeling of fellowship. This may mean that the person who chooses only this kind of activity is ready only for visual impressions. Breasts mean different things to different women, and only the individual woman can define how great a handicap breast surgery is. Langellier and Sullivan  studied It is also important that a CP is able to manage timing, in the sense of the phase in the process at which contact is made with others who have experience or when the person is ready to confront others' experiences. Great sensitivity towards individual needs is required and there are individual variations as to the optimal time for a meeting, which are related to when the individual is ready to encounter other people's experiences. In terms of being ready, thoughts arise as to the many visual and auditory impressions that cancer patients receive during a treatment session, e.g. in a waiting room and when given treatment, regardless of whether they are ready for these or not.It is conceivable that some individuals do not have any need to meet with other people and share their experiences since recovery can also occur without involving others. However, it is not only the initial need for contact that varies. Women also have different needs in terms of the duration of the contact. For one person, contact with a CP may simply mean guidance in the association's activities while for another a CP can act as a support person for a period of several years and the information-seeking behaviour can also change over time .For the woman who felt great isolation in the illness the meeting with the CP strengthened the feelings of isolation, which demonstrates how the meeting with the CP led to a non-intended consequence. This demonstrates the need for the PABCPs to be meticulous in the choice of CPs; the requirements must not only be that they have been rehabilitated but also that they have substantial knowledge about cancer, treatment possibilities, and psychological reactions to disease. Although self-help may come through a relation with other individuals with a shared experience  the assoThe results also demonstrate that some questions could not be answered by health-care professionals, but rather by individuals with personal experience from cancer. Hence, new knowledge developed in the meeting with the CP  declare that they have no competing interests.CC: study design, data collection, data analysis, and writing the manuscript.MN: supervising the study and participation in writing the manuscript.KN: study design, data analysis, supervising the study, and participation in writing the manuscript.The pre-publication history for this paper can be accessed here: