Drosophila melanogaster is extensively studied as a very powerful model of developmental biology. One main advantage of this system is the ability to study the origin and development of individual sensory cells. However, there remain several discrepancies regarding the organization of sensory organs in each abdominal segment A1-A7.The embryonic and larval peripheral nervous system of We have constructed a web site called FlyPNS  that consolidates a wide range of published and unpublished information about the embryonic and larval sensory organs. It communicates (1) a PNS pattern that solves the discrepancies that have been found in the recent literature, (2) the correspondence between the different nomenclatures that have been used so far, (3) a comprehensive description of each sensory organ, and (4) a list of both published and unpublished markers to reliably identify each PNS cell.Drosophila sensory organs.The FlyPNS database integrates disparate data and nomenclature and thus helps understanding the conflicting observations that have been published recently. Furthermore, it is designed to provide assistance in the identification and study of individual sensory cells. We think it will be a useful resource for any researcher with interest in  Drosophila abdominal larval PNS is composed of a constant number of neurons and associated cells, whose characteristics and positions are reproducible between individuals [The ividuals -5. Its sDespite the extraordinary utility of the PNS for understanding these diverse problems, a prominent current limitation is the lack of agreement in the literature of the exact number, position and nomenclature of sensory neurons. For example, many different names have been used to describe abdominal neurons, reflecting the number of cells within a cluster, their position ,7, theirWe have constructed a website, named FlyPNS, that consolidates a wide range of published and unpublished information regarding the embryonic and larval sensory organs and their associated glial cells. Motoneurons and other glial cells have not been included. The FlyPNS web site is arranged in 6 sections: General references, Nomenclature, Abdominal PNS organization, Sensory organ description, Antibodies and enhancer-traps, Gal4 lines, and a Search option.General references section provides a list of papers on various aspects of sensory organ morphology and development. Subcategories include general descriptions, descriptions of dendritic arborizations, axonal pathways, developmental changes, and functions of multidendritic neurons. Links are provided to PubMed entries for access to abstracts or full-texts of papers.The Abdominal PNS organization section, we have represented the PNS pattern that we have observed , markers, morphology, development and related references. A few unpublished observations relating to maker expression and morphology are also mentioned. A typical sensory organ description is shown in Fig. The Antibodies, enhancer-traps and Gal4 lines pages display a collection of published and unpublished data on expression patterns of antibodies, enhancer-traps and Gal4 lines, with links to the gene/insertion information contained in Flybase when available.The Search option provides a convenient link to the information about any of the organs/markers.Finally, the Drosophila investigators.Much of the discordance in the literature regarding the larval and embryonic PNS pattern seems to be rooted in variation in the precise position of sensory cells with segment, embryo, and time, and a lack of specific markers for some organs. These factors might explain why, for example, some dorsal cluster neurons were often missed in previous publications. We have attempted to resolve remaining ambiguities by (1) identifying the total number of sense organs and neurons in each abdominal segment A1-A7, (2) resolving different nomenclatures used for each organ, and (3) providing a collection of molecular markers and descriptions that provide an unambiguous guide for cell identification. This data compilation will hopefully also increase the accessibility of the fly PNS literature to non-. Comments and additions are welcomed by e-mail to VO.The FlyPNS web site is available from the link under Miscellaneous in the FlyBase Allied Data section PNS: peripheral nervous systemda neuron: neuron displaying a dendritic arborizationThe PNS pattern was established based on the observations of WBG and VO. VO created the FlyPNS web site and WBG made significant improvements and corrections to the web site. Both VO and WBG wrote the manuscript.


A cross sectional study was designed to survey the relationship between anxiety/depression and duration/cause of infertility, in Vali-e-Asr Reproductive Health Research Center, Tehran, Iran.After obtaining their consents, 370 female patients with different infertility causes participated in, and data gathered by Beck Depression Inventory(BDI) and Cattle questionnaires for surveying anxiety and depression due to the duration of infertility. This was studied in relation to patients' age, educational level, socio-economic status and job (patients and their husbands).Age range was 17–45 years and duration and cause of infertility was 1–20 years. This survey showed that 151 women (40.8%) had depression and 321 women (86.8%) had anxiety. Depression had a significant relation with cause of infertility, duration of infertility, educational level, and job of women. Anxiety had a significant relationship with duration of infertility and educational level, but not with cause of infertility, or job. Findings showed that anxiety and depression were most common after 4–6 years of infertility and especially severe depression could be found in those who had infertility for 7–9 years.Adequate attention to these patients psychologically and treating them properly, is of great importance for their mental health and will improve quality of their lives. The impact of infertility on the psychological well being of couples involved has been the object of increasing attention in recent years. It cannot be denied that infertility is a deeply distressing experience for many couples . In lattWe aimed to examine prevalence and severity of anxiety/depression in relation to duration/cause of infertility in Iranian infertile women.Several studies have demonstrated anxiety has a detrimental effect on fertility  and thatInfertility sometimes is accompanied by existential crises and emotional tensions such as anxiety, interpersonal problems, and suppressed anger, unsatisfactory in interpersonal, frustration, inferiority feeling, depression, rejected feeling and unconscious guilt feeling. Those couples with a history of failure in Assisted Reproductive Technique (ART) have shown personality maladjustment .Overall percentage of psychological problem in infertile couples ranges between 25 and 60% . One stuAnother study showed those who had 2–3 years infertility had more depression / anxiety than those who had this problem for a year or more than 6 years. Peak of depression could be seen during third year of infertility. After six years there will be a reduction in psychological symptoms in women. During first three years, infertility is accompanied by signs such as anxiety, depression, loss of self-esteem, impotence and maladjustment of marital status. After 3 years, optimistic attitude would change to despair and at last there will be some emotional changes to adopt a child or live without one, thereafter. Those who have social support, positive personal characteristics, and have a satisfactory life with their spouse show no signs of anxiety/depression .Since most literature on psychological aspects of infertility is from developed countries it was thought that a study from a developing country with a different culture might contribute to existing knowledge on the topic. We aimed to examine prevalence and severity of anxiety/depression in relation to duration of infertility in Iranian infertile women. The results of this study, which included counseling and couple-therapy, are being prepared for infertile couples.The subjects were 370 infertile women who were referred to Vali-e-Asr Reproductive Health-Research Center between January 2001 and January 2002 for treatment of their infertility problems. In this group mean (± SD) age was 28 (± 5.37) and mean (± SD) duration of infertility was 6.36 (± 4.18). Among them 293 (79.2%) women were housewives and the others were working outside.A gynecologist evaluated patients and then they were visited by a psychologist and were informed of the study purposes. Diagnosis of male factor is based on WHO criteria 1999. Menstrual history and mid-luteal progesterone level considers ovulatory factor, diagnosis of endometriosis is done by lapascopy, tubal factor is diagnosed by HSG and cervical facto is detected in Post Coital Test. After obtaining oral consent from each patient, data were collected using BDI , and CatThe test used was a translated and validated Persian version of Beck's depression Inventory. A full 21-items BDI was administered. This scale is a widely used measure for intensity of depression.Each item describes a specific behavioral manifestation of depression. Scores on each item can range from 0, indicating no depressive symptomatology, to 3, indicating a severe level of symptomatology. Total scale scores can thus range from 0 to 63. Scores of 17 or above it indicates of a clinically significant depression. The classification of depression scores involves:1. 0–16 (without depression)2. 17–27 (mild depression)3. 28–34 (moderate depression)4. 35–63 (severe depression)The Cattle inventory is a 40 items self-report measure of anxiety. This test was a translated and validated Iranian version of Cattle's Inventory. Scores can range from 0 to 80, with scores of 28 or above demonstrate anxiety. Classification of anxiety scores involves:1. 0–27 (without anxiety)2. 28–40 (moderate anxiety)3. 41–49 (neurotic anxiety)4. 50–80 (severe anxiety)For each patient the following data were recorded: age, cause and duration of infertility, education and job.Data were analyzed by using statistical SPSS. The relationship between continuous and binary explanatory variables with Beck and Cattle scores were assessed using spearman's rho and unpaired t-test, respectively. In addition, the relationship between categorical responses and explanatory variables were evaluated using chi-square test. For descriptive purposes, we presented frequency tables.Three hundred seventy infertile women were considered in this cross-sectional study. The results of Beck and Cattle inventories showed that 40.8% and 86.8% of these women had depression and anxiety symptoms, respectively Table .Mean scores of depression and anxiety by age groups are shown in figure 2 statistic for this 2 × 9 table (chi-square = 20.643 P = 0.014) showed that prevalence of depression is not equal in these categories. Same analysis for the prevalence of anxiety (anxiety group is consisted of moderate, neurotic and severe anxietic women) in different groups of infertility causes showed no significant difference between them (chi-square = 7.491 P = 0.485)  in all categories of infertility causes. ChiThe correlation between Beck and Cattle scores based on Spearman's Rho was 0.707 (P < 0.001), which shows a significant relation between depression and/or anxiety scores. Then, we checked correlation coefficient between Beck and Cattle scores with quantitative variables like age, education (in years) and especially the duration of infertility.Duration of infertility showed a significant relation with both Beck (r = 0.15, P = 0.004) and Cattle (r = 0.157, P = 0.002) scores. Based on duration of infertility 31(29.2%), 51 (42.1%), 30 (46.8%) and 39 (49.3%) patients had depression in different groups, but there was no significant relationship between duration of infertility and depression  (Table Also 82 77.3%), 110 (90.9%), 58 (90.6%) and 71 (89.8%) of patients had different stages of anxiety and there was a significant relation between anxiety and duration of infertility  . Anxiety and depression were observed more in homemakers comparing to outside employees though we found no significant effect of women's job on Cattle scores (t = 2.943, P = 0.087) Table .Patients participating in this study were from different geographical areas in Iran. The finding of this study provides information about frequency and severity of anxiety/depression in order to duration of infertility in childless women.The prevalence of psychiatric morbidity specially depression and/or anxiety in infertile patients have been assessed in several countries, for example Jones et al 1993) found that, there was mild to moderate depression in 28.3% of infertile women, moderate to severe depression in 7.2% and 1.2% had most severe depression based on BDI 993 found. AnxietyThe most common age for depression and/or anxiety in our study was 21–25 years. In this study, anxiety and/or depression had negative correlation with education. In other words, with the raise of education level, anxiety and/or depression decrease.Results of different studies about relationship of age and education with anxiety and/or depression were not similar. Age and education level have no significant relationship with depression and/or anxiety . AnotherHaving a job may reduce stress from In Vitro fertilization (IVF) . In our Based on previous researches -28, infeBased on our study depression is more common in "unexplained cause" group comparing to other causes and anxiety is more common in "endometriosis" group comparing to other causes, our results is similar to other studies ,3,6,9,13Anxiety and/or depression increases with duration of infertility . A studyIn conclusion it can be suggested that psychological interventions especially in 4–9 years of infertility may prevent the surge in depression/anxiety and could presumably lead to increased pregnancy rates.FM contributed to design and management of the study.NA contributed to study design and data gathering.The initial idea of this study was by MMA.NK contributed in design, writing of the manuscript and analysis of this research.FZ contributed in design, writing of the manuscript and analysis of this research.MS contributed in edition and revision of the article.MJ contributed in edition and revision of the article.The pre-publication history for this paper can be accessed here:


Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension.S100A4 is a small CaCell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B.In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB).In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown. It has operties , stimulaoperties . Moreoveoperties , indicatoperties .Interferon-gamma (IFN-γ) is a pleiotropic cytokine secreted from activated T lymphocytes and natural killer cells. It can activate and suppress a number of target genes, leading to various effects, including inhibition of viral replication, activation of immune cells and induction of cell cycle arrest and apoptosis ,15. ApopSearching for cytokines and signal transduction modulators affecting S100A4 expression, we recently discovered that IFN-γ downregulated S100A4 transcription and induced apoptosis in the human osteosarcoma cell line OHS . In the 4 cells/cm2. After overnight incubation, the culture medium was replaced with medium in presence or absence of IFN-γ and recombinant S100 proteins, and harvested as indicated in the text. Where indicated, protease inhibitors or antioxidants were added to the cell culture simultaneously as IFN-γ.The human osteosarcoma cell line OHS and its anti-S100A4 ribozyme transfected counterpart II-11b have been described previously . Cell liHuman recombinant IFN-γ, L-NMMA, zVAD-fmk, zYVAD-fmk, zVDVAD-fmk, zDEVD-fmk, zVEID-fmk, zIETD-fmk, zLEHD-fmk, zFA-fmk and E64 were purchased from Calbiochem (San Diego, CA, USA). N-acetylcysteine was from Sigma Chemical Co (St Louis, MO, USA).2+-column and dialyzed against phosphate-buffered saline. A satisfactory degree of purity was confirmed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) where a single band was visualized by silver staining. Verification of the identity of the protein was performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI). Human recombinant S100A10 and S100A13 tagged with maltose-binding protein were kindly provided by C. S. Skjerpen .Histidine-tagged mouse recombinant S100A4 was cloned into the pQE30 vector (kindly provided by E. Lukanidin, Institute of Cancer Biology, Copenhagen, Denmark), expressed in E. coli, purified on a Ni® AQueous One Solution Reagent (MTS) (Promega, Madison, WI, USA) according to the manufacturer's manual. The absorbance at 490 nm was recorded using a Wallac 1420 Victor2 Multilabel counter .Cell viability was estimated by measuring metabolic activity using CellTiter 96Protein lysates were prepared in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.1% NP-40 with 1 mM PMSF and 2 μg/ml each of pepstatin, aprotinin  and leupeptin (Roche Diagnostics, Mannheim, Germany). Total protein lysate from each sample was separated on 8–12% SDS-polyacrylamide gel electrophoresis, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA) according to the manufacturer's manual. As a loading and transfer control, the membranes were stained with 0.1% amidoblack. After blockage of non-specific binding sites with 10% non-fat dry milk in Tris-buffered saline with 0.25% Tween (TBST), blots were incubated for 1 h at room temperature with rabbit polyclonal anti-poly (ADP-ribose) polymerase , mouse monoclonal anti-lamin B , mouse monoclonal anti-α-tubulin , rabbit polyclonal anti-caspase-1 , goat polyclonal anti-caspase-3 , mouse monoclonal anti-caspase-8 , mouse monoclonal anti-caspase-9 , rabbit polyclonal anti-caspase-12 , mouse monoclonal anti-NFκB p65  or mouse monoclonal anti-phospho-IκBα (Ser32/36) . After washing, the blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) diluted 1:5000. Anti-lamin B, anti-α-tubulin, anti-PARP, anti-caspase-1, anti-caspase-8, anti-NFκB p65 and anti-phospho-IκBα were diluted in TBST with 5% non-fat dry milk, anti-caspase-3 and anti-caspase-12 were diluted in TBS containing 0.05% Tween and 2% non-fat dry milk and anti-caspase-9 was diluted in TBS with 0.05% Tween, 1% non-fat dry milk and 1% BSA. Signals were visualized using the ECL chemiluminescence substrate (Amersham Pharmacia Biotech, Buckinghamshire, UK).Cells were resuspended in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 10 mM EDTA, 0.5 % SDS and 0.5 μg/ml proteinase K) and incubated for 1 hour at 50°C. High molecular weight DNA was precipitated by adding 1 M NaCl and subsequently incubated overnight at 4°C. Samples were centrifuged for 30 min, 2700 g at 4°C, and supernatants collected. 95 % ethanol was added, and DNA precipitated overnight at -20°C. Samples were centrifuged for 10 min, 10 000 rpm at 4°C, and pellets were washed in 70 % ethanol and re-centrifuged. Thereafter, pellets were air-dried and dissolved in 10 mM Tris-HCl (pH 7.0), 15 mM NaCl, 1 mM EDTA and 0.2 mg/ml RNAse A, and incubated for 1 h at room temperature. DNA was separated by electrophoresis in 1.0 % agarose gel containing ethidium bromide and photographed under ultraviolet illumination.Cell culture medium from OHS and II-11b cells were harvested at 24, 48 and 72 hours, and immediately filtered using a 0.45 μm filter to discard cells in suspension. From each sample, 1.5 ml of medium was subjected to immunoprecipitation using 15 μg of rabbit polyclonal anti-S100A4 (DAKO, Glostrup, Denmark) or 15 μg rabbit polyclonal anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibody binding was carried out in cell culture medium at 4°C overnight. Thereafter, antibody was precipitated using Protein A Sepharose beads for 1 h at 4°C and eluted in loading buffer. Samples were subjected to Western blot analysis as described above using rabbit polyclonal anti-S100A4 diluted 1:300 in TBST.6 II-11b cells were transiently transfected with 10 μg of the NF-κB reporter construct using electroporation (240 V). Thereafter, cells were seeded in 96-well plates at a density of 4.5 × 104 cells/cm2 and incubated in the absence or presence of rS100A4 and zFA-fmk. Cells were harvested 48 h later and assayed for luciferase activity using the Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's manual.The NF-κB reporter construct containing three NF-κB response elements  driving luciferase expression has been described previously . 1.0 × 1All statistical analyses were performed using Student's t-test. P-values less than 0.05 were considered to be statistically significant.We have previously observed that the human osteosarcoma cell line OHS is more sensitive to IFN-γ induced apoptosis than the S100A4-ribozyme transfected counterpart II-11b . To examPreviously, we have shown that treatment of OHS cells with IFN-γ induced a significant increase in the fraction of apoptotic cells, while the cell cycle distribution remained unchanged . Thus, iWhen performing the OHS cell culture experiments, we observed that, when keeping the concentration of IFN-γ constant, decreasing the volume of cell culture medium or increasing the number of cells resulted in increased cell death (data not shown). This observation suggested that the cells might secrete a soluble factor with impact on the level of apoptosis induced by IFN-γ. Since the II-11b cells are derived from the OHS cells by transfection with an S100A4-specific ribozyme, this finding prompted us to investigate whether S100A4 is secreted from these cells, and whether extracellular S100A4 could cooperate with IFN-γ in induction of apoptosis. Fig. In order to investigate whether extracellular S100A4 could increase apoptosis induced by IFN-γ, recombinant S100A4 (rS100A4) was added to the cell culture medium of OHS and II-11b cells in addition to IFN-γ. Addition of rS100A4 alone had no effect on cell viability at concentrations ranging from 400 pg/ml to 20 μg/ml Fig. . In factCaspase-mediated apoptosis is the most important program of cell death. In order to investigate whether IFN-γ activated the caspase cascade in our cell system, we have followed two different approaches: detection of activated caspases by Western blotting and treatment of cell cultures with caspase inhibitors. Using specific antibodies detecting both the proenzyme and the active form of caspase-1, -3, -8, -9 and -12, we were not able to detect any activation of caspase-1, -3, -8 or -12 Fig. . This coThe caspase inhibitors used in the experiments above were solved in dimethyl sulfoxide (DMSO), and therefore DMSO was included as a negative control. Surprisingly, a significant inhibition of IFN-γ-induced apoptosis was observed using 0.25 % DMSO Fig. . One of in vitro [The pan-caspase inhibitor zVAD-fmk has previously been shown to bind the cysteine protease cathepsin B and to suppress cathepsin B activity in vitro . Since cin vitro , zVAD-fmin vitro ; thus, win vitro . Interes2+-binding protein S100A4 sensitizes human osteosarcoma cells to IFN-γ-induced apoptosis. Furthermore, we have shown that the IFN-γ-mediated cell death is independent of caspases, but possibly mediated by reactive oxygen species. Finally, we have demonstrated that extracellular S100A4 can activate NF-κB through increased phosphorylation of IκBα, but whether this NF-κB transactivation is required for apoptosis induction remains unknown.IFN-γ induces apoptosis in a variety of different cell types, but some cells are less susceptible or even resistant to IFN-γ-mediated apoptosis. The reasons for this variation have not been clearly elucidated, and given the biological importance of IFN-γ, a better understanding of the mechanisms of IFN-γ-induced cell death is warranted. In the present study, we have demonstrated that the Ca2+ or other divalent cations [Several of the S100 proteins are released into the extracellular space, mostly by unknown mechanisms ,31,32. T cations .Addition of 20 μg/ml rS100A4 (1.5 μM) to the cell medium of II-11b cells was required to induce apoptosis upon IFN-γ-treatment. Comparably, micromolar amounts of S100B and S100A8/A9 were needed to induce cell death ,34. Alsoet al demonstrated that S100A4 interacts with the tumor suppressor protein p53 to enhance p53-dependent apoptosis. The proposed mechanism was based on modulation of p53-dependent transactivation of target genes [et al., in preparation). These findings, in addition to the present work, suggest that secreted S100A4 could sensitize IFN-γ-signaling, and this possibility is currently under investigation. Previously identified sensitizers of IFN-γ-signaling include type I interferons (IFN-α/-β) [et al. discusses, based on their experiments using peripheral blood mononuclear cells, that other factors probably also exist to sensitize IFN-γ-signaling. Interestingly, S100A4 is strongly expressed by T lymphocytes , but whether lymphocytes also secrete S100A4 remains to be investigated.In many cases, stimulation of tumor cells with a single proapoptotic signal is not sufficient for induction of programmed cell death. On the other hand, a number of cytokines and other proapoptotic signal substances can increase the cells' sensitivity to other apoptosis-inducing agents, thus initiating the cell death program. IFN-γ induces apoptosis in synergism with a wide variety of such agents, and particularly members of the tumor necrosis factor (TNF) superfamily have been studied, e.g., TNF-α , FasL 3, LIGHT  and TRAIet genes . The ostet genes , thus, SFN-α/-β)  and IFN-FN-α/-β) . Hu et aet al. suggest that zFA-fmk has a promoter-specific NF-κB inhibitory effect [Our results suggest that S100A4-mediated NF-κB activation might be required for triggering the apoptotic process induced by IFN-γ. To support this hypothesis, we made attempts to inhibit NF-κB signaling by treatment with SN50, a peptide that contains the p50 NLS and selectively blocks nuclear translocation of NF-κB, but this lead to an increased fraction of apoptotic cells. Schotte y effect , whereasy effect .IFN-γ-induced apoptosis in OHS cells was shown to be caspase-independent by two different approaches: (i) no active forms of a number of caspases were detected; and (ii) caspase inhibitors were not able to suppress cell death triggered by IFN-γ. Even though the caspase cascade was not activated, well-known hallmarks of apoptosis were identified. DNA fragmentation and Lamin B cleavage have previously been shown to occur independent of caspase activation ,45, wherIn conclusion, we have demonstrated that extracellular S100A4 induces NF-κB transactivation and possesses proapoptotic activity through sensitization of IFN-γ-induced apoptosis, but whether activation of NF-κB is required for the observed sensitization of cell death is still unknown. Even though the precise physiological role of the apoptosis sensitizing effect of S100A4 awaits further studies, it highlights yet another area of research in the attempts to elucidate the complex biological roles of S100A4.None declared.KBP carried out the cell culture experiments, immunoprecipitations and western blots used in the detection of S100A4 in cell culture medium. In addition he did the dose-response experiments in II-11b cells, all the experiments using caspase inhibitors and NAC, western blot analysis of caspase activity, and drafted the manuscript. KA carried out dose-response experiments in OHS cells, and in addition all the cell culture experiments used in subsequent western blot analysis of caspase- and NF-κB activity and DNA fragmentation analysis. In addition she did transient transfections and all cell culture experiments using other S100 proteins and anti-RAGE antibodies. ØF participated in the design of the study. GMM conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We have demonstrated that moderate alcohol consumption (15 g/d, 30 g/d) for 8 weeks resulted in significantly increased levels of serum estrone sulfate and DHEAS in 51 postmenopausal women in a randomized, placebo-controlled trial. We now report on the relationships between serum estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) levels after 4 weeks of moderate alcohol supplementation, and compare the results to the 8 weeks data to elucidate time-to-effect differences.Postmenopausal women (n = 51) consumed 0 (placebo), 15 (1 drink), and 30 (2 drinks) g alcohol (ethanol)/ day for 8 weeks as part of a controlled diet in a randomized crossover design. Blood samples were drawn at baseline, at 4 weeks and at 8 weeks. Changes in estrone sulfate and DHEAS levels from placebo to 15 g and 30 g alcohol per day were estimated using linear mixed models.At week 4, compared to the placebo, estrone sulfate increased an average 6.9% (P = 0.24) when the women consumed 15 g of alcohol per day, and 22.2% (P = 0.0006) when they consumed 30 g alcohol per day. DHEAS concentrations also increased significantly by an average of 8.0% (P < 0.0001) on 15 g of alcohol per day and 9.2% (P < 0.0001) when 30 g alcohol was consumed per day. Trend tests across doses for both estrone sulfate (P = 0.0006) and DHEAS (P < 0.0001) were significant. We found no significant differences between the absolute levels of serum estrone sulfate at week 4 versus week 8 (P = 0.32) across all doses. However, absolute DHEAS levels increased from week 4 to week 8 (P < 0.0001) at all three dose levels.These data indicate that the hormonal effects due to moderate alcohol consumption are seen early, within 4 weeks of initiation of ingestion. Epidemiological evidence consistently shows a positive association between alcohol, even low to moderate intake, and breast cancer risk . HoweverA reanalysis of nine prospective studies shows that levels of endogenous sex hormones are strongly associated with breast cancer risk in postmenopausal women . Clear eDetails of the Women's Alcohol Study (WAS) was previously published . BrieflyBlood for hormone analyses was collected after an overnight fast at weeks 4 and 8. Serum was separated, and aliquots were frozen at -70°C. In the samples taken after 4 weeks of alcohol supplementation, we only measured serum estrone sulfate and DHEAS, as they were the only two hormones that were significantly elevated at 8 weeks [Hormone concentrations were transformed using natural log. Changes in hormone concentrations from placebo to 15 g and 30 g of alcohol per day were estimated at 4 and 8 weeks using linear mixed models including a random intercept and alcohol levels as fixed effects treated as two indicator variables. Separate models used alcohol as a continuous variable to test for trend. Regressions to evaluate the effect of several baseline covariates on the precision of the parameter estimates for alcohol consumption included age, BMI, and years since menopause modeled as continuous fixed effects; assignment order, dietary period, hysterectomy, and race were modeled as indicator variables. Effect modification by race, assignment order, dietary period, age, BMI, and years since menopause were assessed by likelihood ratio tests of improvement in the model fit after addition of cross-product terms to models that included main effects for alcohol and the characteristic being evaluated. All tests of statistical significance were two-sided. Statistical analyses were performed using S-PLUS (S-PLUS version 6.1 for Windows. Seattle (WA): Insightful Corporation; 2002.)Table The effects of alcohol supplementation on serum estrone sulfate and DHEAS levels did not vary with age, BMI, race, or years since menopause. These hormone concentrations also did not differ among the three dietary periods, and the order of the assignment to the treatment regimens did not modify the associations with either of the two hormones.At 8 weeks of alcohol intake, the 15 g dose versus the placebo increased serum estrone sulfate and DHEAS by 7.5% and 5.1% respectively, whereas the 30 g dose compared to the placebo, increased levels of estrone sulfate and DHEAS by 10.7% and 7.5% respectively. When comparisons using linear mixed models are made, after controlling for alcohol intake across all doses, there was no statistically significant difference between the absolute levels of serum estrone sulfate at week 4 versus 8 (P = 0.32). However, controlling for alcohol intake absolute DHEAS levels increased between weeks 4 and 8 (P < 0.0001). The increase in DHEAS between weeks 4 and 8 did not occur in just the 15 or 30 g per day groups, however; the increase was similar in each of the three groups. In the 0 g per day group the geometric mean shifted from 55.3 ng/dL at 4 weeks to 59.4 ng/dL at 8 weeks. In the 15 g per day group the geometric mean shifted from 59.7 ng/dL to 62.5 ng/dL, and in the 30 g per day group the geometric mean shifted from 60.4 ng/dL to 63.9 ng/dL. The models did not suggest an interaction between measurement week and alcohol dose in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58).In postmenopausal women moderate alcohol consumption for four weeks resulted in statistically significant increased levels of serum estrone sulfate, the most abundant circulating estrogen, and DHEAS, the steroid hormone with the highest concentration in the blood. Compared to the placebo, serum estrone sulfate levels increased 6.9% (P = 0.24) and 22.2% (P = 0.0006) respectively among women who consumed 15 g and 30 g alcohol per day for four weeks. Serum DHEAS concentrations also increased by 8.0% (P < 0.0001) and 9.2% (P < 0.0001) respectively among women who consumed 15 g or 30 g alcohol per day for four weeks. At week 8, serum estrone sulfate levels increased 7.5% and 10.7% respectively among women who consumed 15 g and 30 g alcohol per day whereas DHEAS concentrations increased 5.1% and 7.5% respectively among women who consumed 15 g or 30 g alcohol per day compared to the placebo [For DHEAS, the absolute levels were statistically (P < 0.0001) higher at week 8 compared to week 4 in all the alcohol groups. This increase in DHEAS between weeks 4 and 8 did not differ regardless of 0 g, 15 g, or 30 g per day dose (P = 0.58). Biological reasons may explain the increase in the 15 g and 30 g per day alcohol dose, but the reasons behind the increases in DHEAS for the 0 g dose need further research. We did not find any evidence for an effect modification between measurement week and alcohol supplementation in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58).These data indicate that the hormonal effects due to moderate consumption of alcohol equivalent to one or two drinks per day are seen early, within 4 weeks of initiation of ingestion. Importantly from a study design perspective, our study also demonstrates that it may be possible to utilize shorter study periods when assessing the effects of alcohol consumption on hormone levels, at least in postmenopausal women. To understand the earliest effects of moderate alcohol intake on hormone levels in postmenopausal women, future studies will have to be designed to assess serum levels earlier than 4 weeks or possibly later than 8 weeks.Postmenopausal women with elevated levels of serum estrone sulfate ,9 and DHAlcohol has many physiologic effects and could influence breast cancer risk through non-hormonal mechanisms as well. Experimental evidence suggests that alcohol interferes with folate absorption, transport, and metabolism, potentially limiting folate stores in the tissues and may interfere with DNA methylation ,13. AlcoIn conclusion, our results provide additional evidence for a mechanism by which moderate alcohol drinking could modify breast cancer risk, indicating that this effect occurs after a short time period (as early as four weeks), and thus provides further support for a causal association.None declared.


Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method.Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated.p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms. Colorectal cancer is the second most common cause of cancer-related mortality in Western countries, with about 1 million new cases every year diagnosed world-wide and 500,000 patients dying from the disease . In addiOver the past decade, induction of apoptosis has emerged as a major event in tumor cell response to cytotoxic agents (for a recent review see ). This vextrinsic pathway is initiated by ligation of transmembrane receptors to activate membrane proximal "activator" caspases, which in turn cleave and activate downstream "effector" caspases. The intrinsic pathway requires disruption of the mitochondrial membrane and the release of mitochondrial proteins, two events that are regulated by the opposing actions of pro- and antiapoptotic Bcl-2 family members. "Intrinsic stresses", such as those produced by DNA-damaging agents, activate the intrinsic apoptotic pathway; the multifunctional transcription factor p53 is thought to be part of a "fast track" connection between nuclear DNA damage and the intrinsic pathway machinery [p53 regulates multiple responses to genotoxic stress by transcriptional activation or repression of a number of genes encoding proteins involved in cell cycle control (p21WAF1/Cip1), DNA repair (gadd45), and apoptosis (e.g. Bax, Bcl2 and survivin) [p53 and in the p53 pathway can produce multidrug resistance in vitro and in vivo, and reintroduction of wildtype p53 into p53 null tumor cells can re-establish chemosensitivity [p53 status is not a universal predictor of treatment response, in part because not all drugs absolutely require p53 for their apoptotic function [Two separable, although extensively cross-talking, pathways leading to apoptosis have been characterized ,8. The eachinery . p53 regurvivin) . Mutatiositivity . p53 stafunction  and in sfunction . Still, function  and the function ).N-oxyl  analysis of the relationship between p21 status and sensitivity to treatment with the cytotoxic anticancer agent doxorubicin (DOX) in p53-positive and -negative colon cancer cell lines; (b) design of treatment strategies based on the use of small molecules able to modulate p21 status; for the present study we have used a low molecular weight, stable nitroxide radical, 4-hydroxy-2,2,6,6,tetramethylpiperidne-N-oxyl (Tempol, TPL) and cell culture reagents were purchased from Sigma-Aldrich srl. , unless otherwise indicated; doxorubicin (DOX) was kindly provided by Dr. A Suarato .Standard chemicals, including 4-hydroxy-2,2,6,6-tetramethylipiperidine-2 and 95% humidity.The human colon carcinoma cell lines HCT116, HT29 (obtained from ATCC, Rockville, MD) and HCT116 p21-negative cells (HCT116 p21-/-), kindly provided by Dr B. Vogelstein , were maintained in DMEM medium supplemented with 10% FBS (Mascia Brunelli); the HCT116-E6 cell line, obtained from HCT116 cells by transfection with pCMVneo-E6 plasmid (provided by Dr B. Vogelstein) containing the HPV16-E6 human gene, was maintained in ISCOVE medium supplemented with 10% FBS and geneticin (500 μg/ml). All the cell lines were cultured at 37°C, in an atmosphere of 5% CO25 or IC50values obtained for each cell line according to schedule (b). At the end of the treatment period, 50 μl of MTT (2 mg/ml in PBS) were added to each well at 37°C for 3 h and the reduction of MTT by viable cells was measured colorimetrically at 570 nm, using a Universal Microplate Reader EL800 (Bio-Tek Instruments). IC25 and IC50 values  were calculated according to the median effect equation and analysis of the interaction between DOX and TPL was performed as described by Chou & Talalay [The effects of DOX and/or TPL on cell growth were assessed by the MTT assay . Briefly Talalay .Transfection of HCT116 cells with the pCMVneo-E6 plasmid was performed by electroporation as described by Yanez and Porter , using aThe expression of p53 and p21, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, or after 24 h exposure to TPL (1.0 and 2.5 mM), was evaluated by Western blot analysis of total protein extracts (lysis buffer: NP40 1%, leupeptin 10 μg/ml and aprotinin 10 μg/ml in TBS). Protein concentration in the cellular lysates was determined by the BCA assay (Pierce, Rockford, IL, USA). 15 μg of protein extract/lane were loaded onto 11% polyacrylamide gels and separated under denaturing conditions. Protein samples were then transferred onto nitrocellulose membrane and Western blot analysis was performed by standard techniques. using a mouse anti-p53 monoclonal antibody  and a rabbit anti-p21 polyclonal antibody . Proteins were visualized using peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Amersham Pharmacia Biotech) and the ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia Biotech).Densitometric analysis was performed using the Scion Image software (Scion Corporation, Frederick, MD).5 cell samples were analyzed and data were processed using the CellQuest software (Becton Dickinson). The percentage of apoptotic cells in each sample was determined based on the sub-G1 peaks detected in monoparametric histograms.The presence of apoptotic cells in HCT116, HT29, HCT116-E6 and HCT116 p21 -/-, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, was evaluated by flow cytometric analysis, using a Becton Dickinson FACScalibur flow cytometer. Cells were detached by trypsinization, washed in phosphate-buffered saline (PBS) and fixed in ice-cold 70% ethanol for 20 min at -20°C. After an additional wash in PBS, DNA was stained with 50 mg/ml propidium iodide in PBS in the presence of RNAse A (30 U/ml) at 37°C for 30 minutes. 5 × 1050 values and apoptotic cell percentages in the four different cell lines were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations an relative statistical analysis were performed as described by Chou and Talalay [combination index (C.I.) can be calculated from dose-response curves obtained following exposure to DOX and/or TPL as single agents and in combination. C.I. values approximating 1.0 indicate additive interactions between the two agents; C.I. < 1.0 indicate synergy and, conversely, C.I. > 1.0 indicate antagonism.Comparisons of IC Talalay . Accordi50 values calculated from these data are reported in table resistance index (R.I.) is calculated as the ratio between the IC50 value obtained for each cell line and that obtained for HCT116 cells. The HT29 cell line, which carries a mutant form of the p53 gene, is significantly more resistant to the cytotoxic action of DOX than the HCT116 cell line, carrying a wild-type p53 gene . HCT116-E6 cells are 2-fold more resistant to DOX than HCT116 cells , while HCT116 p21-/- cells are 14-fold more resistant to DOX than HCT116 cell line , in spite of the presence of a wild type form of p53.Figure M for 1 h followed by 23 h in drug-free medium), in the four cell lines tested; panels B and D report the densitometric analysis of p53 and p21 immune reactive bands, respectively. In the absence of drug treatment, it is possible to observe that HT29 cells show higher p53 but lower p21 protein levels, as compared with the HCT116 cell line. DOX treatment induces a dose-dependent increase in both p53 and p21 levels in HCT116 cells, whereas in HT29 cells p53 protein levels are not significantly modified by the treatment and p21 protein levels are only detectable when 25 μg of protein extract/lane are loaded onto the gel (instead of the 15 μg loaded for the other cell lines), and even then only at the highest DOX concentration used. As expected, HCT116 E6 cells do not show detectable p53 levels, both under baseline conditions and following DOX treatment; in contrast in HCT116-E6 cells p21 levels are increased by DOX treatment in a dose-dependent fashion, although to a lesser extent than in HCT116 cells. HCT116 p21-/- cells show higher baseline expression levels of p53 compared with HCT116 cells and DOX treatment in this cell line enhances p53 expression to a an even greater extent than in HCT116 cells line. As expected, p21 is undetectable in HCT116 p21-/- cells, and DOX treatment does not modify the intracellular levels of this protein.Figure Figure 25 and IC50 values obtained for the four cell lines after 24 h of continuous TPL exposure followed by 72 h in drug-free medium. TPL can be observed to inhibit cell growth in all four cell lines; although no significant differences can be detected among IC50 values, HCT116 p21-/- cells appear to be less responsive than the other three cell lines. Figure Table 25 and IC50 values obtained for each cell line, on DOX cytotoxicity. The cells' response to DOX is expressed as the IC50 values derived from dose/response curves obtained after 1 h exposure to DOX with or without pretreatment with TPL (24 h), followed by 72 h in drug-free medium. Analysis of cytotoxicity data shows a synergistic interaction (C.I.<1) between DOX and TPL for both TPL concentrations in HCT116 cells and in HCT116-E6 and HT29 cells at the lower concentration; only additive effects (C.I. ≈ 1) can be observed in HCT116 p21 -/- cells. IC50 values for DOX and TPL according to the three different schedules are reported in table Figure p53 status is not unequivocally related to the response to DOX: in fact, while p53-deficient cells (HT-29, HCT116-E6) are indeed less responsive than the p53/wt parental HCT116 cell line, the highest resistance index was obtained for HCT116 p21-/- cells, harboring two wildtype p53 alleles. As expected, treatment with DOX leads to p53 upregulation in the cell lines expressing wildtype p53; this effect has been thoroughly documented in colon cancer cells as well as in tumor cell lines derived from other tissues, and has been attributed to phosphorylation and subsequent stabilization of p53, possibly through activation of DNA-dependent protein kinase or ATM (ataxia-teleangectasia mutated) kinase . In see e.g. ) and cansee e.g. , the rolsee e.g. ,25. In asee e.g.  or by losee e.g. ,27-30. Hsee e.g. -34 and tsee e.g. -37. The see e.g. , but stiInterestingly, the situation outlined by our results does not seem to conform to either view: in fact, while induction of p21 in p53-proficient and -deficient cell lines is associated with increased response to drug treatment, this was not accompanied by a parallel increase in apoptotic cells, as no significant differences in apoptosis were observed between HCT116 cells and the 14-fold resistant HCT-116 p21-/- cell line  p21 induction may significantly contribute to the response of colon adenocarcinoma cells to DOX treatment; and 2) small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.The author(s) declare that they have no competing interestsThe pre-publication history for this paper can be accessed here:


Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM.+ or AG-EB+) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukn (IL)-1β, IL-2, IL-4, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and IL-10. They can be sub-divided into glycophorin A positive (Gl A+) and erythroblast antigen positive (AG-EB+) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-β1 and TNF-α production were found between Gl A+ and AG-EB+ BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-γ and IL-2 production specifically by the AG-EB+ ENC. Thus, Gl A+ and AG-EB+ ENC produce IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1 and TNF-α. Gl A+ ENC also produce IL-10.We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl ACytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM. Haematopoesis is regulated by lymphoid and non-lymphoid cells through a complex network of paracrine and autocrine mechanisms involving cytokines, growth factors and their receptors. However, although stromal -6, endotExperiments on ENC isolated from mouse spleen undergoing erythroid hyperplasia, and on cells separated from single erythroid colonies, have revealed that mRNAs for cytokines such as IL-1α, IL-1β, IL-4, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), TGF-β1 and IFN-γ are present ,18. Prod+ BFU-E cells) and on colony-forming units – erythroid (CFU-E) (up to 36% of AG-EB+ CFU-E cells) [+erythroblasts). Later in development, AG-EB expression decreases, and only 4% of reticulocytes carry AG-EB [The aim of the current work was to confirm previous observations by studying the production of the main hemo- and immunoregulatory cytokines, IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and TGF-β1, in human ENC from BM. Furthermore, we wished to determine whether different subsets of ENC (AG-EB+ and Gl A+ erythroid cells from human BM) might account for altered expression levels. Expression of both these markers is specific to erythroid cells; they are absent from the membranes of other blood-forming cells. AG-EB is expressed in a proportion of BFU-E cells (up to 4% of AG-EBE cells) . Its expry AG-EB . In contry AG-EB ,27. ShemIn this article we show which human BM ENC produce the cytokines IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1, TNF-α and IL-10.+ cells, whereas Gl A+ secrete more TNF-α (p < 0.05).Erythroid cells were separated according to the presence of the surface markers AG-EB and Gl A. BM erythroid cells carrying AG-EB produce the cytokines IL-1β, IL-2, IL-4, TNF-α, IFN-γ and TGF-β1  or Glycophorin A (Gl A) . Monoclo6 cells were placed in culture medium containing 2 U/ml Epo (Boehringer Mannheim, Germany) for a 24-h incubation.1 × 105/ml and then cultivated in MethoCult H4433 medium (Stemcell Tech. Inc. Vancouver, B.C.) for 14 days. Colonies were visually characterised as CFU-E using an inverted microscope. After cultivation, the colonies were harvested and washed twice to remove methyl cellulose. The resulting cellular suspension (1 × 106/ml) was cultivated for 24 h in RPMI-1640 with 10% horse serum, then supernatants were collected. Samples were stored at -20°C.Cells from the enriched erythroid population were maintained as described above ,20 at 2·+, AG-EB+ and CFU-E cells) were characterised as erythroblasts and were haemoglobin positive.Cell purity was assessed using Nocht-Maksimov staining of smears  and staiQuantitative determination of cytokines was performed by the electrochemiluminescent immunoassay -52 usingFor TGF-β1 detection, samples were treated with HCl to obtain intact and easily detectable proteins as described .All polyclonal and monoclonal antibodies were purchased from R&D Systems (Abington, UK). The following antibodies were used: against recombinant IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1 polyclonal and monoclonal: anti-hIL-1β #AB-201-NA, anti-hIL-1β #MAB201, anti-hIL-2 #AB-202-NA, anti-hIL-2 #MAB202, anti-hIL-4 #AB-204-NA, anti-hIL-4 #MAB204, anti-hIL-6 #AB-206-NA, anti-hIL-6 #MAB206, anti-hIL-10 #AB-217-NA, anti-hIL-10 #MAB217, anti-hTNF-α #AB-210-NA, anti-hTNF-α #MAB210, anti-hIFN-γ #AB-285-NA, anti-hIFN-γ #MAB285, anti-hTGF-β1 #AF-101-NA, anti-hTGF-β1 #MAB240. No antibody used displayed cross-reactivity with other cytokines.3 and 0.1% BSA .Recombinant cytokines were purchased from R&D Systems (Abington, UK): rhIL-1β #201-IL, rhIL-2 #202-IL, rhIL-4 #204-IL, rhIL-6 #206-IL, rhTNF-α #210-TA, rhIFN-γ #285-IF, rhTGF-β1 #240-B-002; and PeproTech, Inc. (Rocky Hill, NJ): rhIL-10 #200-10 was used for calibration curves. All the recombinant cytokines were diluted to 2 μg/ml in PBS, pH 7.4, supplemented with 0.05% NaNResults are presented as means ± SEM. Statistical significance was determined using the nonparametric Mann-Whitney U-test. P < 0.05 was considered to be significant.IBK, NJD, ANZ and MIL carried out the selection of donors, sternum BM sampling and analysis of myelograms. TVI, ANS and SVK carried out all of the experiments and drafted the manuscript. SVS participated in design and coordination of the research and revision of the manuscript. ANS participated in the preparation and revision of the manuscript. VAK conceived of the study. All authors read and approved the final manuscript.


In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. Adenovirus vectors are commonly utilized in cancer gene therapy experiments. They readily infect numerous tumor cell types and are easily manipulated allowing for transgene expression. In an eThe E1 region genes of the common serotypes of adenovirus optimize their own replication in target cells and interact with many cell cycle associated gene products. The E1a region genes promote transition of cells into S phase through their interaction with the retinoblastoma protein (pRB). pRB in Various heat shock proteins have been shown to be necessary for efficient adenovirus replication. The avian adenovirus CELO requires the induction of HSP70 and HSP40 for production of viral proteins. CELO muAdenovirus E1 region genes from serotype 5 and 12 have previously been shown to induce HSP72 expression . HSP72 iOne of the difficulties with studying E1 adenovirus deletion mutants is the lack of appropriate animal tumor models which allow for permissive growth of these viruses. Common animal tumor models for glioblastoma are the cell lines 9L and RT2. Previously, it has been shown that replication competent E1 deletion mutants can transduce 9L cells and result in transgene expression but there is a block against virus replication. In thisPrior to studying the efficacy of ONYX-015 to result in tumor cell lysis, we determined the optimal dose necessary for tissue transduction. Adenovirus infection efficiency is predicated on the expression of adenovirus surface receptors. Using aThe dramatic difference in tumoricidal effect between HSP72 transfected and control tumor cells is best observed by cytopathic effect. In Figure When HSP72-transfected cells were infected with a low dose (MOI 100) of ONYX-015, there is no loss of cell viability. However, comparison of cell numbers to GFP-transfected cells or AD-GFP infected cells showed a dramatic cytostatic effect  supplemented with 10% (v/v) heat inactivated fetal bovine serum , 2 mM L-glutamine and 100 units/ml Penicillin and 1000 ug/ml Streptomycin (Invitrogen). 9L and RT2 cells were transfected with the plasmids pEGFP-N2 (Clontech) or pHSP72 using Lipofectamine (Invitrogen) according to the manufacturers protocol followed by selection in 0.2 mg/ml G418 (Invitrogen). 293 cells were obtained from American Type Culture Collection  were used for virus propagation and purification.9L and RT2 cells are rat glioma cell lines with different derivations. 9L cells are chemically induced gliosarcoma cells while RTpHSP72 was created by ligation of the DNA Polymerase blunt ended 2.3 KB Bam H1-Hind III fragment from plasmid pH2.3 (ATCC) into the blunt ended Xho1-XbaIA vector pEGFP-N2.4 cells/well in 96 well flat-bottomed tissue culture plates. Virus titer from combined supernatant and freeze-thawed cell lysate was determined by serial 10-fold dilution into different rows of the titer plate. After 10 days, wells were scored for cytopathic effect. The titer was calculated using the formula T = 101+d(S-0.5) where d is the Log 10 of the dilution and S is the sum of the ratios of positive wells in each row. A more lengthy description of this method is provided in the adenovirus application manual at .Creation of ONYX-015 has previously been described. The vir2. Cells were subsequently washed with phosphate buffered saline and then cultured in complete medium.Cells were infected when 50% confluent with the dose of adenovirus given in the appropriate table. The infection was performed in serum free DMEM media for 90 minutes at 37°C and 5% COCell viability was determined by the standard Trypan-blue exclusion test. Cells were washed with PBS and then lifted from tissue culture plates with Trypsin-EDTA solution (Invitrogen). Cells were stained with 0.2% Trypan blue solution for 5 minutes and then counted on a hemacytometer.JM performed all of the experiments outlined in this study. JAK independently replicated results for validity. JAK and MRS contributed equally to the molecular cloning of the plasmids and adenovirus propagation and purification. All authors participated in maintenance of cells in culture. The hypothesis, study design and statistical analysis were all performed by MRS. MRS drafted the entire manuscript with editing and approval by all of the authors.


The essential difference between cancer cells and normal cells is that cancer cells evolve. Most cancers arise from a single cell through a sequential evolutionary process of mutation and selection. Cancer cells harbor mutations in a number of critical genes that, at various stages during the evolution of the tumor, provide those cells with a selective advantage. Many of the phenotypes, or physical outcomes, conferred by these mutant genes are subverted from a normal cell's repertoire, including proliferation, invasion, migration, loss of differentiation, and loss of apoptosis (programmed cell death); other phenotypes, such as immortalization, are novel. Tumor evolution is thought to adhere to Darwinian principles, with mutations arising randomly within an individual cell, followed by selection for mutant clones with favorable traits.Support for this idea stems from the observation that end-stage tumors have mutations in a number of genes. But linking mutations in particular genes with defined stages is difficult for most human cancers and especially so for the last and deadliest stage: when cancer disseminates throughout the body during metastasis. It's unclear, for example, whether there are mutations in particular genes or sets of genes that enhance metastasis. That is, do metastatic lesions develop through a continuation of Darwinian evolution, or is metastasis an intrinsic property of the primary tumor, meaning that further genetic evolution is not required? It is also unclear whether there is a “preferred” sequence of mutations, such that selective pressure for particular mutations depends on preexisting mutations.Ras and the loss of the tumor suppressor p53 cooperate to transform cells.Since the earliest days of research on oncogenes—genes that can cause a cell to become cancerous—it has been known that certain oncogenic mutations cooperate to transform normal cells into cancer cells. For example, an “activating” mutation in the oncogene Ras. Using a well characterized mouse model of squamous cell carcinogenesis, which generates a form of skin cancer, the authors examine both the functional and evolutionary relationships between three cancer genes that play major roles in most human cancers: Ras and the tumor suppressors Arf and p53.The paper by Christopher Kemp and his colleagues at the Fred Hutchinson Cancer Research Center sheds light on some of these questions, and many of the issues center around the most notorious oncogene: Ras is the initiating genetic event in this cancer model, while mutation of p53 occurs later, during the benign to malignant transition; and expression of mutant Ras in cells activates p53 via signaling through the protein encoded by Arf.Two seminal early observations set the stage: mutational activation of p53 expression in tumors with Ras mutations is dependent on the presence of Arf. Thus, during the early benign stages of tumor growth, Ras activates Arf, which in turn activates p53, thereby inhibiting tumor progression. This provides strong selective pressure in favor of cells with mutations in either Arf or p53, and these mutations are indeed observed as the tumors progress to malignancy. That Arf and p53 function as tumor suppressors was confirmed by demonstrated accelerated tumor progression in mice lacking either Arf or p53. This answers a longstanding question concerning the nature of the signal that activates p53 during autochthonous tumor development: Mutation of Ras not only initiates tumor development but, through its intracellular signaling through Arf and p53, directly influences the subsequent evolutionary trajectory of the tumors. In this view, secondary evolutionary events are determined by the preexisting genetic lesion, as a result of direct signaling interactions.Kemp et al. confirm that this pathway is active in “autochthonous” tumors—which grow and develop where they are initiated—by showing that Arf or p53 show accelerated metastatic dissemination, a phenomenon rarely seen in mouse squamous cell cancer models. Thus both benign and malignant tumors lacking these tumor suppressors are at high risk for metastasis. As Ras is well known to confer many phenotypes required for the metastatic process, it appears that Ras, together with loss of its inhibitors, Arf and p53, may be sufficient to drive this process. More direct evidence that metastasis does or does not require further genetic evolution awaits.The authors go on to show that tumors lacking


Splice variants of 461 transcription factor loci were analyzed using a new database of splice variants in the mouse transcriptome, MouSDB3, providing quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs. Analyzing proteins in the context of all available genome and transcript sequence data has the potential to reveal functional properties not accessible through protein sequence analysis alone. To analyze the impact of alternative splicing on transcription factor (TF) protein structure, we constructed a comprehensive database of splice variants in the mouse transcriptome, called MouSDB3 containing 461 TF loci.Our analysis revealed that 62% of these loci in MouSDB3 have variant exons, compared to 29% of all loci. These variant TF loci contain a total of 324 alternative exons, of which 23% are in-frame. When excluded, 80% of in-frame alternative exons alter the domain architecture of the protein as computed by SMART (simple modular architecture research tool). Sixty-eight % of these exons directly affect the coding regions of domains important for TF function. Seventy-five % of the domains affected are DNA-binding domains. Tissue distribution analyses of variant mouse TFs reveal that they have more alternatively spliced forms in 14 of the 18 tissues analyzed when compared to all the loci in MouSDB3. Further, TF isoforms are homogenous within a given single tissue and are heterogeneous across different tissues, indicating their tissue specificity.Our study provides quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs. Analyses described here reveal the presence of tissue-specific alternative splicing throughout the mouse transcriptome. Our findings provide significant biological insights into control of transcription and regulation of tissue-specific gene expression by alternative splicing via creation of tissue-specific TF isoforms. Alternative splicing is a widespread mechanism involved in regulation of gene expression, which enables production of many structurally and functionally different forms of proteins from a single gene, adding to the complexity of the genomes -3. DiffeAn expanding body of expressed sequence data from the human and mouse genomes indicates that alternative splicing is an important mechanism in creating protein diversity, and adds to functional complexity encoded in eukaryotic genomes. Earlier studies indicate that at least 50% of the genes in the human genome are alternatively spliced . ExampleComprehensive analysis of alternative splicing is essential to understand fully the proteomes of organisms . SeveralTranscription is a critical process that specifies the mRNAs and the proteins expressed within a cell. Expression of a given gene is dependent on the interactions of different transcription factors and their cofactors with the regulatory regions of that gene. These transcription factors are in turn regulated by processes that include interaction with other proteins and signaling cascades .Alternative splicing is a mechanism that regulates transcription factor (TF) activity by generating a variety of protein isoforms from a single gene. As noted by Lopez, alternative splicing can affect TF structure in two primary ways : alteratIn this paper, we use an integrated approach to analyze DNA and protein sequence data jointly to determine the potential effect of alternative splicing on protein structure and function. We perform a detailed analysis of tissue-specific distribution of alternatively spliced mouse TFs to gain biologically meaningful insights into regulation of gene expression by alternative splicing.For our joint DNA-protein analysis described here, we developed MouSDB3 , which iOur cluster analysis revealed that out of the 461 TF clusters, 62% are variant, compared to 29% of all genes in MouSDB3  are in-frame. Only 11% of cassette exons are expected to be multiples of three and in codon position 1 randomly. The twofold difference between expected and observed numbers indicates a bias towards in-frame cassette exons. The exons which are a multiple of three and in codon position 2 and 3 comprise 10% and 7%, respectively. When deleted, these exons introduce an amino-acid substitution to the sequence. As exons which are a multiple of three starting at codon position 1 are enriched and do not introduce an amino-acid substitution when deleted, our study focuses on these exons only.As shown in Figure et al. [SMART ,16 and Pet al. , who shoPart two of our analysis assessed the tissue distribution of alternatively spliced transcription factors. We chose 18 tissues from the existing libraries in MouSDB3 on the basis of the fact that they contain both variant and invariant transcripts annotated as TFs. There are a total of 1,413 library names in MouSDB3 imported from expressed sequence records in GenBank and dbEST. Of these, 328 are ambiguous in that they list several different tissues or cell types for a single library, such as 'mixture of brain and testis' or no tissues at all, such as 'embryo or carcinoma'. For the work described here we did not include tissue information from such ambiguous libraries. There are a total of 95 libraries in MouSDB3 for which there are TF transcripts. In addition, to account for library ambiguities within these 95 libraries, we pooled different parts of a tissue into one library. For example, the term 'brain' corresponds to all parts of the brain found in MouSDB3, including cerebellum, thalamus, hippocampus and 16 other libraries. When analyzing the tissue distribution of all genes, only the libraries that contain TF transcripts have been used.Transcript counts within variant loci for 18 pooled libraries indicated that in 14 of the 18 analyzed tissues, the proportion of TFs that are variant is higher than the proportion of all genes that are variant Figure . This fi2 values above 1 on the graph). In salivary gland, skeletal muscle, urinary bladder and testis, the fold-differences are 8.7, 5.6, 3.8 and 3.0-fold respectively. Spinal cord, liver, adipose tissue and eye also exhibit more than twofold differences. These values are independent of the sampling depth of the transcripts from these tissues, as illustrated in Figures x-axis as in Figure For each of the 18 tissues in Figure p-value = 0.04). This is true for both transcription factors and the rest of the genes in the mouse transcriptome.We analyzed the presence of different isoforms of transcription factors within and across these 18 tissues. For this analysis we consider transcripts with coding sequence information only. We ignore variation due to 5' and 3' truncation of transcripts. We consider only cassette exons within coding sequences when assessing the differences between isoforms. Within a cluster we compute homogeneity and heterogeneity within a single tissue by checking for the transcripts from the same library and comparing the cassette exons within their coding sequences. If all transcripts from the same tissue contain the same cassette exons with same splice sites they are termed 'homogeneous within'. If the cassette exon distribution within the coding sequences of these transcripts differ, they are termed 'heterogeneous within'. We compute 'homogeneity across' and 'heterogeneity across' tissues in the same way by taking into account transcripts within the same clusters but from different libraries. As shown in Figure p-value = 0.02). Figures Through integrated analyses of DNA and protein sequences for TF genes, we show that alternative splicing of TFs are more prevalent in the entire mouse transcriptome and in specific tissues when compared to alternatively spliced forms of all the genes. In 78% of the tissues analyzed, higher proportions of TFs exhibit alternative splicing compared to all the genes in the mouse transcriptome. This result, along with the finding that 62% of TF loci are variant, indicates the widespread impact of alternative splicing on regulation of TF function.We also show that alternative splicing changes TF structure by adding or deleting domains. This study reveals that 80% of alternatively spliced TFs have different domain architectures due to introduction of an in-frame cassette exon by alternative splicing. Of the altered domains, 75% have a role in DNA binding. These findings provide quantitative evidence for the role of alternative splicing in controlling the presence of domains in the proteins. They also suggest that alternative splicing might regulate TF activity by changing the architecture of the DNA-binding domains of these proteins.Our analyses revealed that within a single tissue there generally is only one TF isoform, and that across tissues, isoforms differ. This finding indicates tissue specificity of alternatively spliced TFs and suggests that TFs might regulate gene expression in a tissue-specific manner by having different isoforms in different tissues. These findings further indicate the role of alternative splicing in regulation of tissue-specific gene expression. Activation and repression of different sets of genes within different tissues can be regulated through variant TF isoforms created by alternative splicing. These findings will significantly aid further understanding of control of transcription and tissue-specific gene expression. In addition, our study shows that all variant loci in the mouse transcriptome display isoform homogeneity within single tissues and heterogeneity across tissues. This finding demonstrates the presence of tissue-specific alternative splicing across the mouse transcriptome and greatly expands the knowledge on the tissue specificity of alternatively spliced genes.Overall, our study provides quantitative evidence for the effect of alternative splicing on protein structure and sheds light on how alternative splicing might regulate transcription factor function in a tissue-specific manner. This, in turn, reveals the contribution of alternative splicing to regulation of gene expression via tissue-specific TF isoforms. The work described here implies that future high-throughput screens of gene expression analyses should be sensitive to multiple alternatively spliced forms of TFs. Because gene-expression arrays are intended to measure transcription, the next generation of arrays should contain probes specific to all known isoforms of genes represented on the arrays. Given that alternatively spliced exons are highly conserved across species ,22, it wMus musculus entries. All expressed sequences were mapped to a region of the University of California Santa Cruz (UCSC) February 2003 version mm3 of the mouse genome assembly using BLAT [For this analysis, we constructed a database of alternatively spliced mouse transcripts called MouSDB3 , using ting BLAT . BLAT toing BLAT . This waing BLAT  version We identified in-frame cassette exons and extracted from MouSDB3 nucleotide and amino-acid sequences for transcripts containing these exons. The selected amino-acid sequences were then analyzed with SMART ,30 to coFrom MouSDB3, we then extracted library information for the transcripts within clusters and their annotations. We used these data to compute the tissue distribution of variant transcripts as reported in Results. All scripts and README files used to carry out this data-gathering process are available upon request from the Laboratory of Computational Genomics of The Rockefeller University.


Pharmaceutical company representatives (PCRs) influence the prescribing habits and professional behaviour of physicians. However, the skills for interacting with PCRs are not taught in the traditional medical school curriculum. We examined whether an innovative, mandatory workshop for third year medical students had immediate effects on knowledge and attitudes regarding interactions with PCRs.Surveys issued before and after the workshop intervention solicited opinions  from third year students (n = 75) about the degree of bias in PCR information, the influence of PCRs on prescribing habits, the acceptability of specific gifts, and the educational value of PCR information for both practicing physicians and students. Two faculty members and one PCR led the workshop, which highlighted typical physician-PCR interactions, the use of samples and gifts, the validity and legal boundaries of PCR information, and associated ethical issues. Role plays with the PCR demonstrated appropriate and inappropriate strategies for interacting with PCRs.The majority of third year students (56%, 42/75) had experienced more than three personal conversations with a PCR about a drug product since starting medical school. Five percent (4/75) claimed no previous personal experience with PCRs. Most students (57.3%, 43/75) were not aware of available guidelines regarding PCR interactions. Twenty-eight percent of students (21/75) thought that none of the named activities/gifts  should be restricted, while 24.0% (8/75) thought that students should be restricted only from sporting events. The perceived educational value of PCR information to both practicing physicians and students increased after the workshop intervention from 17.7% to 43.2% (chi square, p = .0001), and 22.1% to 40.5% (p = .0007), respectively. Student perceptions of the degree of bias of PCR information decreased from 84.1% to 72.9% (p = .065), but the perceived degree of influence on prescribing increased (44.2% to 62.1% (p = .02)).Students have exposure to PCRs early in their medical training. A single workshop intervention may influence student attitudes toward interactions with PCRs. Students were more likely to acknowledge the educational value of PCR interactions and their impact on prescribing after the workshop intervention. Pharmaceutical company representatives (PCRs) influence the prescribing habits and professional behavior of physicians . DespitePhysicians in training may be particularly susceptible to marketing strategies from PCRs. Restricting interactions between physician in training and PCRs is one approach to eliminating adverse effects of contacts with PCRs . HoweverWe sought to examine whether a single workshop intervention had immediate effects on the attitudes of third year medical students regarding interactions with PCRs. The goal of the workshop was to increase the student's knowledge and awareness of ethical issues surrounding the PCR encounter, and to improve the students' interactions with representatives by fostering discussion on the profit motive in pharmaceutical marketing, PCR marketing techniques, and appropriate interactions with the PCR, issues necessary for critical thinking about the potential conflict of interest. Unique to our intervention was the participation of a PCR who role played a typical PCR encounter and who offered a perspective on marketing from the perspective of industry. Our findings have implications for institutions considering strategies for controlling PCR interactions and for medical educators seeking to develop curricula for marketing in medicine.During the ambulatory internal medicine clerkship of the third year medical school curriculum, students were required to attend a ninety minute workshop entitled "Appropriate Encounters with Pharmaceutical Representatives". These workshops took place three times during the calendar year 2001 for three different student groups. To the best of our knowledge, there were no other organized learning experiences in the medical school curriculum about interactions with pharmaceutical representatives, either before or during the study period. We did not seek approval by the institutional review board for ethical research practice at our institution, because at that time the study was conducted, approval of student education projects was considered unnecessary.Two faculty members interested in the subject (JW, CO), and a regional manager of pharmaceutical representatives from a major pharmaceutical company facilitated the ninety minute workshop. The workshop began by soliciting student opinions regarding the characteristics of typical interactions with PCRs. After a list of characteristics was compiled, each characteristic was discussed in more detail and compared with previous personal experiences with PCRs. Salient points of the subsequent discussion included the usefulness of patient assistance programs, the use of samples and gifts in PCR marketing strategies, the validity and legal boundaries of information provided by the PCR, and the ethical and legal aspects of physician-industry relations. The final segment of the workshop involved two student volunteers who role played a typical PCR encounter in the office. After discussion of the first role play, a second role play between one faculty member (CO) and the PCR demonstrated desirable characteristics of the PCR encounter.A pre-intervention survey handed out and collected prior to the beginning of the workshop solicited information about the number of previous personal experiences with PCRs, and whether the student was previously aware of guidelines  for appropriate interactions with PCRs. Using five point Likert scales, the survey solicited student attitudes about the educational value of PCR information for practicing physicians and for medical students, the degree of bias in PCR information, and the degree of influence of PCRs on prescribing habits. One additional question solicited the acceptability to students of specific gifts  from PCRs. A post-intervention survey with the same attitude questions was administered and collected as students left the workshop. The available data comes from three groups – the third, or last student group of academic year 2000–1 and the first two student groups of academic year 2001–2.Students were characterized by gender, age, and number of previous personal contacts with PCRs (None, 1–3, 4–6, >6). For the purposes of understanding the Likert scale responses for student attitudes, we collapsed Likert scores into three categories – scale responses of 1 or 2 to signify disagreement, a scale response of 3 to signify neutral, and a response of 4 or 5 to signify agreement with the attitude question. We compared student attitudes toward the educational value of PCR detailing for medical students with the perceived value for practicing physicians using the Pearson chi square test.The association between previous personal PCR experience and attitudes about the educational value of PCR detailing was explored using analysis of variance. We also compared attitudes before and after the workshop intervention using the Pearson chi square test and a dichotomous variable a response of 4 or 5 (versus 3 or lower) on the Likert scale.A total of 75 students attended one of the three mandatory workshops on "Appropriate Encounters with PCRs". One student did not complete a post-intervention survey. The mean age of students was 26.4 (± 2.4), and males (56.1%, n = 41) outnumbered females. Fifty-six percent of students (42/75) had experienced more than three personal conversations about a pharmaceutical product with a PCR since starting medical school. Five percent (4/75) claimed no previous personal experience with PCRs. There was no association between the number of PCR contacts, and either gender, age or time of the academic year.The pre-intervention survey showed that PCR detailing was of educational value to 22.1% (18/75) of students with no perceived difference in educational value to medical students versus that to practicing physicians (chi square, p = .40). While students agreed that the degree of bias from PCR information was substantial (86.7%, 65/75), only 44.0% (33/75) of students felt that pharmaceutical representatives were influential with regard to physicians' prescribing habits. No relationship between number of previous personal PCR contacts and educational value to medical students was demonstrated (ANOVA p = .08)Forty-three percent (32\75) of students reported awareness of available guidelines regarding PCR interactions. Fifty percent (16/32) of those students reported familiarity with medical school guidelines, 21.8% (7/32) with federal government guidelines, and 40.6% (13/32) with professional society guidelines. When asked which drug company sponsored activities/gifts targeting medical students should be restricted, 28.0% of students (21\75) thought that none of the named activities/gifts  should be restricted. Twenty-four percent (8/75) of students thought that only sporting events should be restricted.Figure Professional relationships with PCRs begin early in a physician's career. Because physicians typically underestimate the influence of pharmaceutical marketing on prescribing practices, countering this naivete early is warranted. Because students do not yet have prescribing privileges, the effect of PCR detailing on the prescribing decision is less relevant than how PCR contact shapes professional values . SeveralOur data show that students have early exposure to PCRs, perhaps earlier than previously suspected. Only five percent of third year students at this institution had not yet experienced PCR detailing, and many had experienced greater than six such encounters. Although resident physicians understandably have more PCR contacts, on average at least three PCR encounters per month ,16, to oIn this study few students were aware of existing guidelines for PCR interactions. Furthermore, students perceived information from PCRs to have educational value, and a value equivalent to that for practicing physicians. That students felt no more or less susceptible to marketing influence than practicing physicians is a marked contrast to the opinions of many educators that physicians in training need special protection from marketing influences ,2,17.Several interventions have been proposed for educating physicians-in-training about pharmaceutical marketing practices. Shaughnessy et al described a single brief seminar of marketing concepts followed by regular structured evaluation of PCR sales presentations throughout the following year . DespiteSimilar to these interventions, our workshop intervention targeting third year medical students was a one time intervention and brief in duration. Our intervention differed in that the workshop (1) took place during the clinical clerkship year, (2) made use of practicing PCRs and physicians, and (3) encouraged a distanced but amicable relationship with the PCR. In contrast to many educators who oppose PCR contact for trainees, we encouraged respect for the individual PCR and the success of the pharmaceutical company's business model. We were concerned enough about the growing influence of pharmaceutical marketing on trainees to stage this workshop, but we were careful to remember our goals of encouraging critical thinking about the topic rather than simply condemning the PCR contact. As an example, we emphasized the legal limits regarding what the PCR could and could not say to the physician. This may explain, in part, why attitudes toward PCR information improved after the intervention, at the same time that perceived degree of influence on prescribing increased. Teaching students the "rules of the game" in PCR encounters may explain why students thought that PCR information had more educational value after the workshop.The limitations of this study should be recognized. First, our survey took place at a single academic medical center affiliated with a private hospital. Student attitudes might be different at a state supported institution or at institutions where there are restrictions on the activities of PCRs. Second, students may have answered the post-intervention survey in a socially desirable manner. Respect for the PCR and the business model was presented as a balanced perspective toward marketing in medicine. Third, how immediate changes in student perceptions will ultimately translate into durable attitude changes and prescribing practices was not a goal of this study. We are not naïve enough to be certain that a one time intervention on any subject matter related to ethical issues will change student attitudes in a way that is durable. However, our goal as educators should be to make the students think critically, and demonstrating self-reported attitude changes is a necessary first step toward more durable change.Some academic institutions have chosen to ban PCRs from the academic learning environment. McCormick et al showed that restricting access to PCRs during residency training was associated with less informational dependence on the PCR and a decreased frequency of PCR contact after training . UnderlyThe challenge for medical educators is how to incorporate this increasingly important knowledge domain into training programs. What aspects of marketing strategy need to be taught and how? The growing emphasis on social justice and professionalism should encourage the appropriate distance from and respect for marketing pressures in medicine and add support for this curricular element in medical education . Our modMedical students have exposure to PCRs early in their medical education, at least in this setting. Students perceived information from PCRs to have moderate educational value, and a value equivalent to that for practicing physicians. A brief workshop intervention can have a measurable immediate effect on student attitudes.PCR – pharmaceutical company representativeThe author(s) declare that they have no competing interests.JW conceived of the study, participated in its design and coordination, and drafted the manuscript.CO participated in design and coordination of the study, conducted the intervention, participated in data interpretation, and manuscript revision.Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: