Food allergies are a major component of the burden of allergic disease. Accurate risk assessment for prediction of future clinical reactivity or clinical tolerance is limited by currently available techniques. Recent studies suggest that constitutively elevated global serum levels of IL-10, a cytokine that down-regulates both Th1 and Th2 cytokine production, may be useful in identifying human clinical tolerance to foods.Determine the usefulness of constitutive IL-10 levels as a marker of clinical tolerance to peanut in children and adults.107 subjects who were clinically tolerant to peanut and 94 subjects who were clinically allergic to peanut participated. Plasma was analyzed via ELISA to quantify the frequency of individuals with constitutive IL-10 levels and the intensity of those responses. The data were then stratified by age, gender and clinical status to assess the utility of this putative biomarker in specific at-risk groups. All 201 subjects had readily quantified plasma IL-10. Levels were no higher in subjects who were clinically tolerant to peanut than those in individuals clinically allergic to peanut. Stratification by age, gender or both did not improve the capacity of IL-10 levels to identify clinical tolerance to peanut.Plasma IL-10 levels are neither a useful biomarker of clinical tolerance to peanut nor a potential tool for identification of clinical tolerance to peanut in humans. Peanut allergy is a leading cause of fatal anaphylaxis Erroneously classifying patients as low risk for clinical reactivity to peanut presents obvious problems, while erroneously classifying them as high risk based on low or moderate sensitization to peanut leads to a negative psychosocial impact and reduced quality of life Identification of novel biomarkers that would reliably allow determination of clinical status and/or disease severity is a major focus in many areas of biomedical research. Most of this work has concentrated on identification of biomarkers that associate with disease pathogenesis or exacerbation rather than clinical tolerance. Markers with sufficient sensitivity and specificity to offer clinical utility have been sought for cancers et al. In a recent clinical study involving 12 DBPCFC-negative subjects and 29 adults who were clinically allergic to hazelnut, Alonso While IL-10 is clearly an important component of immune regulation, given the wide variety of innate and adaptive immune cells that produce IL-10, the extensive range of environmental stimuli and the many individual antigens to which humans are exposed daily, it seemed unlikely to us that constitutively elevated IL-10 levels would provide a useful tool to discriminate between clinical tolerance and food allergy to any single antigen. Here, in more than 200 adults and children who were either clinically tolerant or clinically allergic to peanut, we quantified endogenous IL-10 levels to assess the potential utility of elevated IL-10 as a reliable biomarker of clinical tolerance to peanut.This study was approved by the University of Manitoba Research Ethics Board. Before enrolment, written informed consent was obtained from 56 adults and from parents of 145 children. Subjects were divided into study groups based on their age and clinical reactivity to peanut. Peanut allergic subjects (History positive/SPT positive: Hx+SPT+) had anaphylaxis upon peanut exposure within the past 5 years, had peanut skin prick tests  of >3 mm wheal in comparison to the negative saline control and frequently had peanut specific ImmunoCAP values >0.35 kUa/L. Many of these peanut allergic subjects also had positive skin tests or clinical symptoms to additional unrelated allergens. 94 subjects were recruited fitting this definition. One hundred and seven clinically tolerant, unsensitized subjects (History/SPT negative: Hx−SPT−) were recruited on the basis of a detailed history consistent with a lack of clinical reactivity to peanut, negative skin prick tests to peanut, and peanut specific IgE ImmunoCAP results <0.35 kUa/L. These participants took no precautions to avoid peanut, and ingested it without developing any symptoms or signs.et al. Fifty and 20 ml of peripheral blood were obtained by venipuncture from adults and children, respectively. Plasma was isolated by centrifugation. Human plasma IL-10 cytokine levels were quantified in an assay optimized to provide sensitivity <5.0 pg/mL with interassay variability of 5–10%  essentially as described in Stefura The median ages and range of the study populations were as follows: control children , control adults , peanut allergic children  and adults . To determine if plasma IL-10 differed significantly between populations who were clinically tolerant to peanut versus those exhibiting clear clinical histories of symptoms and signs after peanut ingestion, positive SPTs to peanut allergen, and elevated peanut specific IgE, the plasma from each group was analyzed to quantify constitutive IL-10 levels using a high sensitivity ELISA. No difference was evident in the frequency of constitutive IL-10 producers between clinically tolerant and peanut allergic populations. IL-10 was detectable in all 201 subjects, with levels ranging 12 to 9860 pg/ml. Moreover, no quantitative difference in IL-10 levels was evident between the two clinical groups  Figure 1Food allergy prevalence is strongly influenced by age and gender. Therefore, we carried out a subanalysis stratifying the populations to test the hypothesis that in vivo IL-10 levels could provide a useful cytokine biomarker to diagnose clinical tolerance to peanut within specific at-risk groups. The putative effect of gender on IL-10 levels in the two phenotypes was analyzed, as shown in Given that peanut allergy, unlike most food allergies, is typically life long, we also sought evidence of age-related differences in plasma IL-10 levels Figure 3We report that elevated plasma IL-10 levels, suggested as a useful biomarker of clinical tolerance to tree nuts in a study of 41 subjects In many countries, peanut allergy poses the greatest risk of anaphylaxis of all food allergies Reports suggesting that serum IL-10 might act as a biomarker for clinical tolerance are reasonable based on extensive studies in murine and human systems. IL-10 is capable of suppressing production of Th1 and Th2 associated cytokines, and there is extensive evidence suggesting a role for elevated IL-10 in immune tolerance Subanalyses focussed on gender also failed to demonstrate utility for this approach in diagnosis of clinical tolerance to peanut. While recent evidence shows that the severity of symptoms elicited by peanut or tree nut anaphylaxis does not differ by gender In the past, attempts at using biomarkers that are not specific to the disease or specific allergen(s) in question have often shown a lack of usefulness in predicting future events. Total IgE was once measured for the diagnosis of allergy, until it was shown that these levels are poorly predictive of clinical status because they vary substantially depending on gender, race, and other variables, making decision point cut-offs impossible to establish Among allergen-specific biomarkers, peanut specific IgE levels are useful for identification of patients at lowest risk of clinical reactivity to peanut  and for identification of those at highest risk for clinical reactivity to peanut . However, they are less useful in the many peanut-sensitized patients who have values in the range of 0.35–14 kUa/L One limitation in the study design used here is that DBPCFC were not performed on the 200 subjects for ethical and practical reasons. Peanut non-allergic subjects reported that they regularly ate peanut. Diagnoses of peanut allergy were made by experienced pediatric allergists based on recent convincing histories of peanut induced anaphylaxis, peanut specific IgE, and peanut skin prick test results. While some misclassification is possible, in our view, the overlap in range of IL-10 values between the populations, spanning almost three orders of magnitude, makes it extremely unlikely that this parameter would serve as a useful diagnostic marker of clinical tolerance with acceptable positive and negative predictive values.The primary focus in this study was testing the hypothesis that globally enhanced plasma or serum IL-10 levels could provide a sensitive and specific tool for determination of human clinical tolerance to food antigens. For peanut allergy it did not. The mechanisms that are responsible for ongoing clinical tolerance to peanut in the 97–99% of the general population that is not clinically allergic to peanut remain unclear. Thus, the broader question of what combination of mechanisms is responsible for establishing and maintaining oral tolerance to foods in most of the human population remains controversial. Evidence suggests the participation of multiple mechanisms, including elevated antigen-specific  IL-10, TGFb and/or IL-35 production, antigen-driven CTLA4 expression, alterations in tryptophan metabolism, anergy of food-specific T cells and activity of one or more of the 15 regulatory T cell subsets putatively identified to date In summary, we demonstrate that constitutive plasma IL-10 levels do not serve as a useful biomarker to discriminate clinical tolerance to peanuts from peanut allergy in humans. Further research is required to identify clinically useful markers by distinguishing factors that differ quantitatively between those who are peanut allergic and those who ingest peanut without allergic reactions. With greater understanding of how tolerance is achieved and maintained, identification of such biomarkers may be possible.


To the Editor: Hantaviruses cause 2 severe and often fatal human diseases, hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Rodents are the natural hosts for hantaviruses that cause HFRS and HCPS, and humans are usually infected by aerosolized virus-contaminated rodent excreta –infected humans contains viral RNA , HTNV (strain 76-118), or PUUV (strain Kazan E6) for 1 hr , we observed a slight effect on HTNV, even though we saw no effect on ANDV and PUUV. ANDV was the only virus that resisted higher concentrations of saliva (25% and 50%), and an antiviral effect was clearly observed on HTNV and PUUV at these saliva concentrations .Oligoryzomys longicaudatus), the natural host for ANDV (Our finding that ANDV is less sensitive than HTNV and PUUV to the antiviral effect of human saliva might explain why ANDV, but not HTNV or PUUV, is transmitted between humans. Saliva might be the preferred route of transmission for ANDV between humans, as it is for the long-tailed rice rat (


Sc) form results in imbalance of iron homeostasis in prion disease affected human and animal brains. Here, we show that selective deletion of PrP in transgenic mice (PrPKO) alters systemic iron homeostasis as reflected in hematological parameters and levels of total iron and iron regulatory proteins in the plasma, liver, spleen, and brain of PrPKO mice relative to matched wild type controls. Introduction of radiolabeled iron (59FeCl3) to Wt and PrPKO mice by gastric gavage reveals inefficient transport of 59Fe from the duodenum to the blood stream, an early abortive spike of erythropoiesis in the long bones and spleen, and eventual decreased 59Fe content in red blood cells and all major organs of PrPKO mice relative to Wt controls. The iron deficient phenotype of PrPKO mice is reversed by expressing Wt PrP in the PrPKO background, demonstrating a functional role for PrP in iron uptake and transport. Since iron is required for essential metabolic processes and is also potentially toxic if mismanaged, these results suggest that loss of normal function of PrP due to aggregation to the PrPSc form induces imbalance of brain iron homeostasis, resulting in disease associated neurotoxicity.Despite overwhelming evidence implicating the prion protein (PrP) in prion disease pathogenesis, the normal function of this cell surface glycoprotein remains unclear. In previous reports we demonstrated that PrP mediates cellular iron uptake and transport, and aggregation of PrP to the disease causing PrP-scrapie (PrP KO) transgenic mouse lines have been futile since the mice do not develop an overt phenotype other than resistance to prion disease KO mice since these do not up-regulate Doppel, a PrP homologue that induces cerebellar degeneration Sc) form is also likely to alter neuronal function, partially explaining the pathogenesis of prion disorders and justifying the focus KO mice show altered circadian rhythm and sleep pattern, increased susceptibility to neuronal damage by oxidative stress and cerebral ischemia, neurotoxicity by expression of Doppel and N-terminally truncated PrP, increased predilection to seizures, motor and cognitive abnormalities, reduced synaptic inhibition and long term potentiation in the hippocampus, altered development of the granule cell layer, mis-regulation of the cerebellar network, and age-dependent spongiform change with reactive astrogliosis KO mice to colonize when transplanted to irradiated recipient mice Prion protein (PrP) is a ubiquitously expressed cell surface glycoprotein linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Although several lines of evidence support the obligate role of PrP in animal and human prion disorders, relatively less is known about the normal function of this protein Sc) form induces an imbalance of iron homeostasis in prion disease affected human, hamster and mouse brains in vivo. Using the Zurich 1 PrPKO and matched wild type (Wt) mice expressing normal levels of PrP as experimental models, we demonstrate that PrPKO mice show a phenotype of systemic iron deficiency and altered iron homeostasis compared to Wt controls. The underlying cause of this abnormality is impaired transport of iron from the duodenal epithelium to the blood stream, and a similar defect in iron uptake by parenchymal cells of various organs and hematopoietic precursor cells. The iron deficient phenotype of PrPKO mice is rescued by re-introducing PrP on the PrPKO background, indicating a functional role for PrP in iron uptake and transport. Since iron is an essential element that is necessary for survival but can be extremely toxic if mis-managed Sc form, the principal agent responsible for prion disease associated neuronal death.Recently, we demonstrated that PrP mediates iron uptake and transport in human neuroblastoma cells KO and matched Wt controls. In iron deficiency, we expect low levels of total iron, decreased levels of iron storage protein ferritin, and increased levels of iron uptake proteins Tf and TfR in the affected tissue. To evaluate these parameters, equal quantity of protein from brain homogenates of PrPKO and Wt mice was fractionated by SDS-PAGE and immunoblotted  are increased by 42.2 and 24.0% respectively in PrPKO samples relative to matched Wt controls .To evaluate if the phenotype of iron deficiency in PrPcontrols . The totKO mice arises from inefficient uptake and/or transport of iron from ingested food, defective uptake by target cells of different organ systems, or a combination of both processes.Subsequent studies were directed at determining whether the iron deficiency in PrPKO mice was evaluated by introducing radioactive iron into the stomach, and quantifying 59Fe uptake by various organs after defined time points of chase. The following organs were evaluated: 1) stomach with attached duodenum, jejunum, and a small piece of ileum, 2) liver, 3) spleen, 4) brain, 5) femurs, and 6) tibial bones. Washed upper gastrointestinal tract was vacuum-dried and exposed to X-ray film , plasma, organs, and bones were counted in a γ-counter to quantify 59Fe incorporation.The uptake and transport of ingested iron from the intestinal lumen to the blood stream and target organs of Wt and PrPray film , and aft59Fe in PrPKO samples at each time point compared to Wt controls , littermate PrP+ and PrPKO, Wt, and PrPKO mice were evaluated for iron uptake and transport by introducing equal amounts of 59FeCl3 by gastric gavage as in KO mice are observed at this time point  inefficient transport from duodenal enterocytes to the blood stream, and 2) impaired uptake by target cells of various organs, including the hematopoietic precursor cells. We previously reported that PrP functions as an iron uptake and transport protein in human neuroblastoma cells in vitroIn this report, we demonstrate that the absence of PrP induces systemic iron deficiency in PrPIt is surprising that the mere absence of PrP alters iron metabolism in both enterocytes and hematopoietic cells since distinct processes regulate iron uptake and transport at these sites. In the intestine, absorption of non-heme iron, the form used in this study, is mediated by the divalent metal transporter 1 (DMT1) at the apical (AP) plasma membrane of duodenal epithelial cells. Here, duodenal cytochrome b (DcytB) functions as a ferric reductase and reduces Fe(III) iron to the Fe(II) form for transport to the cytosol by DMT1. Absorbed iron is transported through the cytosol by poorly characterized mechanisms and is exported from the basolateral (BL) membrane by the concerted effort of ferroportin (FPT) and hephaestin, a multicopper oxidase that oxidizes Fe(II) iron to the Fe(III) form for uptake by Tf in the systemic circulation 59FeCl3) in the duodenal enterocytes of PrPKO mice that persists until eleven days of chase when almost all 59Fe from Wt controls has been transported out. Most of the 59Fe accumulates in ferritin within these cells, a surprising observation given the state of iron deficiency in these mice. Ferritin protein expression is also up-regulated in PrPKO enterocytes, probably reflecting a response to increased iron influx from the AP plasma membrane, decreased transport from the BL membrane, or a combination of the two processes. Although limited amounts of 59Fe are released into the blood stream of PrPKO mice during the first hour of chase, the amount is significantly lower compared to Wt controls. Furthermore, PrP is expressed on the BL surface of MDCK cells, a polarized epithelial cell line, and inter-cellular junctions of mouse enterocytes KO mice excrete relatively less 59Fe in their feces despite the iron loaded state of their enterocytes (unpublished observations). It is likely that this response reflects an attempt to conserve and utilize the absorbed iron by PrPKO mice due to their chronic state of iron deficiency.Based on our observations, it appears that PrP mediates iron export from the BL membrane of enterocytes to the blood stream rather than uptake from the intestinal lumen. This conclusion is supported by our observations demonstrating accumulation of orally administered radioactive iron  mice due to a mutation in the intestinal ferroxidase Absence of PrP also compromises the ability of hematopoietic precursor cells to take up iron from plasma Tf, an interesting observation since these cells acquire most of their iron by the Tf/TfR pathway. A similar phenotype of iron deficiency has been reported in neuroblastoma cells expressing lower levels or mutant forms of PrP, another cell line that utilizes the Tf/TfR pathway for acquiring iron KO mice show an earlier and a significantly higher spike in 59Fe uptake by the spleen and long bones relative to Wt controls. Since RBCs consume the majority of plasma iron and the spleen and long bones are the principal sites of erythropoiesis, these findings reflect an abnormally high rate of erythropoiesis in PrPKO mice. The presence of 59Fe labeled cells in the peripheral blood of PrPKO mice after four hours of chase is surprising given the time it takes for RBCs to mature, and may reflect the binding of 59Fe-Tf to TfR positive immature erythrocytes. However, while Wt RBCs continue to incorporate 59Fe, PrPKO RBCs show only a modest increase even after eleven days of chase. These results suggest that the erythropoiesis in PrPKO mice is ineffective despite adequate supplies of iron in the spleen and long bones, probably due to inefficient uptake by the hematopoietic precursor cells. The alternate possibility, i.e., increased turnover of RBCs in the spleen or intravascular hemolysis is unlikely based on the following observations: 1) 59Fe counts did not increase in the spleen of PrPKO mice at any time point after four hours of chase, the main organ where RBCs are turned over and the released iron recycled, 2) PrPKO RBCs did not show a decline in 59Fe counts during eleven days of chase given that the normal half-life of mouse RBCs is ∼15 days, 3) 59Fe from hemolyzed RBCs was not detected in the urine, and 4) markers of hemolysis such as hemoglobinuria were not detected in PrPKO mice (unpublished observations). Furthermore, the kinetics of 59Fe uptake from the plasma of Wt and PrPKO mice suggests decreased uptake by target organs rather than increased hemolysis in the latter. The precise biochemical pathway by which PrP facilitates iron uptake by the parenchymal and hematopoietic precursor cells is difficult to define from our data. However, based on our observations from neuroblastoma cells PrPKO mice is mild since overt signs of anemia were not detected except for a mild reduction in hemoglobin levels, a significant reduction in serum iron, serum ferritin, and transferrin saturation, and an increase in the total iron binding capacity and the number of circulating reticulocytes. This observation is surprising given the obvious decrease in brain iron content, a privileged organ that is affected last by iron deficiency KO mice in all major organs, including the hematopoietic system. Introduction of PrP on the PrPKO background reverses the iron deficient phenotype completely, indicating that PrP is an integral component of the iron homeostatic machinery that feeds into the major pathways of iron uptake and transport.The iron deficiency in PrPIn conclusion, this report demonstrates a significant role of PrP in maintaining systemic iron homeostasis. Specifically, PrP modulates iron transport from the duodenum to the blood stream and uptake by cells of the hematopoietic system, brain, liver, and spleen. Since major proteins and pathways of iron transport and uptake utilized by enterocytes and hematopoietic cells differ significantly PrP-specific monoclonal antibody 8H4 (against residues 145 to 180) was obtained from Abcam  and from Drs. Man-Sun Sy and Pierluigi Gambetti (Case Western Reserve University). Rabbit anti-human ferritin antibody that cross-reacts with H and L-chains of mouse ferritin was obtained from Sigma , ferritin H-chain specific antibody was from Santa Cruz , ferritin L-chain specific antibody was also from Santa Cruz , anti-Tf was from GeneTex , anti-TfR from Zymed Laboratories Inc , and horseradish peroxidase (HRP)-conjugated secondary antibodies were from GE Healthcare . All other chemicals were purchased from Sigma.KO mice in the FVB background (FVB/0/0Prnp) were originally obtained from George Carlson, (McLaughlin Research Institute). The PrP+ transgenic mice expressing wild type murine PRNP were created by microinjection of the half-genomic PrP clone 0/0Prnp eggs. The transgenic mice were screened by PCR and maintained via breeding with FVB/0/0Prnp mice. The genotype of littermate PrPKO and PrP+ mice were determined by PCR of DNA from ear punches.The PrPKO mice maintained under similar conditions were fasted overnight with water ad libitum, and 20 µCi of 59FeCl3 diluted in 0.2 ml of PBS was administered orally with an olive-tipped gavage needle. The mice were chased on normal chow for 1 h, 4 h, 24 h, 48 h and 11 days. At each chase time point, a set of Wt and PrPKO mice were euthanized, and blood was collected by cardiac puncture in heparinized vials. Plasma and red cells were separated by centrifugation at 5,000 rpm for 5 min. Stomach and upper gastro-intestinal tract was separated and rinsed with cold PBS until clean. The samples were placed on a filter paper, vacuum dried, and subjected to autoradiography. The brain, liver, spleen, and femur were collected, washed with PBS, and snap frozen on dry ice. The organs were weighed, and radioactivity was counted in a γ-counter. Selected samples of brain were fixed in 5% phosphate buffered formalin for 24 h, and 700 µM thick sections were cut using a Leica vibrotome. Cut sections were briefly air-dried and exposed to X-ray film.All procedures with mice were performed according to the guidelines established by the Animal Resource Center of Case Western Reserve University, and were based on protocols approved by the IACUC committee. Three to six month old age- and sex-matched Wt and PrPNative gradient gel electrophoresis, autoradiography, and immunoblotting were performed essentially as described by Vyoral et al. Brain, liver, and spleen homogenates (10%) prepared in lysis buffer were resolved by SDS-PGE and subjected to western blotting using specific antibodies as described in previous reports Snap frozen liver and spleen sections (7 µm) were immersed in acidified potassium ferrocyanide solution (4%) for 20 min followed by washing with distilled water. Sections were then counterstained with 1% Neutral Red for 2 min and mounted.In-gel iron estimation of protein bands and the iron content of liver and spleen homogenates were performed by Ferene-S staining essentially as described KO mice were assessed by incubating 10 µM thick brain sections prepared from frozen samples essentially as described by Moos et al. Tf binding sites in the brains of Wt and PrPKO mice as described by Hanazawa et al. 59FeCl3-citrate complex . Differences were considered significant at p<0.05.A minimum of 3 mice in each group were analyzed for all experiments, and the experiments were repeated at least 3 times. The results are expressed as mean±standard error of mean (SEM). Statistical analysis was done by unpaired Student's t test when comparing Wt with PrP


In contrast to previous reports, we only detected a small proportion of ovarian cancers with hedgehog target gene expression, suggesting that identification of the tumors with activated hedgehog signaling activation will facilitate chemotherapy with hedgehog signaling inhibitors.The hedgehog (Hh) signaling pathway regulates many processes of development and tissue homeostasis. Activation of hedgehog signaling has been reported in about 30% of human cancer including ovarian cancer. Inhibition of hedgehog signaling has been pursued as an effective strategy for cancer treatment including an ongoing phase II clinical trial in ovarian cancer. However, the rate of hedgehog signaling activation in ovarian cancer was reported differently by different groups. To predict the successful for future clinical trials of hedgehog signaling inhibitors in ovarian cancer, we assessed hedgehog pathway activation in 34 ovarian epithelial tumor specimens through analyses of target gene expression by  Ovarian cancer is the most deadly type of gynecological cancers. Epithelial ovarian cancer is the major ovarian malignancy consisting of five histological subtypes: serous, mucinous, endometrioid, transitional and clear cell ,2. Overain-situ hybridization, immunohistochemistry, RT-PCR and real-time PCR  signaling pathway regulates many processes of development and tissue homeostasis ,5. In thPTCH1, Gli1, HIP1. Tumors with expression of two hedgehog target genes are regarded as hedgehog signaling activated tumors. PTCH1 expression was detected in 9 of 34 (~26%). In in-situ hybridization analysis, most positive staining of PTCH1 was seen in the cancer tissues  signal but the sense probes did not show staining, indicating the specificity of in-situ hybridization. Further analysis did not reveal association between PTCH1 expression and tumor subtypes, stage or other characteristics  and 7 of 34 (~21%) cancers respectively. Like PTCH1, most positive staining of GLI1 and HIP1 was shown in cancer  with any tumor characteristics. The result of RT-PCR shown in Figure in-situ hybridization. Further analysis indicated that PTCH1 co-expressed with GLI1 (p = 0.0033), but not with HIP1 expression. HIP1 expression was only detected in 2 specimens which expressed PTCH1 or Gli1, indicating that HIP1 is not highly expressed in ovarian tissues or is silenced in cancers as reported in other studies [9 of 34 ~6% and 7 9 of 34 ~6% and 7  studies ,12. OnlySHH was found in 11 of 34 (~32%) ovarian cancers  in 17 ovarian cancers. Although we found SMO expression in 4 (~24%), and Su(Fu) in 4 (~24%) tumors respectively. Most tumors with elevated expression of Gli1 and PTCH1 had no expression of SMO, suggesting that SMO expression was not responsible for hedgehog signaling activation in ovarian cancer.In addition to SHH, we also detected expression of in situ hybridization was confirmed by semi-quantified RT-PCR showing in Figure SHH and SMO by in-situ hybridization and RT-PCR. In summary of our data, we found only a small proportion of ovarian cancer specimens with elevated hedgehog signaling activation. Our data also showed that elevated SHH expression is not always associated with Hh target genes expression. Based on our data, we caution that clinical trials with hedgehog signaling inhibitors in ovarian cancer may be successful in selected patient population with elevated Hh target genes.The result of in-situ hybridization analyses. Our data suggest that it is necessary to identify the right population of ovarian cancer patients in the clinical trials with hedgehog signaling inhibitors. In addition, we found that even in the tumor with elevated expression of hedgehog target genes Gli1 and PTCH1, expression of SHH is not necessarily high, suggesting other mechanisms of hedgehog signaling activation in the cancer. The fact that hedgehog signaling activation is not associated with any particular subtypes of ovarian cancer suggests that the morphological classification of ovarian cancer may not reflect the molecular pathogenesis of this disease. It will be of interests to establish an animal model to study hedgehog signaling-mediated carcinogenesis in ovary.Using several approaches, we showed that the percentage of hedgehog signaling activation is low in ovarian cancer. We only found 20% of tumor specimens with detectable expression of two hedgehog target genes, as indicated in real-time PCR and in-situ hybridization, real-time PCR and immunohistochemistry. Although it is possible that our results may under estimate the frequency of hedgehog signaling, we predict that screening of Hh signaling activated tumors will significantly improve the successful rate of clinical trials using Hh signaling inhibitors. It is also possible that the involvement of Hh signaling in human cancers may be context dependent, occurring in some tissues or cell lines but not in others. Evidence suggests that Hh signaling may be involved in maintaining cancer stem cell proliferation [Different results have been reported on hedgehog signaling activation in ovarian cancer ,9. Thereferation ,14.Taken together, our findings suggest that activation of the Hh pathway is not frequent in ovarian cancer. The fact that SHH expression is not correlated with Hh target gene expression suggests that there are other mechanisms responsible for Hh pathway activation. Our studies predict that targeted inhibition of the hedgehog pathway may be only effective in a small percentage of ovarian cancer patients.patched 1; Shh: sonic hedgehog; SMO: smoothened.HIP: hedgehog:interacting protein; Su(Fu): suppressor of fused; PTCH1: human homologue of The authors declare that they have no competing interests.in-situ hybridization and immunohistochemistry, performed the statistical analysis and drafted the manuscript. SH and YB participated the in-situ hybridization and immunohistochemistry. JH, XZ and NH performed the real-time PCR analysis of ovarian cancer. JX and HZ designed and planed the experiment, drafted the manuscript. All authors read and approved the final manuscript.LY carried out the Dr JX is a professor at Indiana University Wells Center for Pediatric Research and IU Simon Cancer Center, with a focus on hedgehog signaling in human cancer. He was involved in the initial linking of hedgehog signaling to human cancer in 1996 and was the first to report activated SMO mutations in human cancer. Professor HZ from Shandong University has expertise in developmental biology and interests in linking developmental pathways to human diseases. In the last few years, these two groups have reported links of hedgehog signaling to several cancer types, including liver, esophageal, colon and gastric cancers using comprehensive analyses of hedgehog target gene expression.Materials and methods.Click here for fileAdditional table.Click here for file


Earlier models of plant-herbivore interactions relied on forms of functional response that related rates of ingestion by herbivores to mechanical or physical attributes such as bite size and rate. These models fail to predict a growing number of findings that implicate chemical toxins as important determinants of plant-herbivore dynamics. Specifically, considerable evidence suggests that toxins set upper limits on food intake for many species of herbivorous vertebrates. Herbivores feeding on toxin-containing plants must avoid saturating their detoxification systems, which often occurs before ingestion rates are limited by mechanical handling of food items. In light of the importance of plant toxins, a new approach is needed to link herbivores to their food base. We discuss necessary features of such an approach, note recent advances in herbivore functional response models that incorporate effects of plant toxins, and mention predictions that are consistent with observations in natural systems. Future ecological studies will need to address explicitly the importance of plant toxins in shaping plant and herbivore communities. By definition herbivores depend on plants to survive. The need to obtain suitable food in sufficient amounts drives innumerable herbivore behaviors; for example, movement decisions often are related to the distribution and abundance of plant resources . By the When focusing on optimal diet choice by herbivores, ecologists traditionally have relied on linear programming or linear dynamic programming methods ,8. GivenConsumer-resource interactions at the population level can be modeled using equations that relate the rate of resource intake by a consumer to resource abundance . These sEucalyptus trees produce 1,8-cineole, a monoterpene that serves as a potent deterrent to herbivorous marsupials such as brushtail possum, Trichosurus vulpecula [Larrea tridentata) in the western United States produces phenolic resins containing nordihydroguaiaretic acid, which limits intake by desert woodrats, Neotoma lepida [Betula) in boreal North America produce the triterpene papyriferic acid as a deterrent to feeding by snowshoe hares, Lepus americanus [Populus tremuloides) produces coniferyl benzoate, a phenylpropanoid ester that inhibits feeding by ruffed grouse, Bonasa umbellus [A problem with conventional plant-herbivore models is their failure to incorporate factors related to plant quality into decelerating functional responses. For many herbivores, plant toxicity plays an important role in diet choice ,15. Indeulpecula . Creosota lepida . Tree biericanus . Althougericanus , plant tumbellus .In addition to interspecific differences, production of toxins varies ontogenetically within plants, and among individuals and populations within species. Intraspecific variation in chemical defense often contains strong genetic components -23. WhenJuniperus monosperma), detoxification costs are comparable to energy needed for reproduction [One consequence of feeding on plants containing toxins is that rates of ingestion may be limited by an herbivore's ability to avoid toxins or detoxify food rather than to mechanically process food. Not surprisingly, herbivores have developed a host of physiological and behavioral mechanisms to deal with plant secondary metabolites . Physioloduction . For rufoduction . In the oduction .C(N):In light of the widespread nature of plant toxins and their influence on herbivores, a new approach to linking herbivores to their food base is needed if we are to understand implications for herbivore populations and plant communities. Traditional functional responses for vertebrate herbivores have not considered the role of plant toxins. At least three analytical modifications should be considered when incorporating the effects of toxins on plant-herbivore dynamics. Toxin-mediated functional responses should (1) explicitly account for the negative effects of plant toxins on herbivore growth; (2) permit herbivores to regulate intake of toxins; and (3) allow for intake of multiple plants that are detoxified with independent pathways. Recently, progress has been made in the first two areas ,43. Specwheref(N) is the traditional Holling Type 2 functional response in which N is plant biomass, e is resource encounter rate, h is handling time for each plant, and σ is the fraction of encountered food items that are ingested, thereby allowing herbivores to regulate their intake. The second factor in C(N) explicitly accounts for the negative effect of toxins. The parameter G stands for the ratio M/T, where M is the maximum amount of toxicant per unit time that the herbivore can tolerate and T is the amount of toxicant per unit of plant biomass. The factor 4 simplifies the peak value of C(N) as a function of N. In the limit that 1/G << 1, the effect of the toxicant can be viewed as purely a slowdown in feeding rate. For example, in that limit C(N) above is an approximation ofThe term G.So in (2), the presence of toxin simply results in an effective increase in the handling time that is proportional to 1/Related functional response models have been formulated to examine how plant growth is limited when the presence of one resource interferes with another resource or is toxic . In otheN1 and N2 represent the concentrations of nutrients limiting plant growth, Cmax is the maximum possible rate of nutrient-limited plant growth, and K1 and K2 are constants reflecting the stoichiometry of the two nutrients in the plant. Note that in the limiting case, as N1/N2 approaches zero, (3) reduces to the traditional Michaelis-Menten equation for N1, with K1 as a half-saturation coefficient [N2, causes growth rate to increase in (3), whereas in (2) an increasing concentration of toxin in food relative to the rate of toxin ingestion the herbivore can tolerate, 1/G, decreases growth rate. In both equations, this change in C is due to a change in size of the third term in the denominator.fficient . More im/G is large (and hence each gram of plant is quite toxic to herbivores), the functional response can represent a more serious deterioration of the herbivore's ability to feed or survive. Analysis has demonstrated the critical importance of σ to herbivore dynamics; in the presence of a toxin, selection should act strongly to regulate intake below the herbivore's detoxification threshold [The toxin-determined functional response (1) differs from plant-nutrient models (3) because toxins can do more than reduce feeding rate. Specifically, when 1hreshold .Alces alces) and snowshoe hares feeding on woody plants. Under conditions of the multi-species model, herbivores switch feeding on plant species to avoid saturating detoxification systems. One consequence of toxin-induced switching is that herbivores are predicted to spend a disproportionate amount of time foraging on less abundant plant species, resulting in depensatory mortality that can limit invasion by more palatable species (Figure Because most vertebrate herbivores are generalists, a toxin-mediated functional response should be able to consider multiple plant species. Fortunately, the single-species framework  extends s Figure . ConsistEvidence for the importance of plant toxins as determinants of herbivore functional response is indisputable. Recent modeling efforts implicate toxins as potentially key drivers of change in plant communities and herbivore populations. Future models should consider the role of resource patchiness and tri-trophic interactions on plant communities. For instance, adaptive foraging by herbivores is hypothesized to have important effects on ecosystem processes such as nutrient cycling rates, and predators may alter herbivore effects by changing their density or behavior . How do All authors shared in the conception and organization of this commentary, including review of pertinent literature. RKS wrote the initial draft. All authors read and approved the final manuscript.


Revealing the dispersal of dengue viruses (DENV) in time and space is central to understanding their epidemiology. However, the processes that shape DENV transmission patterns at the scale of local populations are not well understood, particularly the impact of such factors as human population movement and urbanization. Herein, we investigated trends in the spatial dynamics of DENV-2 transmission in the highly endemic setting of southern Viet Nam. Through a phylogeographic analysis of 168 full-length DENV-2 genome sequences obtained from hospitalized dengue cases from 10 provinces in southern Viet Nam, we reveal substantial genetic diversity in both urban and rural areas, with multiple lineages identified in individual provinces within a single season, and indicative of frequent viral migration among communities. Focusing on the recently introduced Asian I genotype, we observed particularly high rates of viral exchange between adjacent geographic areas, and between Ho Chi Minh City, the primary urban center of this region, and populations across southern Viet Nam. Within Ho Chi Minh City, patterns of DENV movement appear consistent with a gravity model of virus dispersal, with viruses traveling across a gradient of population density. Overall, our analysis suggests that Ho Chi Minh City may act as a source population for the dispersal of DENV across southern Viet Nam, and provides further evidence that urban areas of Southeast Asia play a primary role in DENV transmission. However, these data also indicate that more rural areas are also capable of maintaining virus populations and hence fueling DENV evolution over multiple seasons. Dengue virus (DENV) is the cause of the most common vector-borne viral disease of humans, and is at particularly high prevalence in parts of Southeast Asia. Most studies of DENV transmission have focused on very local or international movement patterns, and have not explored how DENV moves through an endemic region. To address this issue, we employed newly developed phylogeographic methods to study patterns of spatial spread in 168 full-length DENV-2 genome sequences collected during a hospital-based study in southern Viet Nam, focusing on the Asian I genotype that recently emerged in this region. This analysis revealed that the urban population of Ho Chi Minh City plays a central role in the dispersal of the virus, and that DENV in this city tends to move along a gradient of population density. In addition, human movement between urban and rural areas was the most likely explanation for the rapid diffusion of DENV across southern Viet Nam following its introduction into Ho Chi Minh City. After reaching more rural areas, some virus lineages were maintained there for a number of years. These results therefore indicate that virological surveillance is necessary in both urban and rural populations. Flaviviridae) that exist as four antigenically distinct viruses or serotypes (DENV-1 through DENV-4) and show complex immunological interactions within a human host and at the epidemiological scale Dengue viruses (DENV) are mosquito-borne RNA viruses  and dengue shock syndrome (DSS) ranging from 36 to 405/100,000 population per year between 1998 and 2008 In recent years, a number of studies have utilized gene sequence data to investigate the spatial and temporal dynamics of DENV in a restricted geographic area To this end, the aim of this study was to reveal trends in the spatial dynamics of DENV-2 in southern Viet Nam by using full-length genome sequence data obtained from patients admitted to a tertiary referral hospital in HCMC. These data provide a unique opportunity to investigate the spatial dynamics of different lineages of DENV-2 within a geographical region characterized by high endemicity. By using recently developed phylogeographic methods we address the following key questions: (i) Does HCMC, as the primary urban center of the region, act as a source population for dengue viruses circulating in southern Viet Nam? (ii) Within HCMC, are the highest density population areas acting as foci of viral dispersal throughout the city and the region? (iii) At this scale, does DENV dispersal adhere to a predictable population- or density-based model of transmission?DENV-2 genome sequences were obtained from dengue patients enrolled into a prospective clinical and virological study of dengue at the Hospital for Tropical Diseases in Ho Chi Minh City, Viet Nam. These data and the sampling, serotyping, virus isolation, and sequencing methods used have been described elsewhere http://tree.bio.ed.ac.uk/software/). Of these, three were initially excluded from this analysis; two sequences were missing spatial or genetic data, and one sequence was obtained from a subject who identified his or her home as being far outside of the study area .From 2001 to mid-2008, 187 full DENV-2 genome sequences were obtained from hospitalized dengue cases in southern Viet Nam, and their complete coding regions  were manually aligned using Se-AL v2.0a11 (available from 2 and a population of nearly 20 million (∼532 persons/km2) As a large hospital-based study that likely included only severe cases, we would not reliably expect to capture clustering at the finest spatial and temporal scales, including direct chains of transmission. To identify such fine-scale clustering, pairwise genetic distances between the 184 remaining sequences were determined using the HyPhy package, employing the HKY85 model of nucleotide substitution with global branch length estimation parameters 4 model of nucleotide substitution  phylogenetic trees were inferred for the DENV-2 genome sequences of the American/Asian and Asian I genotypes separately using a Bayesian Markov Chain Monte Carlo (MCMC) method implemented in the BEAST package (v1.5.2) 2, ‘Urban HCMC’ - population density <15,000/km2 and >2500/km2, and ‘Suburban HCMC’ - population density <2500/km2; 66 possible diffusion pathways overall), and (iii) by geographic proximity and population density-based district groupings within HCMC and by province .To assess the overall degree of spatial admixture and geographical structure among DENV-2 lineages in this region, we calculated values of the association index (AI) http://tree.bio.ed.ac.uk/software). To account for the potential effects of sampling bias, data from highly sampled geographic areas were randomly subsampled with replacement to create smaller data sets from each geographic location, and analyses were repeated 10 times for each subsampling scheme.The strength of support for viral exchange between individual locations at each of the spatial levels was inferred using a geographically-explicit Bayesian MCMC approach implemented in BEAST Because pathogen dispersal across geographic areas is often thought to be influenced by factors such as distance and human population density, we integrated both distance and population-based priors into the phylogeographic analysis to mimic the effects of these factors on the DENV population. To calculate the distances between the defined populations, centroids for relevant geographic regions were determined using R (version 2.9.1) ijC) between community i (of size iP) and community j (of size jP) were calculated using the relation:d) between the two geographic areas To obtain prior estimates corresponding to these distance and population-based parameters, simple gravity model calculations providing estimates of the movement of populations   tests of phylogeographic structure rejected the null hypothesis of no association between sampling location and phylogeny at all of the spatial levels tested for the Asian I DENV-2 genotype in southern Viet Nam . Hence, The MCC phylogeny of the Asian I genotype with BSSVS reconstructed ancestral locations (10 provinces) of the internal nodes reveals that viruses from nearly all provinces are dispersed throughout the phylogeny, most notably HCMC and DT, as well as LA and TG . AlthougWhen Asian I viruses were categorized according to population density in HCMC and by province elsewhere , various patterns emerged. The highly populated area of HCMC showed two strongly supported pathways of diffusion: between the Superurban and Urban districts, and between the Urban and Suburban districts (BF>30). In contrast, viral exchange between Superurban and Suburban HCMC was not supported. Significant diffusion pathways were also observed between regions of HCMC and provinces both distant and adjacent in the Mekong Delta region, while one significant pathway was detected between two adjacent provinces west of HCMC  revealed that samples from most geographic locations (both within and outside of HCMC) were dispersed throughout the phylogeny of the Asian I genotype . Within Importantly, the general effects of the number and scale of spatial parameters on phylogeographic model inference are currently under investigation, such that the results of this finest-scale analysis, particularly those that are inconsistent with earlier analyses, should be considered as provisional. It is also possible that these changes reflect general trends in mixing between HCMC and other communities within the region that become clearer as population characteristics of the large urban area of HCMC are taken into account in the analysis. These uncertainties not withstanding, it is interesting to note that the new connections observed at this finest spatial level are compatible with a previously undetected transmission network among the provinces east and north of HCMC, largely industrial areas that are geographically isolated from the Mekong Delta provinces showing consistent viral exchange with HCMC.Finally, although some movement patterns are clearly suggestive of gravity-like dynamics, and particularly within HCMC (see above), distance and gravity model-informed priors did not result in an overall better fit to the data than the default constant rate priors  Major urban areas of Southeast Asia have previously been proposed to play central roles in DENV epidemics, harboring the greatest viral genetic diversity and population sizes sufficiently large to allow sustained outbreaks that may subsequently spread to more rural areas, and potentially acting as harbingers of epidemic dengue activity in a given season Notably, its role as the center of commerce and industry in the region makes HCMC a major acceptor of human in-migration from more rural areas, particularly to districts labeled ‘urban’ and ‘suburban’ in this analysis in situ evolution, was clearly detected among viruses isolated from residents of Dong Thap, Tay Ninh, and Vung Tau. Dong Thap province in particular yielded several small monophyletic groups, as well as one well-supported clade that contained a sequence from An Giang, its neighbor to the west. This suggests that locales that are relatively geographically isolated from highly populated urban areas may experience some population subdivision similar to that observed at a smaller spatial scale in northern Thailand Despite the relatively small sample size, phylogenetic clustering of viruses, itself an indicator of potential Within HCMC, the patterns of DENV movement between the three regions of varying population density are consistent with a gravity model of virus dispersal, with viruses moving down (or up) gradated population density categories even though all three regions share borders. This trend of movement across a gradient of population densities is largely upheld in the finer-scale analysis within the city, with 13 of 15 significant viral diffusion pathways in the city detected between areas of similar population density or one degree removed, and the majority of viral movement occurring between adjacent districts. Using a similar phylogeographic approach, Balmaseda et al. also observed viral movement between adjacent neighborhoods in a cohort study in Managua, Nicaragua, with some exchange occurring between more distant neighborhoods, likely attributable to transportation networks and migrant workers moving within the community In sum, our study indicates that DENV moves relatively freely among human populations in southern Viet Nam, over both long and short distances, and hence suggests a major role for anthropogenic factors and urban areas as drivers of DENV dispersal in Southeast Asia. However, the relative isolation of some areas directly adjacent to well-connected areas is not well understood, such that it is difficult to make strong conclusions on the predictability of DENV transmission dynamics within this highly endemic region; spatio-temporal variation in seropositivity may play a significant role in preventing new viral lineages from being established in some populations, and the contribution of this factor should be investigated further in future studies. Importantly, neither distance-based nor gravity models were able to explain the full complexity of the transmission dynamics, although a gravity model showed a slightly better fit to our data than did the distance-based models for both the provincial and population density-based analyses. The improved fit of the model upon the integration of population data provides further evidence that human population movement is an important factor acting on DENV dispersal in the region. Thus, it is possible that increased information on short- and long-term human population movements between rural and urban areas may provide a model with improved predictive power for estimating the future spatial spread of DENV over this area. The use of transportation information, including distances by land (such as road distance) and water travel, as well as relative costs of travel between these areas, may also increase the power of these models to determine important viral migration pathways. In the absence of this information, DENV transmission dynamics within and across cities and rural areas are difficult to predict, as many factors, including human population densities and movement, population immunity, mosquito densities and dispersal, and the seasonality of dengue transmission intensity, as well as other unknown factors, may affect the rates of virus dispersal and establishment of new transmission networks in a locality. As these data represent the initial results of an ongoing study, the isolation of additional DENV sequences over the coming years will allow us to investigate the spatial relationships among viral lineages circulating within the country in greater detail, and may enable us to determine how specific population movement patterns such as seasonal migration and international travel affect the dispersal of viral lineages throughout the region.Figure S1Genetic and geographic distances among complete coding region sequences of DENV-2. Each pair is represented as a point, with the color of the point indicating the year of sampling. (a) Asian I DENV-2. Colors represent: 2003, black; 2004, blue; 2005, cyan; 2006, green; 2007, yellow, 2008, red; all years, grey. (b) American/Asian DENV-2. Colors represent: 2001, black; 2002, blue; 2003, cyan; 2004, green; 2005, yellow, 2006, red; all years, grey.(3.48 MB EPS)Click here for additional data file.Figure S2Results of the phylogeographic analysis on subsampled populations of DENV-2, Asian I genotype. (a) Phylogeographic analysis by province. (b) Phylogeographic analysis by urban levels within HCMC and by province outside of HCMC.(0.97 MB EPS)Click here for additional data file.Figure S3MCC phylogeny of the Asian I genotype in southern Viet Nam (2003–2008) according to district group (within HCMC) or province of sampling. Tips are colored by district group (within HCMC) or province of sampling. Internal branches are colored based on the reconstructed ancestral state as estimated by the reversible diffusion model. Branches colored black indicate Bayesian posterior probabilities less than 0.85. Estimated support for the reconstructed ancestral state is indicated by open (>95%) and closed (>85%) diamonds.(0.48 MB EPS)Click here for additional data file.Figure S4MCC phylogeny of the American/Asian genotype in southern Viet Nam (2001–2006) according to province of sampling. Tips are colored by province of sampling. Internal branches are colored based on the reconstructed ancestral state as estimated by the reversible diffusion model. Branches colored black indicate Bayesian posterior probabilities less than 0.85. Estimated support for the reconstructed ancestral state is indicated by open (>95%) and closed (>85%) diamonds.(0.31 MB EPS)Click here for additional data file.Figure S5MCC phylogeny of the American/Asian genotype in southern Viet Nam (2001–2006) according to population density (within HCMC) or province of sampling. Tips are colored by urban level (within HCMC) or province of sampling. Internal branches are colored based on the reconstructed ancestral state as estimated by the reversible diffusion model. Branches colored black indicate Bayesian posterior probabilities less than 0.85. Estimated support for the reconstructed ancestral state is indicated by open (>95%) and closed (>85%) diamonds.(0.36 MB EPS)Click here for additional data file.Table S1GenBank accession numbers, year, and province of sampling of DENV-2 genome sequences used in this study.(0.19 MB DOC)Click here for additional data file.Table S2Results of geographic diffusion model for DENV-2, Asian I genotype; locations given by province.(0.05 MB DOC)Click here for additional data file.Table S3Results of geographic diffusion model for DENV-2, Asian I genotype; locations given by urban level within HCMC or province.(0.06 MB DOC)Click here for additional data file.Table S4Results of spatial diffusion model for DENV-2, Asian I genotype; locations given by district group within HCMC or province.(0.10 MB DOC)Click here for additional data file.Table S5Model exploration for the Asian I genotype at two geographic levels.(0.04 MB DOC)Click here for additional data file.


Pain, which afflicts up to 20% of the population at any time, providesboth a massive therapeutic challenge and a route to understanding mechanisms inthe nervous system. Specialised sensory neurons (nociceptors) signal theexistence of tissue damage to the central nervous system (CNS), where pain isrepresented in a complex matrix involving many CNS structures. Geneticapproaches to investigating pain pathways using model organisms have identifiedthe molecular nature of the transducers, regulatory mechanisms involved inchanging neuronal activity, as well as the critical role of immune system cellsin driving pain pathways. In man, mapping of human pain mutants as well as twinstudies and association studies of altered pain behaviour have identifiedimportant regulators of the pain system. In turn, new drug targets for chronicpain treatment have been validated in transgenic mouse studies. Thus, geneticstudies of pain pathways have complemented the traditional neuroscienceapproaches of electrophysiology and pharmacology to give us fresh insights intothe molecular basis of pain perception. Noxious environmental stimuli, tissue damage, and disease all evoke pain. Theavoidance of painful stimuli, the protection of damaged tissue to promote healing,and the amelioration of disease-evoked pain (preferably through resolution of thedisease itself) have obvious survival value. The evolutionary utility of pain-evokedbehavioural responses is confirmed by apparent conservation of some mechanisms inall animals with nervous systems. The existence of specialised mammalian sensoryneurons that respond to tissue damage (nociceptors), first proposed by Sherrington acentury ago, has been clearly demonstrated in humans and mice, where mutationsleading to loss of responsiveness to the trophic factor nerve growth factor (NGF)result in the loss of nociceptive neurons and a pain-free phenotype http://www.europeanpainnetwork.com/). New drugs acting on targetsvalidated in mouse and man by genetic studies show promise for many of the presentclinical problems. This therapeutic challenge is complemented by the fact that painalso provides a wonderful model system to understand how the nervous system works.This area is perhaps the most exciting for geneticists who want to addressmechanisms of perception and consciousness, neuronal signalling, synapticplasticity, and integrative aspects of nervous system function.In this review we focus on genetic approaches to the mechanisms involved inactivating nociceptive neurons, human genetics of pain perception, and geneticvalidation of pain targets leading to new drugs. The application of moleculargenetics to the problem of pain has provided some remarkable insights over the past15 years Heterologous expression studies have identified a number of sensoryneuron–associated genes that have been invoked as sensors fordifferent sorts of damaging stimuli.+ currents, and reduction of electrogenic pumpactivity. Recent work suggests that TRPM8 andTRPA1 may play important roles in the detection of noxiouscold, and that the inhibition of an as-yet-unidentified backgroundK+ channel may be important. In addition to currentresearch focusing on transduction by primary sensory neurons, recent reportshave suggested that epidermal cells, particularly keratinocytes, may beresponsible for the transduction of certain stimuli, synapsing with primarysensory neurons  is not inactivated by cooling, in contrast toall other voltage-gated sodium channels, provides an insight into themechanisms behind the detection of noxious cold V1.8 reflects the vital importance of the ability to detectnoxious stimuli in cold environments, particularly relevant to cold-bloodedanimals. A more detailed treatment of the mechanisms involved in cold painis given in The discovery that the voltage-gated sodium channel NaTRPV1,expressed in sensory neurons and activated by both heat and red hot chilipeppers (the capsaicin receptor), caught the public imagination andsuggested that the mechanism for noxious thermosensation had been identifiedThe identification of a cation selective ion channel, ACCN3encoding ASIC 3, as described by TRPV4SCN9A and SCN10A), do have amechano-insensitive phenotype Sensory neurons express ion channels that can be directly activated bymechanical stimuli, and evidence has been presented that such channelsunderlie noxious mechanosensation SCN11A, is found in a subset ofdamage-sensing neurons, and is activated close to the resting membrane potential.Knock-out studies have confirmed that the encoded channel NaV1.9 does notsupport action potentials but plays a key role in setting pain thresholds, as it isregulated by inflammatory mediators SCN9A, is essentialfor peripheral pain and had been analysed in mouse knock-outs SCN9Adie, probably because they are unable to feed. However, nociceptor-specific nullmutants show a loss of acute mechanical and inflammatory pain SCN9A human mutants that have a defectiveNaV1.7 channel appear to be completely pain free but are otherwisenormal. This observation SCN9A, resulting in lowered thresholds of activation, result inerythermalgia, a chronic inflammatory condition. Rarer mutations that impede theinactivation of the channel seem to cause acute paroxysmal pain SCN10A (NaV1.8), a specific marker for nociceptiveneurons, is a major contributor to electrogenesis in primary pain pathways, and isan important target for inflammatory mediators. It is also essential for cold painTissue damage depolarises sensory neurons, but the transmission of information to theCNS requires the recruitment of voltage-gated sodium channels to propagate actionpotentials, and cause neurotransmitter release (mainly glutamate) into the CNS. In the Drosophila Para have been correlatedwith functional changes. Liu et al. Regulation of sodium channel expression, both transcriptionally andposttranscriptionally, is an important element in determining neuronal excitability.A short sequence found upstream of neuronal sodium channel genes  was identified and named NRSE (Neuron restricted silencing element)or RE-1 (repressor element 1) SCN3A(NaV1.3) channel exists as an embryonic or adult spliced form, withdifferent exons that code for the S3 and S4 segments in domain one of the ratchannel. A similar pattern is present with SCN9A, where somedifferences in biophysical properties and the effects of cAMP on splice variantshave been described V1.8 is enhanced bytreatment with NGF, suggesting that this neurotrophin may regulate trans-splicingevents in these cells In mammals, mutually exclusive exon usage also occurs. The COMT that influence the activities of endogenous opioids isdiscussed in Neural correlates of consciousness have been the focus of much attention, especiallywith the advent of functional imaging. Pain perception seems to involve a variety ofdiscrete CNS structures COMT, the gene coding for catechol-O-methyltransferase (COMT).COMT mediates the inactivation of catecholamine neurotransmitters, includingdopamine, adrenaline, and noradrenaline, and reduced COMT enzymatic activityappears to result in increased pain sensitivity and temporal summation of painCOMT. This SNP results in thesubstitution of valine by methionine at position 158 , andis associated with reduced thermostability and a 3–4-fold reduction inCOMT activity. Humans homozygous for the met158 allele reported veryslightly increased sensory and affective ratings of pain in response to muscularinfusion of hypertonic saline compared to heterozygotes, while ratings forval158 homozygotes were lower than heterozygotes. Correspondingvariations in regional, pain-related µ-opioid responses were observedusing positron emission tomography, and were taken to explain the differences inpain perception Many SNPs affecting pain perception have been identified in recent years.Possibly the best-characterised of these are the SNPs present in158met) in the coding region, two in the promoterregion, and one at the end of the 3′ UTR; no other SNPs exist in thecoding region at frequency >0.15%. Two of these  were found to be associated with altered painsensitivity, present in the same haploblock. The haplotype at this haploblockwas found to have a significant effect on pain perception, with haplotypesdesignated as high, low, or intermediate pain sensitivity . This was found to correlate inversely with levels of COMTactivity. Different combinations of these haplotypes were strongly associatedwith variations in experimental pain sensitivity. Additionally, a reduction inrisk of a common painful musculoskeletal condition, temporomandibular jointdisorder, was observed in the presence of even a single low-sensitivityhaplotype. The mechanism by which these COMT haplotypes influence activity wasrecently uncovered using in silico modelling of mRNA secondary structures. Itwas found that the haplotypes differed with respect to mRNA local stem-loopstructures, and that the most stable structure resulted in lower protein levelsand thus activity. Further evidence was provided by site-directed mutagenesis,restoring normal levels of activity by eliminating the stable structure A broader investigation of the influence of SNPs in the COMT gene on painsensitivity provided a more comprehensive picture. Diatchenko et al. Altered pain states, such as inflammatory hyperalgesia and neuropathic allodynia, arediscussed in P2RX7), an ATP receptor absent from neurons, is essentialfor altered inflammatory or neuropathic pain http://paingeneticslab.ca/4105/06_02_pain_genetics_database.asp.Clinical problems associated with the pain system occur when pain thresholds aredramatically altered so that noxious insults became extremely painful or non-noxious stimuli, such as light touch or mild cold, alsobecome very painful . Hyperalgesia is commonly found in inflammatorypain conditions where damaged tissue is infiltrated with immune system cells,and a variety of soluble mediators that act on peripheral neurons are released.Allodynia may occur when nerves themselves are damaged—this isneuropathic pain, which occurs in situations such as diabetic neuropathy,HIV-associated neuropathy, or following physical trauma. Studies have identifieda number of critical regulators of altered pain states, including genes notnormally expressed in the nervous system, but present in cells of the immunesystem that also are activated in situations of tissue damage. For example, P2X7 act directly on sensory neuronsand through cascades of second messengers and kinases, altering the sensitivityof both primary transducing receptors and sodium channels required for actionpotential transmission A wide variety of pain assays have been developed for the mouse, examining differentpain modalities. Models of inflammatory and neuropathic pain are alsowell-described, and may have more relevance to human clinical conditions than assaysof acute pain. These models have been discussed in detail by the Mogil group Mus musculus means that the extent ofgenetic contributions to disease can be evaluated. For example, work on behaviour inresponse to various pain modalities The variety of strains of loxP recombination system to effect conditionalgene deletion, has been productive loxP-flankedsequences upon chemical induction of Cre expression SCN10A, the gene encoding the voltage-gated sodiumchannel NaV1.8, was recently reported to have almost completeinsensitivity to cold pain For the investigation of the function of specific DNA sequences, transgenic and genetargeting techniques have proved useful. Examples of transgenics that have generatedinsights into pain pathways are numerous, and include reports of modulation of painbehaviour by the overexpression of the neuropeptide galanin Substantial variations in both pain sensitivity and susceptibility to chronic painfulconditions occur between individuals in both human and animal populations These natural variations in propensity to pain result from a combination ofenvironmental and genetic influences on pain-sensing systems. Environmental factors,such as early exposure to acute painful stimuli, can have long-term effects onnociceptive thresholds in both animals and humans. Perinatal painful events such ascircumcision without anaesthesia, for example, have been reported to increasesensitivity to pain in later life Gender differences in pain perception have been reported by many studies, in bothanimals and humans. For example, women are more likely to suffer from a varietyof chronic pain disorders, including fibromyalgia, complex regional painsyndrome, and trigeminal neuralgia. Experimentally, pain thresholds for pressurepain and electrical stimulation have been shown to be lower for females than formales, while less variation has been observed for thermal pain stimuli. For adetailed treatment of gender differences in pain perception, see the recent andcomprehensive review from Greenspan et al. Heritability studies using sensory testing of twins can identify the importance ofgenetic contributions to pain traits, and SNP association studies have correlated anumber of genes with altered pain behaviour see . In contV1.7-encoding SCN9A gene as an essential componentof human pain provides an exciting new analgesic drug target.Despite the fact that most of these syndromes involve cell death, and are thusunappealing in terms of drug development, they have provided important insights intoaspects of pain signalling. For example, HSAN-3 demonstrates the essential role ofNGF-dependent sensory neurons in the pain process, whilst the recent discovery ofthe NaKCNJ9 (GIRK3) has beenidentified as a locus affecting analgesia from multiple drug classes Several studies have attempted to evaluate the relative contributions of genetics andenvironment to variations in pain sensitivity, in both animals and humans.Comparisons of pain thresholds  within and between 11 inbredlaboratory mouse strains revealed genetic contributions of between 30%and 76% In humans, the genetic contribution to variation in pain sensitivity has beeninvestigated using twin studies, comparing correlation of pain thresholds betweenmonozygotic (shared genetic and environment) and dizygotic (shared environment)twins. Two recent papers investigating responses to experimentally induced painreported genetic contributions to sensitivity to the majority of pain modalities.Norbury et al. In addition to experimentally induced pain, several studies have investigated thecontribution of genetic variability to differences in severity of and susceptibilityto chronic pain conditions. For example, a recent study, using 15,950 pairs oftwins, found a genetic contribution of about 50% to the likelihood ofdeveloping fibromyalgia, a relatively prevalent chronic pain condition While these studies have been useful in defining the importance of genetic variationin differences in pain perception, they do not provide mechanistic explanations ofthese differences. Analysis of clinically presenting, congenital disorders of painsensation can define genes central to the ability to detect noxious stimuli, butrely on the resulting phenotype being relatively conspicuous. Quantitative traitlocus (QTL) mapping  can be used to determine moreprecisely the genetic location underlying this variability. SNPs are traditionallydefined as having an allele frequency of >1% or>0.5% in the population, although this is no longer a strictrequirement. They may occur within any part of the genome: coding and noncodingsections of genes, and in intergenic regulatory or undefined regions. Coding regionSNPs which do not alter the resulting amino acid are termed synonymous. Althoughsynonymous SNPs do not alter the sequence of protein produced, they may still resultin major changes in function. For example, they may affect levels of transcription,splicing, mRNA stability, or regulatory RNA expression. One mechanism by which thismay occur is through effects of synonymous SNPs on cotranslational folding The study of pain-related SNPs in the human has proved problematic, due to thetendency of different data sets to yield conflicting conclusions; in many cases, afinding from one has been contradicted by those from others. Here, we present aselection of the positive associations that have been reported, with the caveat thata number of these findings have not been replicated in other investigations.COMT, which are discussed in OPRM1 , which mediates the physiologicalanti-pain effects of endorphins, have been linked to variations in experimental painsensitivity OPRM1 (frequency ∼10%) affected both subjectivepain reporting SNPs in several genes have been found to have substantial impacts on painsensitivity. These are summarised in MC1R contains polymorphismsassociated with both pain sensitivity and µ-opioid analgesia in humans andmice The melanocortin-1-receptor gene GCH-1)in pain sensitivity. Excess BH4 is thought to increase pain due to increased nitricoxide synthesis. A pain-protective haplotype (probably consisting of five SNPs in aregulatory region of the gene) of GTP cyclohydrolase was identified in about15% of the population studied, shown to decrease both persistentpost-surgical and acute mechanical experimental pain. Subjects heterozygous orhomozygous for the pain-protective haplotype were found to show reduced upregulationof the GCH1 transcript in response to cAMP, resulting in lowerlevels of BH4, suggesting that altered GCH1 transcriptionalmodulation underlies the decreased pain sensitivity observed in these individuals.In a study going from initial pathway identification to human pain variation, Tegederet al. TRPA1 gene encoding a channel activated bycold, mustard oil, and possibly mechanical stimulation in nociceptors TRPA1deletion on cold pain in the mouse FAAH), which inactivates the endocannabinoidanandamide, may also contribute to variation in pain sensitivity Genetic variations in the CYP), which play a key role in the metabolism of many drugs,can affect the efficacy of opiates, and NSAIDs. Reduced activity of cytochromes caneither reduce or enhance analgesic efficacy, depending on the activity of themetabolites compared to the original drug. For example, one metabolite of the opioidtramadol, O-desmethyltramadol, is a considerably more potent agonist of theµ-opioid receptor than tramadol, meaning that low metabolisers of tramadoldisplay reduced analgesia, despite an increased half-life of tramadol being observedMDR1), which is a majordeterminant of morphine bioavailability, can alter the efficacy of morphine painrelief A range of genetic variations have been identified that alter the effectiveness ofanalgesic drugs. In particular, polymorphisms of the cytochrome P450 enzymes for analgesic drugs that are under development. Chronicpain in the absence of disease is an obvious form of dysfunctional sensation andprovides a useful model system for understanding other nervous system disorders that involve pathological responses to external sensations.Studies of the affective component of pain perception, combined with functionalimaging, genetically manipulatable animal models, and new genetic insights into painmechanisms offer considerable hope for our understanding not only of “painin the brain”, but other aspects of human sensation, consciousness, andbehaviour.Substantial progress has been made in pain genetics recently. Just as a massiveresearch attack on cancer genetics in the 1980s is now paying dividends in terms ofdrug treatment, so the genetic description of key signalling molecules and mediatorsin the pain system has provided many new targets (e.g.,


Critical limb ischemia (CLI) is an advanced form of peripheral artery disease which is responsible for approximately 100,000 amputations per year in the US. Trials to date have reported clinical improvement and reduced need for amputation in CLI patients receiving autologous bone marrow or mobilized peripheral blood stem cells for stimulation of angiogenesis. While such treatments are currently entering Phase III trials, practical and scientific pitfalls will limit widespread implementation if efficacy is proven. Hurdles to be overcome include: a) reduced angiogenic potential of autologous cells in aged patients with cardiovascular risk factors; b) invasiveness/adverse effects of bone marrow extraction and G-CSF mobilization, respectively; and c) need for on-site cellular manipulation. The Endometrial Regenerative Cell (ERC) is a mesenchymal-like stem cell derived from the menstrual blood that is believed to be associated with endometrial angiogenesis. We discuss the possibility of using allogeneic ERCs as an "off the shelf" treatment for CLI based on the following properties: a) High levels of growth factors and matrix metalloprotease production; b) Ability to inhibits inflammatory responses and lack of immunogenicity; and c) Expandability to great quantities without loss of differentiation ability or karyotypic abnormalities. Critical limb ischemia (CLI) is classically defined as chronic ischemic rest pain, ulcers, or gangrene due to proven occlusive disease . It is dThis condition is a major cause of morbidity and mortality: Approximately 20–45% of patients require amputation, and 1-year mortality is estimated to be as high as 45% in patients who have undergone amputation . CurrentAngiogenesis therapy has been described as a "biological bypass", the idea being that through administration of agents capable of inducing collateralization, a more natural type of "bypass" can be achieved. Indeed it has been observed that ischemic muscles secrete angiogenic factors in response to hypoxia and that to some extent natural angiogenesis does occur in animal models of CLI and in humans ,6. One oAnother approach involved use of the cytokine fibroblast growth factor-1 (FGF-1). Given that FGF-1 is considered "upstream" of VEGF, it is believed to stimulate numerous angiogenic processes so as to result in creation of more mature vessels . Indeed,It is known that additive and/or synergistic effects are observed in terms of neoangiogenesis when several angiogenic factors are combined. For example, Cao et al demonstrated synergy between administration of PDGF-BB and FGF-2 in terms of increasing blood vessel formation and function in the femoral artery ligation model in rats and rabbits . SimilarIn conclusion, while administration of angiogenic factors to patients with CLI does induce some benefit in early trials, data from randomized trials to date do not support widespread use. The transfection of upstream transcription factors such as HIF-1 alpha is a promising approach since it mimics natural angiogenesis in that a plurality of growth factors are induced following transfection ,28. HoweCellular therapy for CLI is based on the rationale that delivery of endothelial progenitor cells into areas of ischemia may result not only in differentiation of the cells into endothelium and thus contribute to angiogenesis, but also produce various growth factors capable of stimulating angiogenesis through paracrine interactions with adjacent cells. One of the first observations that bone marrow contains high numbers of endothelial progenitor cells was by Ashara et al using infusion of bone marrow cells in which the beta-galactosidase marker was expressed under control of endothelial cell-specific promoters such as Flk-1 and Tie-2. EPC derived from the genetically marked bone marrow were shown to promote physiological , and pathogenic (cancer) angiogenesis. The same study demonstrated that administration of bone marrow cells into muscles of mice with induced hindlimb ischemia led to neoangiogenesis mediated in part by the transferred cells .A more extensive investigation of bone marrow administration of treatment of a model of CLI utilized the rat femoral ligation model and assessed not only perfusion but also the functional endpoint of treadmill running. Administration of syngeneic bone marrow in six locations proximal to the area of ligation caused a significant increase in perfusion, as well as treadmill performance in the treated rats . A study9 cells in the gastrocnemius muscle of the ischemic leg using approximately 40 injections in a volumes of 0.75 ml. Statistically significant increases in ankle brachial index, trancutaneous oxygen pressure, pain free walking time, and amelioration of rest pain were detected at 4 and 24-week follow-up. The investigators also demonstrated production of proangiogenic cytokines by the injected cells. Other groups also reported similar therapeutic results: Nizankowski et al treated 10 CLI patients with condensed bone marrow stem cells and observed improvement using laser doppler flux and percutaneous oxygen partial pressure, as well as decrease in pain severity. Interestingly no correlation was found between cell number injected and effect [Bone marrow transplantation has been clinically used for hematopoietic reconstitution for more than 40 years, thus procedures for bone marrow extraction and manipulation are routine. In 2002 Tateishi-Yuyama et al  used thed effect . Durdu ed effect . Numeroud effect ,38, as wd effect -41. A red effect .Thus, autologous bone marrow therapy for CLI appears to be a promising solution to the current lack of treatments in patients ineligible for endovascular/surgical interventions. However, this is limited by the need for anesthesia during the bone marrow extraction procedure, which is dangerous in the CLI population since numerous co-morbidities exist . MobilizAdditionally, there are suggestions in the literature that endothelial progenitors from bone marrow of patients with peripheral artery disease may be suboptimal in comparison to other progenitor sources ,46. SpecDr Taylor's group demonstrated that various endometrial cells are actually derived from bone marrow. Through studying endometrial stromal cells isolated from female recipients of male bone marrow, they reported positive staining for the Y-chromosome in a substantial number of cells . ReportsCells resembling some phenotypic characteristics of ERC appear to be similar to the menstrual blood derived mesenchymals described by Miyoshi's group . These cN. peroneus for reproducing a neurotrophic ulcer-like injury was performed. Mice were divided into 2 groups of 8. Immediately after induction of injury, 1 million ERC were injected into the hind-limb muscle below the area of ligation. Cells were also injected on day 0, day 2 and day 4. ERC where injected in a volume of 200 microliters of saline. By day 14, necrosis was observed in legs of 8 control mice. 8 mice treated with ERC had intact limbs, with 2 displaying signs of impeded walking. Figure The endometrium undergoes rapid angiogenesis in a controlled manner monthly. Upregulated production of angiogenic factors including PDGF, EGF, and VEGF have been described both in the mouse and human endometrium ,56. SomeMesenchymal stem cell populations appear to possess active immune modulatory properties in vitro and in vivo. Specifically, bone marrow derived mesenchymal cells have been demonstrated to inhibit ongoing mixed lymphocyte reaction (MLR) , induce In preliminary experiments we have demonstrated that addition of ERC to ongoing mixed lymphocyte reactions results in suppression of proliferation Figure , inhibitClinical entry of novel cellular therapies requires demonstration of safety and reasonable possibility of improving the patient's condition. Previous studies involving long term follow up of animals treated with ERC, as well as karyotypic normality of these cells after extended passage (68 doublings) have demonstrated lack of tumorogenicity . In factNeil Riordan and Thomas Ichim are shareholders and management of Medistem Inc (OTCBB:MEDS).All authors read and approved the final manuscript. MP, HW, AP, SK, NA, KC, AM, AP, EC, TI, and NR concieved experiments, interpreted data, and wrote the manuscript.


KISS1, lack of αvβ3-integrin and low levels of RHOC.Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines , and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts. Melanoma arises from melanocytes, specialized cells in the skin responsible for synthesizing and distributing the pigment melanin. This tumor is one of the most aggressive malignancies, marked by elevated capacity to metastasize and by high drug resistance [for review see -3]. Melain vitro the characteristics representing the original state of the tumor stage from which they are derived . Most sequences (94–95%) corresponded to annotated mRNA sequences. The sequence redundancy within each library is low, 288 different genes are represented in the RGP library (non-redundancy of 82%) and 217 in the Met library (non-redundancy of 59%). Additional File ABCB5 , ALS2CR7 (10 clones) and MBOAT1 (3 clones). DCN encodes decorin, a secreted protein involved in cell growth regulation and apoptosis induction in tumors [DCN mRNA only in the WM1552C cells and at very high levels, indicating that these libraries represent genes highly differentially expressed. ALS2CR7, a candidate gene of amyotrophic lateral sclerosis 2, encodes a putative protein kinase, based on Gene Ontology prediction, with no characterized function. As shown in Fig. ALS2CR7 mRNA expression was detected in all eight cell lines analyzed, all four metastatic cell lines presented equivalently low signals and the highest levels were detected in WM1552C, confirming the differential expression of this gene identified in the RGP SSH library. The MBOAT1 gene encodes a hypothetical transmembrane protein containing an O-acyltransferase domain, also with no characterized function and as predicted by its occurrence in the RGP library, we confirmed that its mRNA expression is higher in WM1552C  was used as probe for Northern blot hybridization , PLP1 (24 clones) and HLA-DRA (55 clones). MITF is a transcription factor involved in melanocyte differentiation and survival and even though it is represented by only one sequence in the Met library, its differential expression between the RGP WM1552C and the metastatic cell lines was confirmed by Northern blot  that also compare the frequency of genes in each functional class with the expected frequency based on the distribution of all human genes throughout the GO functional classes. The GO functional classes that are significantly enriched in the RGP and Met libraries, in comparison to the distribution of all predicted human genes, are listed in Tables We also analyzed the chromosome location of all genes/ESTs identified in the SSH libraries Fig. . InteresLUM (lumican), DCTN6 (dynactin 6) and DNCI2 (dynein intermediate chain 2)  against tumor samples (primary and metastatic melanomas) for each set of genes  Fig. . The Metnes Fig.  and 5C. DCN is preferentially overexpressed in most skin and nevi samples and in 2 (out of 5) primary melanomas, confirming that its expression seems to decrease with melanoma progression, although a subset of melanoma samples also presented an overexpression of this gene  did not exhibit expression pattern clearly associated to any specific stage of the melanoma progression in this study , or reveal novel transcripts, since they match only genomic DNA, mapping in introns or intergenic regions  being common to both libraries, indicating that these collections represent a transcriptional content that distinguish between the RGP cells WM1552C and a group of four metastatic cell lines, and may reflect distinct transcriptional profiles of these two stages of melanoma progression  validated by Northern blots encode proteins of unknown function, both highly expressed in WM1552C compared to VGP and metastatic cell lines making them interesting candidates for further investigations. This is the first evidence for the expression of these genes in melanocytic cells, and points towards a role in melanoma development.Two of the RGP genes , several lines of evidence suggest their involvement in tumorigenesis. HLA-DRA encodes the α chain of the HLA-DR, one of the MHC class II molecules that, in contrast to MHC class I molecules, are not normally expressed by nonprofessional antigen-presenting cells (APC). Functional MHC class II molecules, key initiators of an immune response by activating CD4+ naïve T cells, are heterodimeric proteins composed of α and β chains encoded by separated genes (A and B). Melanocytes from normal skin and common nevus are negative for HLA class II molecules [HLA-DRA gene were the most abundantly sequenced clones from the Met library. However, sequences corresponding to the HLA-DRB gene were not detected in the same library, suggesting that HLA-DRA may not form a functional antigen-presenting molecule in these cells.Among the genes represented in the Met library, many are associated to tumor growth, invasiveness and metastasis, such as TM4SF1 , LAMA4 [1 [LAMA4 , G3BP2 [4 [G3BP2 , CD59 an antigen , and SPPen [SPP1 . Genes eolecules ,67 whileolecules . Althougolecules -73. In colecules . Clones PLP1 in melanoma development, remarkably, we show here that all vertical growth phase and metastatic cell lines exhibit high expression levels of this gene. It is interesting to note that PLP1 expression is regulated by the transcription factor SOX10 [MITF [PLP1 is a transmembrane protein involved in myelinization  whose upor SOX10 , which i10 [MITF , the othMITF encodes a transcription factor required for melanocyte differentiation and survival [MITF mRNA in one of the metastatic cell lines, WM1617, which also showed the highest level of PLP1 expression. The high levels of MITF in WM1617 cells must explain the occurrence of many melanocytic markers (known MITF targets) in the Met library, as TYRP1, TYR, DCT, MLANA. However, differently from PLP1, high levels of MITF mRNA expression were not detected in the other three metastatic cell lines, but rather in cell lines from RGP and VGP tumor stages (WM35 and WM902). Consistent with this result, microarray studies revealed MITF overexpression in a subset of primary and metastatic melanoma samples [PLP1, MITF and the melanocytic markers were all detected as co-regulated genes associated to high proliferation and low metastatic potential in groups of cell lines that included WM1617 and WM35. So it is likely that a set of the genes detected in the Met library is more importantly linked to tumor proliferation/survival than invasion.survival ,81 and w samples  and led  samples  of a claMITF gene was found to be amplified in melanoma and its overexpression induced transformation and drug resistance in BRAF mutant melanocytes [CDK2 [HIF1A [MET [Although MITF is able to induce cell cycle arrest in melanocytes and melanoma cells in a p16 and p21 dependent manner ,83, MITFanocytes . In addies [CDK2 , the hip2 [HIF1A  and the F1A [MET , all of FN1, which codes for the matrix protein fibronectin [ADAM10, which encodes a putative desintegrin metallopeptidase [SGK, which encodes a glucocorticoid regulated kinase [NRP2, which encodes neuropilin 2[C18orf19.Other genes with consistent overexpression in tumor samples in comparison to nevi and skin Fig.  compatibronectin ; genes weptidase , SGK, whd kinase , NRP2, wropilin 2, and alsNPM1 (nucleophosmin), was found at both protein and transcript levels in melanoma cells, compatible with the detection of this gene in our Met library. An accumulation of a specific form of the nucleophosmin protein was also detected in human melanoma cell lines  in comparison to normal melanocytes in another proteomics study [When we looked for the expression profile for RGP and Met genes in the microarray data obtained by Hoek et al , 13 and cs study .Altogether the data shown here strengthen previous evidence for several genes as candidate markers for melanoma progression and suggest that the subtractive libraries described are enriched in cancer-related genes, representing validated tools to be used in future studies for the identification of novel genes or pathways involved in melanoma progression.The author(s) declare that they have no competing interests.JFS designed the study, constructed, and sequenced the SSH libraries, conducted all expression validation and comparisons, and drafted the manuscript. EME conceived the study, participated in its design and coordination and also drafted and revised the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Representative image of agarose gels containing PCR-amplified inserts from 96 randomly selected clones from the subtractive libraries. The images show that most clones from both RGP and Met libraries carry inserts and most of them are ≥ 600 bp in length.Click here for fileComplete lists of the genes identified in the RGP and Met libraries. The tables contain the identifier and annotation of the genes represented in the RGP and Met libraries and indicate the number of occurrences for each gene among the clones sequenced from each library.Click here for fileSchematic representations of the sequence alignments of ESTs from the RGP and Met libraries that match intronic or intergenic regions. The data indicate that some of the sequences identified in the subtractive libraries may correspond to novel transcripts.Click here for fileLists of the genes represented by redundant clones in the RGP and Met libraries. The tables contain the identifier, Gene Ontology annotation and available information about role in cancer related processes for the genes represented by redundant clones in the RGP and Met libraries.Click here for fileSearch for the expression profile of the genes identified in the RGP and Met libraries in a melanoma microarray study performed by Hoek et al. . Lists oClick here for fileSearch for the expression profile of the genes identified in the RGP and Met libraries in the proteomics and SAGE analyses performed by de Souza et al. . Lists oClick here for file