The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer. It derives from the proepicardium that gives rise to the vasculature and interstitial cells of the heart during embryogenesis, a process that is mandatory for normal development Beyond its role during cardiac development, the epicardium also mediates cardiac regeneration after injury in lower vertebrates. Specifically, the zebrafish epicardium supports cardiac regeneration through epithelial to mesenchymal transition (EMT) and subsequent migration into the myocardium to form new vasculature Although some of the genes involved in epicardial function have been identified and characterised Using this approach, we identified numerous genes that have not previously been implicated in the function, homeostasis, or regeneration of the epicardium or the heart. Our results provide several markers of the steady state adult epicardium, and changes of epicardial signature gene expression in response to LCA injury that underscore the involvement of the epicardium after cardiac damage. Epicardial signature gene expression patterns were altered in a model of enhanced cardiac regeneration by delivery of a localized transgenic IGF-1 propeptide, which reactivated genes implicated in the effective regenerative response of other vertebrate species. This study provides a wealth of information on gene expression patterns in this important tissue, which will prove potentially valuable in future studies of epicardial function in cardiac homeostasis and infarction models.Using LCM technology, we isolated the left ventricular epicardial layer of frozen murine hearts as described in As a separate quality control, we also compared the epicardial sample to left ventricle or whole heart tissues This analysis uncovered numerous epicardial signature genes such as uroplakin 1b, 3b, glycoprotein m6a, dermokine or basonuclin that have not previously been implicated in the function, homeostasis or regeneration of the epicardium or the heart. Importantly, our list of epicardial signature genes also contained several genes whose expression have previously documented in the epicardium, playing key roles in epicardial development. Among these were RALDH2 (9 fold increase) the earliest retinoic synthetic enzyme in the embryo and implicated in mammalian epicardial contribution to the coronary vasculature and myocardial repair in the zebrafish system Systematic analysis of tissue expression profiles by comparing the list of epicardium-enriched transcripts to relevant gene ontology lists  and compared to those of non-infarcted (sham operated) mice. Epicardial samples and muscle controls for both conditions were obtained using LCM as described above, and changes in expression levels were analyzed six days after myocardial infarction using gene arrays. SOM cluster analysis as described in Interestingly, GO ontology analysis for epicardial signature genes upregulated in the muscle control revealed a distinctly different set of lists  resembliWe also examined a selected subset of epicardial genes most significantly affected by infarction for further analysis at an earlier time point (three days post-infarction). RT-PCR analysis of three and six day post-infarction epicardial samples showed a consistent downregulation of uroplakin 3b, proline rich 15, dermokine, Efemp-1, sulfatase-1, and basonuclin compared to sham operated hearts . ConversRegarding less dramatically affected genes than the epicardial signature subset, we observed a trend towards up-regulation of vimentin, fibronectin, snail2, and MMP-2 as well as a modest down-regulation of desmoplakin in the epicardial transcriptome after injury (data not shown), all of which are associated with cells undergoing epithelial-to-mesenchymal transition We have previously shown that supplemental transgenic expression of a locally acting Insulin-like Growth Factor-1 propeptide (mIGF-1) in cardiomyocytes improved repair of the heart after infarction. Restoration of cardiac form and function in post-infarct mIGF-1 transgenic mice was facilitated by modulation of the inflammatory response Although the most highly expressed genes of the unperturbed epicardium were common to both genotypes, several genes were selectively upregulated in the epicardium of mIGF-1 animals. Comparing wildtype and mIGF-1 samples revealed a number of genes that were highly epicardium specific in the mIGF-1 samples but that were not part of the epicardial signature in the wildtype mice . FurtherTo test this possibility, we compared the expression profiles of the wildtype and mIGF-1 hearts six days post infarction. Interestingly, the post-infarction gene expression changes seen in wildtype animals were less evident in the mIGF-1 hearts: unsupervised analysis of mIGF-1 epicardial genes using SOM revealed no cluster with significant changes of expression , indicatThe relative paucity of cell-restricted markers represents a current constraint on physiological and pathophysiological studies of the adult murine epicardium. A transcriptomic approach to elucidating epicardial marker genes is a powerful way to obtain better insight into the expression profile and characteristics of the epicardial layer. Using laser capture microscopy, we developed an analytical approach for the identification of novel epicardial genes, yielding a list of potentially interesting genes for epicardial function that has not been possible with more traditional, less high-throughput approaches. The use of LCM affords the unbiased collection of the entire cell population of the epicardium/subepicardium as opposed to more selective cell sorting or staining-based technologies. Although we confirmed the microarray results for an epicedial signature gene subset at the protein level, any analysis based on RNA-transcripts has its limitations, and more extensive validation of protein expression will be necessary when examining other genes contained in these lists.Other approaches have compared gene expression of epicardial regions to endocardial regions of the ventricular wall of the rat The epicardial transcriptomic analysis presented here highlighted cell adhesion genes characteristic of epithelia, confirming a characterization of the epicardium as an epithelial cell layer covering the heart. On a broader scale, the GO ontology analysis confirms the epithelial character of the epicardium, as exemplified by numerous candidates in the cell adhesion list. Other high scoring GO lists were mainly related to immune response, populated with complement-related genes that are normally expressed in epithelial cells such as keratinocytes of the skin Of the novel epithelial genes identified in this study, dermokine and basonuclin are particularly noteworthy in the context of the adult epicardium. Importantly, these genes have been described as epithelial genes in the literature, but heretofore have not been annotated as such in the GO ontology. Dermokine, a gene of unknown function, has been suggested to have a cytokine function, which supports an epicardial signalling role The transcription factor basonuclin-1 plays a role in epithelial cell differentiation and proliferation and has been studied in a corneal epithelium damage model Downregulation of epicardial genes as well as the loss of adhesiveness after induction of cardiac infarct is consistent with cells undergoing EMT in the epicardium before myocardial invasion, migration and differentiation into endothelial or smooth muscle cells to form new vasculature. The upregulation of vascularisation and epithelial genes in the muscle tissue further supports this interpretation. Indeed, several markers for EMT were affected in the epicardial as well as cardiac transcriptome after injury. Together with activation of thymosin β-4 expression in the epicardium samples Despite some similarities to gene programs activated in the initial repair processes after resection of zebrafish hearts, the mammalian epicardium cannot support effective regeneration of cardiac damage. In this regard, the shift in epicardial gene expression patterns in mice expressing supplemental mIGF-1 propeptide in the myocardium may support the improved regenerative capacities documented in these transgenic hearts All mouse procedures were approved by the EMBL Monterotondo Ethical Committee  and were in accordance with national and European regulations.WT male C57/Bl6 mice were purchased from Charles River Laboratories. Male mIGF-1 transgenic animals were used as described before th intercostal space. The retractors were inserted and the pericardium was cut open to allow access to the heart. The left coronary artery was visualized running form the left atrium towards the apex as a red pulsating vessel th and 5th ribs using 6–0 nylon sutures. The muscles were placed into their original position and the skin incision was closed using a 6–0 nylon suture. Mice were monitored until awakening and body temperature was kept up by red light. After surgery mice were kept individually in double cages and sacrificed three or six days after surgery.Male animals between three and four months of age were anesthetized using 2% isoflurane and endo-tracheal intubation was carried out to ventilate the lungs while the chest was open. An incision of several mm length away from the sternal border towards the left armpit was made. Underlying connective tissue was opened and the two muscle layers underneath were spread and held by retractors without incision into the muscle. The rib cage was then visualized and opened at the 4Hearts were excised after perfusion with 40 ml Phosphate Buffer Saline (PBS) and immediately frozen in OCT at −80°C. PEN membrane coated slides  were activated and prepared as suggested by the supplier. Seven µm thick tissue sections of the left ventricle were obtained and washed for 10 seconds in DEPC-treated water, precooled 70% EtOH and 100% EtOH. Slides were air dried for 1–2 min. and stored at −80°C until processing. For laser dissection, slides were thawed briefly and excess liquid was removed. The epicardial samples, as well as cardiac muscle controls, were independently dissected out using a P.A.L.M. LCM  as shown in For non-infarcted samples (sham operated), epicardial cells were obtained by dissecting the epicardial and subepicardial layer of the left ventricle. For infarcted samples, the infarction was carried out as described in the corresponding section and animals were sacrificed for dissection three or six days after induced infarction. Epicardial cells were dissected partly above and partly below the site of infarction using the infarct-suture site as landmark. Laser capture dissection represents the most reproducible way of collecting epicardial sample without introducing a bias towards certain cell populations (FACS sorting) or compromising RNA quality through prolonged tissue handling (staining). Fragments were catapulted into microtube lids filled with 65 µl RLT buffer  and 5 µl of carrier RNA (4 ng/µl solution).After dissection, total RNA of the epicardial layer of individual hearts were isolated using a standard RNeasy micro kit  protocol. Quality of RNA was tested using Agilent Bioanalyzer 2100 . RIN values were at least above 6 while most samples had a RIN between 7 and 8. For each sample between 0.2 and 2 ng of RNA could be collected.RNA was processed for Affymetrix GeneChip analysis by the gene core facility at the European Molecular Biology Laboratory (EMBL) using a two cycle cDNA synthesis protocol as suggested by the supplier  and a mouse430-2.0 chip  was used for the analysis.Data was processed and analysed using GeneSpring GX 7.3.1 . Gene array raw data was first normalized in order to make multiple chip data comparable using the pre-processing algorithm “GeneChip - Robust Multi-Array Average” (GC-RMA) method to account for background correction, normalization, and summarization. For further normalization values below 0.01 were set to 0.01. Each measurement was divided by the 50.0th percentile of all measurements in that sample. Each gene was divided by the median of its measurements in all samples. If the median of the raw values was below 10 then each measurement for that gene was divided by 10 if the numerator was above 10, otherwise the measurement was excluded. For each experimental group of tissues, samples from four or five animals were used. All array data is MIAME compliant and raw data has been deposited with ArrayExpress (Accession number: E-MEXP-2446).Gene clusters representing similar expression profiles between uninjured epicardial and muscle control samples were identified using unsupervised, SOM clustering (Radius 7.0) with a maximum of 4500000 iterations. SOM clustering can be used to partition genes into clusters of similar gene expression changes across samples. In this analysis, only relative changes in expression levels are considered. Dimensions of 2×2 were chosen as this results in 4 clusters with discernible differences. Unsupervised SOM clustering for post infarction expression changes were carried out using an expanded 2×3 matrix, 10000 iterations, and a radius of 4.0. This expansion was necessary to account for the increased number of possible gene expression patterns that one can envision in the infarcted animals.A list of WT epicardium-enriched genes was created by a parametric t-test using the Benjamini-Hochberg method to correct for multiple testing with a false discovery rate of 0.05 starting from SOM cluster 1 from . An epicFor GO analysis, gene lists were analysed using the GOEAST online tool For further classification of the expression changes after infarction, KEGG analysis was also attempted, but no significantly enriched pathways could be identified.Isolated RNA was reverse transcribed using TaqMan Reverse transcription reagents . Quantitative PCR was carried out using Applied Biosystems gene expression Taqman assays on a ABI PRISM 7700 Sequence Detector . Samples were incubated at 50°C for 10 min, 95°C for 2 min and amplified for 40 cycles . Results were normalized against expression of the 18 S ribosomal RNA.For quantitative reverse transcription PCR, expression was analysed by normalizing against expression levels of 18 S transcripts. To transform Ct values to relative expression values, the following formula was used :Relative expression  = 2∧ -(ΔCt - Ct[stable]).Where: ΔCt : Ct[Gene of Interest] – Ct[18 S]Ct[stable]: Ct of one constant sample across all Ct's for the same gene.Statistical significance was tested using student t-test . Expression values are mean relative expression or mean fold induction ± standard error of the mean (SEM). A p value <0.05 was considered statistically significant unless noted otherwise. .Unfixed mouse heart sections were immersed at room temperature in 100% precooled Acetone for 10 minutes. Samples were washed three times with PBS for 3 minutes each and blocked with blocking solution containing 3% BSA in PBS for 30 minutes. Antibodies were applied at the appropriate dilution , rat anti-GPM6A at 1∶100 ) diluted in 3% BSA in PBS. Sections were incubated with primary antibodies overnight at 4 °C and washed three times in PBS for 5 minutes. Secondary antibody ; GPM6A - Alexa rabbit anti rat 594  was diluted in PBS at 1∶1000 dilution and incubated for 1 hr at room temperature. Slides were washed twice with DAPI  in PBS for 5 minutes and mounted with Permafluor mounting medium . Basonuclin stains were carried out on paraffin embedded sections. Isolated tissues were fixed overnight at 4°C in 4% Formaldehyde/H2O solution. Tissues were gradually dehydrated . For further dehydration slides were incubated twice at room temperature in 100% Xylene and then 1∶1 xylene/paraffin, before they were subjected to 6 changes of 100% paraffin. Tissues were then placed into tissue block molds filled with paraffin. After letting stand over night to harden, 10 µm thick sections were cut using a microtome and placed on SuperFrost Plus microscope slides . Slides were then left to dry at 42°C overnight. Before starting the staining protocol, slides were rehydrated through an ethanol series (100%>70%). They were then washed for one minute in dH2O. For antigen retrieval, the slides were placed in BG antigen retrieval solution  and processed in a pressure cooker “Retriever 2100”  as described in the protocol and left to cool down over night. Sections were incubated in 0.5% H2O2/dH2O for 10 minutes and then incubated in 5% normal horse serum for one hour at RT. After removing the serum, anti-basonuclin antibody (Santa Cruz) was applied at 1/50 dilution and incubated (1 h at RT for basonuclin antibody) in a moisturized chamber and washed three times in PBS. As secondary antibodies, peroxidase conjugated antibodies were used for 1 h at RT. Slides were then washed three times in PBS and were incubated in DAB substrate  as described in the protocol. Slides were then rehydrated through an ethanol series (70%<100%) and mounted after three washes in Xylene.Figure S1Isolation of the epicardial cell layer of murine hearts using laser capture microscopy. A) Hearts were frozen and cut in a cryostat at 7µ¼m thickness, washed in DEPC treated water, 70% EtOH and finally 100% EtOH. B) The epicardial surface of the sections was laser excised (dissected area denoted E) and catapulted into RNA stabilizing solution for later RNA isolation. C) Cardiac muscle (excised area denoted M) was excised to compare gene expression of the epicardial layer to the rest of the heart. .(4.49 MB TIF)Click here for additional data file.Figure S2Comparative analyses of epicardial signature genes. Epicardial data was compared to muscle control, left ventricle or whole heart gene array data. For each of the three control groups, a list of epicardial genes was generated using the same conditions. These lists were compared using a Venn diagram, showing that most genes were overlapping between the different comparison approaches see . Especia(0.16 MB TIF)Click here for additional data file.Figure S3Confirmation of post-infarction gene expression changes. RT-PCR analysis of epicardial signature gene expression changes post-infarct confirmed expression differences between three and six days post infarction were mostly negligible. In contrast, CD5L was upregulated three days post infarction but returned to basal expression levels after six days, highlighting the importance of immune response genes in early post infarction processes. GALDH was used as control. .(0.36 MB TIF)Click here for additional data file.Table S1Full List of epicardial signature genes.(0.04 MB XLS)Click here for additional data file.Table S2Comparative analyses of epicardial signature genes. Data supporting (0.05 MB XLS)Click here for additional data file.Table S3Full GO analysis of epicardial signature genes. Analysis of genes with a p-value cut off of 0.005.(0.02 MB XLS)Click here for additional data file.Table S4GO analysis of downregulated epicardial signature genes after myocardial infarction. Analysis of genes with a p-value cut off of 0.005.(0.01 MB XLS)Click here for additional data file.Table S5GO analysis of upregulated epicardial signature genes in muscle after myocardial infarction. Analysis of genes with a p-value cut off of 0.005.(0.03 MB XLS)Click here for additional data file.Table S6List of mIGF-1 epicardial signature genes.(0.09 MB XLS)Click here for additional data file.


We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed  and haplogroup/sub-haplogroup-defining changes . The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids  harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome.Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds  have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by  Mitochondria, the ‘power plant’ of the cell, have their own distinct genome (mtDNA), whose sequence varies among individuals around the globe. This variation, which was formed by the accumulation of mutations (variants) during the course of evolution, appears to alter the susceptibility to common complex diseases (such as Parkinson's disease and diabetes). However, since the accumulation of mtDNA mutations over time results in the formation of new combinations (genetic backgrounds), it is not clear which of the mutations are functional and which are “evolutionary silent hitchhikers”. Thus we aimed at assessing the functionality of mtDNA genetic variants, focusing on variants within the mtDNA regulatory region, hypothesizing that they could affect mtDNA activity and maintenance. We found that a variant defining mtDNA genetic background ‘J’ significantly increased the transcriptional efficiency and elevated mtDNA copy numbers in cells, as compared to other genetic backgrounds. Hence, mtDNA regulatory region variants can affect mtDNA maintenance, which may partially account for the involvement of this genetic background in disease susceptibility. Our analysis demonstrates, for the first time, the functional impact of a particular mtDNA variant that was fixed during evolution. Moreover, our findings underline the functionality of mtDNA variants in the evolutionary variable regulatory region. Tigriopus Americana revealing that the interaction of mtDNA with the nuclear genetic background is under selective constraint Mitochondria are the major sources for cellular energy, through the process of oxidative phosphorylation (OXPHOS), and thus play a central role in cell life and death. Since mitochondrial DNA (mtDNA) encodes 13 essential proteins of the energy production apparatus and 24 key factors of their translation machinery (i.e. 22 tRNAs and 2 rRNAs) it is not surprising that numerous association studies have demonstrated the involvement of mtDNA genetic backgrounds  in complex human disorders as well as in selective events that transpired during human evolution How thus can one isolate the functional effect of specific mutations from their linked genetic background? Previously, we considered evolutionary sequence conservation to assess the functional importance of all common genetic variants in coding mtDNA, following the logic, that the more conserved the nucleotide position among species, the higher the functional importance In contrast to the mtDNA coding region, most mitochondrial regulatory elements  are subjected to many back-mutations We hypothesized that certain sequence elements across the mitochondrial control region, not necessarily those with high inter-species conservation, are subjected to natural selection within populations. To test this hypothesis we chose to assess the functional potential of genetic variants in the mtDNA control region within sites harboring accepted functionality. mtDNA harbors sequence motifs that are recognized by members of the mtDNA transcription and replication machineries, all of which are encoded by the nuclear genome in vitro transcription by ∼2.5 fold and was associated with more than 2 fold increase in mtDNA copy numbers in mtDNA-less cell lines (rho-0 cells) repopulated with the mtDNA from haplogroup J. The interpretations of these results within the framework of understanding the implications of the phenotypic effects of haplogroup J are discussed.Here, we demonstrate that some single base changes that were fixed within TFAM binding sites during human evolution alter protein binding efficiency. One of these changes not only altered TFAM binding efficiency but also increased the efficiency of http://www.genpat.uu.se/mtDB/) for natural variants in experimentally established protein binding sites and regulatory regions in the mtDNA control region (L), comprising a total of 241 bp in each mtDNA. This screen identified that 77/241 of the total screened sites harbored polymorphic point mutations which can be divided into non-fixed changes  and haplogroups/sub-haplogroup-defining changes (i.e. population-fixed changes), comprising 20/77 (26%) of the variable nucleotide positions.We have screened more than 2500 whole mtDNA sequences from various human populations worldwide  D-loop sequences of primates and other available mammalian sequences in each such sites see . Only foThe LSS sites also showed a pattern of variability, with most nucleotides being invariable and most of the un-conserved nucleotide positions undergoing small number of changes in humans . We beliin vitro mitochondrial run-off transcription assays using partially purified mitochondrial lysates as a source of POLRMT and transcription factors As the first step towards experimentally assessing the functional potential of haplogroup-defining mutations (fixed mutations), we employed Electrophoresis Mobility Shift Analysis (EMSA). Specifically, we tested for the effect of transition variants defining Caucasian haplogroup J (C295T) and a sub-haplogroup of J, J1b2 (C242T), on TFAM binding. These variants occur within two of the four TFAM-binding sites previously mapped by footprinting human mtDNA in vitro transcription results may not only reflect an effect of haplogroup J mutations on mtDNA transcription but also on replication, as these two processes are coupled in the mitochondria . Nevertheless, the C295T nucleotide position underwent only a single fixation event during human phylogeny in the branch leading to haplogroup J, suggesting that, although poorly conserved, the low human variability at this nucleotide position is in line with its functional potential. We thus suggest that the degree of variability of a nucleotide position within humans may serve as a clue for functionality. To experimentally test for the generality of this prediction the functionality of additional variable nucleotide positions should be tested.Our results reveal that some genetic variants that became fixed during human evolution affect protein binding to mtDNA. Moreover, a point variant in a TFAM binding-site (C295T) increased in vitro transcription capacity of the C295T variant occurring in one of the downstream TFAM-binding sites, support the role of this site in mitochondrial transcription. To our knowledge, this is the first indication that TFAM binding downstream of the mtDNA promoter might influence transcription, possibly through a more complex configuration of the promoter or by modulating mtDNA wrapping by TFAM, as previously suggested Four TFAM-binding sites have been mapped in the human mitochondrial genome by DNA footprinting, two of which are important for transcription and are located between the heavy and light strand promoters. The importance of the other two binding sites, localized downstream to the light strand promoter is not known . The altThe tested variants define known mtDNA haplogroups, namely Caucasian haplogroup J and a sub-group of haplogroup J (J1b2). Haplogroup J was previously associated with several complex phenotypes such as Parkinson's disease and longevity  but less successful with most proteins, implying limitations for this approach Our experiments demonstrate the effects of specific point mutations in the mtDNA in an In summary, our analysis sheds light on the functional effect of naturally occurring variants in the mtDNA control region and suggests that such variants contribute to the phenotypic impact of mitochondrial DNA haplogroups.5′GGCGAACATACTTACTAAAGTG 3′ and a reverse primer: 5′GCCCGTCTAAACATTTTCAG 3′). The PCR products were purified using the Wizard SV Gel and PCR Clean-up system (Promega) according to the manufacturer's protocol and then separately cloned into the pGEM T-vector (Invitrogen) using a ligation kit (Roche). The ligated products were transformed into E. coli strain DH5α by heat shock and ampicilin-resistant clones were isolated. Plasmid DNA harboring each of the cloned fragments was isolated and served as templates in an in vitro mitochondrial run-off transcription assay (see below).DNA from healthy individuals previously assigned to haplogroups H, J1 and J1b2 Oligonucleotides corresponding to wild-type or mutant TFAM binding sites  were annEcoR1 resulted in the formation of a linear transcription template from which specific initiation from the LSP promoter resulted in transcripts 223 nucleotides in length. Individual reaction mixtures (25 µl) contained 36 µg of EcoRI-digested template, 10 mM Tris-Cl, pH 8.0, 10 mM MgCl2, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 400 µM ATP, 150 µM CTP and GTP, 10 µM UTP, 0.2 µM α P32UTP , 40 units of RNase OUTTM (Invitrogen) and 2.5 µl of a transcription-competent mitochondrial extract from HeLa cells that was prepared as described previously Mitochondrial run-off transcription reactions were performed www.genpat.uu.se/mtDB), which includes more than 2500 whole mtDNA sequences, representing human populations worldwide. To assign control region variants to specific branches in the human mtDNA phylogeny, sequences containing genetic variants in the screened regulatory elements were compared to the revised Cambridge Reference Sequence Human genetic variants were screened within experimentally established protein binding sites and regulatory regions in the mtDNA control region . Specifihttp://meme.sdsc.edu/meme/intro.html]. The number of times that a mutation event occurred during the mammalian evolution was assessed using available phylogenetic trees  We used a phylogenetic approach relying on haplogroup classification to determine the number of times that a mutation event at a given nucleotide position had occurred during human evolution. Such evaluation assumed that a group of sequences belonging to the same phylogenetic branch (i.e. lineage or haplogroup) represent one mutation event during human evolution. For fixed mutations, we set a cut-off value. Changes that were shared in 5 or more different mtDNA sequences belonging to the same phylogenetic branch were considered fixed. The reason for this cutoff value was, first, to avoid relatively recent fixation events that have yet to be statistically established as the number of whole mtDNA sequences increases. Secondly, branches harboring 2–4 sequences gave relatively low bootstrap values in the global mtDNA phylogenetic tree (data not shown). The conservation of mitochondrial regulatory elements in mammals was estimated using a multiple sequence alignment program [(MEME), 6 , were pre-treated with 0.5 µg/ml actinomycin D for 20 hours, the cells combined with 1.5×106 Wal2A rho zero cells, and fused using a pH-balanced PEG-1450 mixture for 60 seconds. Cells were allowed a 5 day post-fusion recovery period in rho zero growth medium and then selected in a 1 µg/ml 6-thioguanine-supplemented DMEM medium containing 10% dialyzed serum solution for selection. Media were replaced every 4 days or when the media looked depleted until such time as the cells achieved normal growth rates, as determined by periodic cell counts. The cybrids were haplotyped post-fusion to determine successful transfer of the mtDNA. The presence of Wal2a nuclear background was confirmed by microsatellite testing and comparison to the original lymphoblast and Wal2A microsatellite identities. The haplogroup assignment of the cybrid clones was determined using haplogroup specific polymorphisms and by sequencing of the mtDNA control region The Wal2A rho zero nuclear donor cell line, which had been cured of its resident mtDNAs with ethidium bromide treatment TsC was used for normalization of transcript expression levels. Primers for the real time PCR reactions were designed using the Primer 3 software (2O in a 20 µl reaction volume within 384-well plates (Genesee Scientific and Roche). For amplification, reaction mixtures were incubated for 2 min at 50°C and 10 min at 95°C, followed by 45 cycles of three-steps consisting of 15 seconds at 95°C, 20 seconds at 60°C and 15 seconds at 72°C. Following the PCR step, samples were heated from 60°C to 95°C with a ramp time of 20 min to construct dissociation curves and verify that single PCR products were obtained. PCR products were also analyzed by gel electrophoresis to confirm primer specificity. Serial cDNA dilutions were used for primer validation experiments to demonstrate that both target and reference genes had equal amplification efficiencies, according to the standard curve method. The comparative TC method was used for relative quantification of gene expression as described by the real time PCR machine manual. Differences in the CT values (dCT) of the transcript of interest and reference genes were used to determine the relative expression of the gene in each sample. The dCT method was used to calculate fold expression. Experiments were carried out in triplicate for each data point. Roche analysis software versions 2.1 (Applied Biosystems) and Microsoft Excel were used for data analysis.mtDNA transcript levels were determined for each of the cybrids by PCR incorporation of SYBR Green dye into real time PCR products. The rate of dye incorporation was monitored using the Roche LightCycler 480 real-time PCR system. Target genes were normalized according to three reference gene products, namely GAPDH, β-Actin and β2-Microglobulin software . Four miT values of the ND2 and 18S rRNA genes were plotted against this standard curve to provide an absolute copy number level for each gene. The ND2 to 18S rRNA ratios were then calculated for each of the cybrid DNAs. Primers used for amplification of the ND2 and 18S rRNA genes are listed in mtDNA copy number calculations were performed by comparing the ratio of the mtDNA-encoded ND2 gene levels to the nuclear DNA-encoded 18S rRNA gene using real time quantitative PCR amplification (Roche LC480) in three independent experiments per tested DNA. To quantify each gene product, serial 10× dilutions of known cloned PCR fragments were used to create a standard curve. C


CEA mRNA was significantly more often detected patients with MTC (72.7%) than in controls . With an example of a patient with MTC and massive tumour mass in the neck we demonstrate the failure of detection of CT mRNA over a period of 6 months, whereas CEA mRNA could be detected in peripheral blood of this patient. As a consequence, CT mRNA detected by RT-PCR in the peripheral blood can not be recommended as a tumour marker in MTC. However, the use of carcinoembryonic mRNA may provide a significant improvement in diagnosis of recurrent disease in MTC. © 2001 Cancer Research Campaign   http://www.bjcancer.comDetection of local relapse or metastasis in medullary thyroid carcinoma (MTC) continue to pose a major diagnostic challenge. Besides established diagnostic studies such as serum calcitonin (CT) and carcinoembryonic antigen (CEA), molecular detection of circulating tumour cells may be an additional diagnostic tool for the early detection of disease recurrence. We performed reverse transcription-polymerase chain reaction (RT-PCR) on blood samples from patients diagnosed with MTC disease using primers specific for CT and CEA, respectively. CT mRNA was not detectable in peripheral blood of all patients with MTC (


During a 10-day dazoxiben treatment period, persistent erythromelalgia was associated with a significant shortened mean platelet life span of 3.2 days. During subsequent treatment with low dose acetylsalicylic acid daily complete relief of erythromelalgia was associated with inhibition of platelet prostaglandin endoperoxide production and correction of platelet mean life span to normal, 7.9 days. These observations indicate that prostaglandin E2, or another prostaglandin endoperoxide metabolite, is involved in the pathogenesisof erythromelalgia. The presented study does not give one single clue as to the origin  of the prostanoid, but very likely originates from platelets because a very low dose of acetylsalicylic acid (250 to 500 mg every other day), which irreversibly inhibits platelet cyclooxygenase, is highly effective in the prevention of erythromelalgia in thrombocythaemia.Fluid of artificial blisters from erythromelalgic skin areas in primary thrombocythaemia contained a high amount of prostaglandin-E-like activity. Dazoxiben did not alleviate the erythromelalgia in patients with primary thrombocythaemia despite complete inhibition of platelet malondialdehyde and thromboxane B


Local control in malignant salivary gland tumours is dose dependent. High local control rates in adenoid cystic carcinomas could be achieved by highly conformal radiotherapy techniques and particle (neutron/carbon ion) therapy. Considering high doses are needed to achieve local control, all malignant salivary gland tumours probably profit from the use of particle therapy, which in case of carbon ion treatment, has been shown to be accompanied by only mild side-effects.The COSMIC trial is a prospective, mono-centric, phase II trial evaluating toxicity (primary endpoint: mucositis ≥ CTCAE°3) and efficacy  in the combined treatment with IMRT and carbon ion boost in 54 patients with histologically proved  salivary gland malignancies. Patients receive 24 GyE carbon ions (8 fractions) and IMRT (50 Gy at 2.0 Gy/fraction).The primary objective of COSMIC is to evaluate toxicity and feasibility of the proposed treatment in all salivary gland malignancies.Clinical trial identifier NCT 01154270 Malignant salivary gland tumours are rare and a heterogenous group of tumours accounting for about 3-5% of head and neck cancers. High-grade tumours such as mucoepidermiod carcinoma (35%) and adenoid cystic carcinoma 25%) are the most common histological subtypes , charact5% are thRegarding radiation therapy, local control was also significantly improved by the application of high-precision techniques, dose-escalation and high-LET radiation -13.Intensity-modulated radiation therapy (IMRT) as well as fractionated stereotactic RT could already improve local control as compared to conventional RT techniques achieving 3-year PFS rates of 38% .So far, the highest local control rates at 75 - 100% ,13 were Dose escalation  though oIn view of the outcome and low toxicity profile, photon IMRT plus C12-boost has been accepted as the standard treatment and method of choice for adenoid cystic carcinoma in Germany. Due to the possibility to apply higher doses at so far mild side effects, charged-particle therapy does promise improved results for all types of malignant salivary gland cancers.Therefore, this trial basically evaluates relative dose escalation with simultaneous normal tissue sparing by using a combination of C12-heavy ion therapy and IMRT in all salivary gland malignancies including mucoepidermoid carcinoma and adenoid cystic carcinoma. Looking at other head and neck tumors i.e. squamous cell carcinoma of the head and neck (SCCHN), radiation therapy commonly causes pronounced mucositis. With therapy usually about 7 weeks duration and mucositis, improvement of clinical symptoms can only be expected at least 2-3 weeks post completion of therapy. At the occurrence of mucositis CTC °III, pain, loss of taste, and dysphagia causing reduced food/fluid intake and weight loss can potentially cause treatment delays, protraction of treatment and worse outcome. Therefore, evaluation of this particular side effect was chosen as the primary endpoint of the trial. Trial objectives hence assess accompanying acute and late toxicity and focusing on mucositis CTC °III. Secondary endpoints consist of evaluation of efficacy  and toxicity (other than mucositis).Initial results of the pilot project at the GSI/Darmstadt (treatment of skull base ACC) reported mucositis CTC °III or higher in only 6% of cases, with a recent update confirming these findings . AssuminThe COSMIC trial is a prospective, non-randomized phase II feasibility trial evaluating acute mucositis ≥ CTCAE °III (v. 3.0) as the primary endpoint. Mucositis CTCAE °III is defined as confluent ulcerations or pseudomembranes within the oral cavity and bleeding with minor trauma leading to the inability of the patient to adequately aliment or hydrate orally.Combined IMRT and C12-heavy ion boost has previously been established as a treatment of choice in skull-base adenoid cystic carcinoma. However, based on the fact that local control seems to be dose-dependent, all types of salivary gland malignancies in the head and neck will probably benefit from high-LET radiation. Therefore, the combination of IMRT (50 Gy in 2 Gy/fraction) and C12-boost (24 GyE in 3 GyE/fraction) will be tested as to toxicity profile and efficacy.To evaluate feasibility and toxicity (with a focus on mucositis CTCAE °III (v. 3.0)) and efficacy of the treatment.Incidence of mucositis ≥ CTCAE°III (v. 3.0) will be assessed as the primary endpoint of the trial, local control, disease-free survival, toxicity (incl. mucositis CTCAE °I-II and late toxicity at 3 years post RT)Incidence of mucositis CTCAE °III in the initial data was 6% for adenoid cystic carcinoma mostly of the skull base. 29 patients with adneoid cystic carcinoma were treated with 54 Gy IMRT and 18 GyE carbon ions, two of these patients (i.e. 6.9%) showed mucositis CTCAE °III. Inclusion and exclusion criteria in COSMIC correspond to the initial patient selection; hence incidence of toxicity should also roughly correspond. Agreeing to an incidence of 6%, 49 patients have to be treated in order to observe at least one patient with mucositis CTCAE °III or higher with a probability of 95%. Assuming a drop-out rate of 10%, 54 patients need to be recruited.• Histologically confirmed or surgically removed malignant tumour of the salivary glands (head and neck)• Inoperable tumour• G2/3• Macroscopic or microscopic residual tumour (R2/R1) or• ≥T3/T4 or• perineural invasion (Pn+)• written informed consent• pts aged 18 - 80 years• effective contraception for pts in childbearing age (< 12 months post beginning of menopause)• Prior radio- or chemotherapy for tumours of the head and neck• Other previous malignancy within the past 5 years except prior, adequately treated basal cell carcinoma of the skin or pre-invasive carcinoma of the cervix• Significant neurological or psychiatric condition including dementia or seizures or other serious medical condition prohibiting the patient's participation in the trial by judgement of the investigators• Legal incapacity or limited legal capacity• Positive serum/urine β-HCG/pregnancy• Drug abusePatients are immobilized using individual thermoplastic head masks with thermoplastic shoulder fixation. Planning examinations consist of a planning CT scan (3 mm slice thickness) with the patient positioned in the individual fixation device and contrast-enhanced MRI for 3D image correlation.CTV1 (carbon ion boost) includes the macroscopic tumour/prior tumour bed with special focus on the R1-area as well as respective neural pathways to the base of skull . For tumours of the parotid gland, the whole former parotid area is also included in the CTV1, if possible the mandibular joint is kept outside the CTV1. PTV1 consists of a 3 mm margin around the CTV1 but does not extend into critical organs at risk .We prescribe a dose of 24 GyE carbon ions in 3 GyE/fraction (5 fractions per week) to the CTV1, we aim at covering the CTV1 with the 95% prescription isodose. Treatment is given at the HIT (Heidelberg ion therapy centre) after inverse treatment planning in active beam application (raster-scanning method). Daily image guidance consists of orthogonal x-ray controls in treatment position.CTV2 includes CTV1 with safety margins along typical pathways of spread. Only ipsilateral nodal levels (II and III) are includes, however, in case the primary tumour is/was located at midline or crossing midline, we cover bilateral nodal levels II and III. In case there is a pathological lymph node involvement, additional nodal level will be covered as indicated. CTV2 also encompasses the complete surgical operational area. The CTV2 also takes account for set-up variations, hence corresponds to the PTV2 (CTV2 = PTV2). Should the primary tumour be located within the parotid gland, also the parotid duct needs to be within the CTV2.50 Gy IMRT (inversely planned step-and-shoot or tomotherapy technique) in 25 fractions (5 fractions per week) are prescribed to the CTV2 (coverage at least with the 90% prescription isodose) taking into account doses applied by daily image guidance with MV-cone-beam CT.Patients do not routinely receive prophylactic feeding tubes, however if they uncommonly experience significant weight loss we will of course offer feeding tube insertion or parenteral feeding.After obtaining written informed consent, patients are included into the trial and receive their treatment planning investigations. Treatment usually starts with 8 fractions carbon ion (8 × 3 GyE C12) therapy followed by 27/28 fractions of IMRT corresponding to a total dose of approximately 74 GyE. Treatment duration is approximately 6-7 weeks , therapy-associated side effects as well as potential intercurrent therapy is recorded.Patients are also encouraged to undergo regular check-ups incl. full ENT clinical examinations in regular intervals.st and 2nd follow-up visit. Thus, the overall volume of blood samples used for Proteomic/Genomic investigations will be approximately 200 mL. Following parameters/pathways will be investigated:For the proteomic examinations 30 mL venous blood will be collected from each subject prior to RT, at day 29, day 44 or 45, and at the 1• In order to predict the efficacy of the therapy blood will be collected during therapy and follow-up to detect and correlate the levels of well known tumour- and angiogenesis markers  using Enzyme-Linked Immunosorbent Assay (ELISA).• Further, platelet protein content (i.e. tumor angiogenesis growth factors and cytokines) will be analyzed using citrate blood samples and correlated with serum- and plasma- protein results.In order to perform the genomic analysis, patients' blood samples are collected as indicated and RNA, miRNA and DNA isolation will be performed. Based on an established platform, linear RNA-amplification, labelling and hybridization on human genome wide oligo-arrays  are planned. DNA samples are used to identify potential chromosomal aberrations or epigenetic alterations that might predict treatment response. RNA and miRNA samples are further analyzed by real time quantitative RT-PCR to confirm microarray data and to test a subset of clinical predictors.The determinations of proteomic and genomic parameters will be carried out at the Department of Radiation Oncology in Heidelberg. No further genetic investigations on the blood collected during the study will be carried out.Assessment of efficacy will be carried out by evaluation of imaging studies (MRI) at each follow-up. If applicable  tumour response will be evaluated according to the RECIST-criteria (19).The COSMIC trial has been designed by the Department of Radiation Oncology, University of Heidelberg, and is carried out at the Heidelberg Ion Therapy Centre (HIT). It is an investigator-initiated trial; the Department of Radiation Oncology is responsible for co-ordination, overall trial management, registration , database management, quality assurance, monitoring, and reporting.Patients are recruited by the Department of Radiation Oncology.Adverse and serious adverse events are recorded using NCI common toxicity criteria for adverse events (CTCAEAE v. 3.0). Acute radiation effects are defined as effects occurring within 90 days from beginning of radiotherapy. Late effects are defined as effects observed thereafter. Safety analysis is performed with respect to frequency of serious adverse events and adverse events stratified by organ system, severity, causality.Patient accrual is completed with inclusion of the last patient and should extend for approximately 2 years from trial initiation. Regular trial participation for each patient terminates 3 years post inclusion into the trial or the patient's death respectively.• Patient wish• Medical condition necessitating treatment termination and withdrawal of the patient from the trial• Pregnancy• Lack of complianceThe trial can be prematurely closed or suspended by the LKP in following cases:• Medical or ethical reasons relevantly affecting the risk-benefit relationship,• Difficulties in recruitment of subjects suggest unjustifiable prolongation of the study timeline,• Previously unexpected adverse events  occur with unjustifiable frequency,• Expected adverse events occur with an unexpectedly high incidence,• Relevant superiority of patients in one treatment arm of a comparable clinical trial,• Legal authorities' decisionThe Ethics Committee (EC) and the competent regulatory authorities will be informed about premature closure of the trial. Furthermore, the Ethics Committee(s) and competent regulatory authorities themselves may decide to stop or suspend the trial.If the trial is closed prematurely, the trial material such as completed, partially completed, and blank CRFs will be returned to the coordinating investigator.All involved investigators have to be informed immediately about a cessation or suspension of the trial. The decision is binding on all trial centers and investigators.The final protocol was approved by the ethics committee of the University of Heidelberg Medical School (S-263/2009). The trial complies with the Helsinki Declaration in its recent German version, the Medical Association's professional code of conduct, principles of Good Clinical Practice (GCP) guidelines and the Federal Data Protection Act. It will be carried out in keeping with local legal and regulatory requirements. It is also subject to authorization by the German radiation protection authority (Bundesamt für Strahlenschutz: = BfS). Medical confidentiality and Federal Data Protection Act will be followed. Written informed consent is obtained from each patient in oral and written form.Achieving local control in malignant salivary gland tumour remains challenging. Progress has been made by the introduction of novel radiotherapy techniques like fractionated stereotactic RT and IMRT, however, particle therapy (neutron and carbon ion therapy) result in the highest control rates in adenoid cystic carcinoma so far. Compared to neutron RT, carbon ion treatment comes at the cost of only mild side effects and is now permanently available at a new dedicated particle therapy facility (HIT). Considering the fact local control in salivary gland tumours is dose dependent, we believe treatment outcome can be improved for all malignant salivary gland histologies without increasing side-effects by a combination of IMRT (50 Gy) with a carbon ion boost (24 GyE) not only in incompletely resected or unresectable tumours but also in the R1-situation or perineural infiltration. The COSMIC trial was designed to address these issues in a prospective phase II trial. To our knowledge this is the first phase II trail combining particle therapy and IMRT.The authors declare that they have no competing interests.ADJ, MWM, CWK, and JD developed the study protocol and planned the trial. CWK is responsible for statistical considerations/basis of the trial. ADJ, AN, MWM are responsible for conducting and co-ordination of the trial as well as patient recruitment. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/546/prepub


Rat ascites hepatoma AH109A cells (present as a free form in vivo) can aggregate and then develop well-defined tripartite junctional complexes, including intermediate junctions, desmosomes and focal tight junctions, on incubation with a glycoprotein separated from rat ascites hepatoma AH136B cells (forming cell islnds in vivo). The development of binding structures was strongly inhibited by actinomycin D. AH109A cells or rat ascites hepatoma YS cells (present as a free form in vivo) previously treated with the glycoprotein for 24 h, when inoculated i.p., proliferated as free cells in the ascitic fluid, like the untreated cells. AH109A cells actively proliferating in the skin do not form any junctional complexes. The reason for the failure of island formation by AH109A cells or YS cells in vivo is discussed.


However, no information regarding the effects of EPO therapy on the behavior of the general mononuclear-lymphocyte population, Tregs or Th17 cells in EAE has emerged.Beneficial effects of short-term erythropoietin (EPO) therapy have been demonstrated in several animal models of acute neurologic injury, including experimental autoimmune encephalomyelitis (EAE)-the animal model of multiple sclerosis. We have found that EPO treatment substantially reduces the acute clinical paralysis seen in EAE mice and this improvement is accompanied by a large reduction in the mononuclear cell infiltration and downregulation of glial MHC class II expression within the inflamed CNS. Other reports have recently indicated that peripherally generated anti-inflammatory CD435–55 induced EAE. We then determined the effect in vivo of EPO on numbers of peripheral Treg cells and Th17 cells. We found that EPO treatment modulated immune balance in both the periphery and the inflamed spinal cord by promoting a large expansion in Treg cells, inhibiting Th17 polarization and abrogating proliferation of the antigen presenting dendritic cell population. Finally we utilized tissue culture assays to show that exposure to EPO in vitro similarly downregulated MOG-specific T cell proliferation and also greatly suppressed T cell production of pro-inflammatory cytokines.We first determined in vivo that EPO therapy markedly suppressed MOG specific T cell proliferation and sharply reduced the number of reactive dendritic cells (CD11c positive) in EAE lymph nodes during both inductive and later symptomatic phases of MOGTaken together, our findings reveal an important new locus whereby EPO induces substantial long-term tissue protection in the host through signaling to several critical subsets of immune cells that reside in the peripheral lymphatic system. In this study, we characterized the effect of EPO on immunogenesis within the peripheral lymphoid tissues and the impact of EPO therapy on infiltrating inflammatory cells within the CNS target organ using the EAE mouse model. We found that EPO therapy exerts unexpectedly profound biologic effects within the peripheral lymphoid system tissues and acts as a potent molecular check on inflammation by blocking dendritic cell expansion and T cell proliferation while inducing rapid expansion of the Treg cell population and a corresponding reduction in Th17 cells. This comprehensive immune modulating mechanism likely accounts for the bulk of the EPO neuroprotective effects induced in the EAE animal model.40–54 and MOG44–54) have been used to construct peptide/H-2Db dimeric complexes to quantify the encephalitogenic CD8 T cell subsets 35–55 immunized mice could be quantified by flow cytometry. As expected, less than 0.5% of normal mouse spleen mononuclear cells stained positive with the MOG44–54/H-2Db dimer  for 48–72 hrs. Culture supernatants were collected for cytokine quantification and the remaining cell population was reacted with peptide/dimer complexes and anti-CD8 antibody to determine the number of MOG specific T cells. EPO (1 U/ml) induced a substantial reduction (*p<0.005) in major pro-inflammatory cytokines  . A mild 35–55 immunization) was also effective in delaying disease onset and reducing disease severity 44–54 dimer binding and EPO treatment significantly reduced the MOG-specific T cell population in EAE mice  or PBS sham treatment over the same period for a total of 6 days. DILNs and spleens were collected from mice on day 7. We next determined if EPO had similar immunomodulating effects on the peripheral lymphoid tissues when EPO treatment was started at the same time as the mice received MOG peptide immunization. Mice were immunized with MOGBrief treatment with EPO during the EAE induction period for 6 days significantly delayed onset of clinical neurologic deficit compared to sham treated EAE mice (P<0.05). This brief course of EPO treatment induced a neuroprotective effect that was sustained over a long term clinical follow-up period of 45 days . Both Tr40–54-specific T cells in EAE animals after treatment with either EPO or sham PBS, fresh MNCs from DILNs of MOG-immunized mice were obtained at different time points after initiation of either EPO or sham PBS treatment. We found sustained reductions in Treg cells during the early EAE induction period, the symptomatic phase as well as the late chronic phase in sham treated EAE animals compared to normal controls (p<0.01). The reduction in peripheral Treg cells in sham treated EAE was more apparent on day 7  and on day 16  than later in the disease course. EPO treatment induced a significant expansion in Treg cells within the peripheral lymphoid tissue that could be detected at a very early stage  on paraffin-embedded spinal cord sections. During the EAE induction phase, we found increased numbers of inflammatory cells within spinal cords shortly before onset of paralysis in sham treated EAE mice (about day 7–10), In contrast far fewer infiltrating inflammatory cells were found in spinal cords from EPO treated EAE mice. The number of CNS infiltrating inflammatory cells was 4–5 times greater in sham treated EAE mice compared to EPO treated animals when animals were studied either at their peak clinical neurologic deficit or at later stages of EAE (day 45). Detailed analysis of the inflammatory cells infiltrating the CNS again revealed higher numbers of Treg cells in EPO treated animals . The incSignificantly increased Th17 cells within the CNS were found at the time animals developed clinical disease, and higher numbers of Th17 cells in spinal cords were notably associated with more severe clinical neurologic deficits in EAE animals. EPO treatment reduced infiltrating Th17 cells in the diseased CNS  as previEAE is a commonly studied animal model of acute central nervous system inflammatory demyelinating disease, and it is believed that autoreactive T cells and proinflammatory cytokines play key roles in the early inductive phase and later paralytic stages of EAE pathogenesis 35–55 has been previously used to identify the key peptide epitopes for pathogenic T cells and two major MOG peptide epitopes within MOG40–54 have been found to be pathogenic for EAE induction 44–54/H-2Db specific CD8 T cells, while much higher doses (100 U/ml) had only minor or no effect. A similar J-shaped dose-response curve following EPO exposure has been previously observed in MHC class II expression on a mouse EOC microglial cell line Traditionally CD4 T cells have been considered to be the critical element in EAE pathogenesis, but recent studies have indicated that CD8 cells may also play an important role in EAE induction and disease progression 44–54 dimer positive pathogenic T cells. There were no significant alterations in Th2-type cytokines such as IL-4 and IL-5 in EPO treated samples compared to controls. The in vitro EPO-mediated reduction in proinflammatory cytokines was also dose dependent.Since proinflammatory cytokines, especially IL-2, IL-6 and TNF-α, play significant roles in EAE pathogenesis The EPO-mediated non-specific and specific immune modulating effects on peripheral lymphoid tissues in EPO-treated EAE animals likely contributes to their less severe clinical course. Anti-inflammatory regulatory T cells have been found to be an important CD4 cell subpopulation for controlling the development of autoimmune disease 35–55 induced EAE mice was very similar to the Th17 cell kinetics following proteolipid protein 139–151 peptide induced EAE in SLJ/J mice 40–54 specific T cells, reside mainly in the peripheral lymphatics during the EAE induction phase after immunization. Later, when increased numbers of infiltrating inflammatory cells invade the CNS (12–20 days post MOG-immunization), Th17 cells, together with MOG-specific T effector cells, were easily detected in diseased spinal cords. EPO treatment abrogates the proliferation of peripheral Th17 cells during both the early induction phase as well as at later stages of EAE, and EPO treatment inhibits Th17 cell formation within the spinal cord during both acute and convalescent phases of EAE.The other important subpopulation of CD4 helper T cells recently recognized to be a critical player in inflammatory autoimmune disease pathogenesis is the IL-17 cell. IL-17 is a potent proinflammatory cytokine and overproduction of IL-17 has been associated with a variety of human autoimmune diseases and their corresponding animal models. The kinetics and organ distribution of Th17 cells in our MOGIt is unknown by what mechanism EPO suppresses expansion of the Th17 cell subset in EAE animals. Since IL-6 has been reported to promote differentiation of naïve CD4 T cells into Th17 cells in vitro, the level of IL-6 secretion by inflammatory cells could be playing a crucial role for in vivo development and expansion of Th17 cells The mechanism and precise signaling pathway by which EPO suppresses the immune response in the peripheral lymphoid tissues is also unknown. Since CD 11c expressing dendritic cells are pivotal in the control of developing immune responses, it may be that the EPO treatment induced massive 20-fold reduction in lymphoid dendritic cells coupled with marked down-regulation of MHC II expression on the surface of mononuclear cells in EAE is sufficient to block full development of the autoimmune process. This notion is supported by reports on 3 genetic conditions of mice where excessive DCs accumulate in the lymphoid tissues ). These animals have greatly expanded dendritic cell populations; show exaggerated immune responses and subsequently develop systemic autoimmunity Beneficial therapeutic effects of exogenously administered whole molecule EPO have been described in several animal models of neurologic injury, including occlusive cerebral vascular disease, acute brain trauma, epilepsy, as well as in both autoimmune arthritis and EAE Other EPO molecular preparations, such as an asialo-form of EPO, carbamylated EPO (CEPO), or certain EPO mutants, have been reported to have protective effects in animals following experimental traumatic spinal cord injury or acute stroke without provoking an increase in red cell mass One of the surprising newly recognized consequences of EPO therapy in humans that has emerged over the past several years has been the unexpected negative effect of EPO therapy on survival in cancer patients In conclusion, we investigated the mechanism behind the neuroprotective effects of EPO in classic autoimmune EAE animals and found that EPO has striking pleiotropic immunomodulatory effects on the peripheral lymphatic tissues as well as on the CNS target organ. EPO appears to restrain the overwhelming autoimmune response in MOG-immunized EAE animals and restores it to a more balanced immune status. Our studies suggest that multiple alterations including a reduction in total autoreactive T cells, DCs, MHC expression as well as an alteration in the balance between Th17 cells and expanded Treg cell populations within peripheral lymphoid tissue and CNS all contribute to EPO-mediated neuroprotection.C57BL/6 (8–10 weeks old) female mice were purchased from Charles River Laboratories and maintained in a conventional facility. The studies were conducted in accordance with the Animal Component of Research Protocol guidelines at the VA Hospital, East Orange, NJ, US.35–55 dissolved in distilled water and emulsified with an equal volume of complete Freund's adjuvant supplemented with 4 mg/ml mycobacterium tuberculosis H37Ra C57BL/6 mice were immunized subcutaneously at the tail base with 100 µl (200 µg) of MOG peptideEPO  in 2,000 U/ml vial stock was stored at 4°C. Administration of EPO was initiated either at the time of immunization or delayed until day 7 post-immunization. Animals were treated with intravenous EPO (5000 U/kg/day×3 days followed by 1000 U/kg/day×3 days) in freshly prepared saline or sham treated for 6 days with an equal volume of saline.At various time-points , animals were sacrificed, their draining inguinal lymph nodes and spleen removed for erythrocyte-depleted single-cell suspension preparations. The total MNCs were quantified subsequently. Mononuclear cells from the spinal cord were isolated following a published protocol by Korn T et al Cell surface staining of mouse single cell suspensions was obtained with mAbs directed against CD3, CD4, CD8, MHC class I, II and CD11c . Flow cytometry was performed on a BD FACScan machine (BD Biosciences). For intracellular IL-17 or Foxp3 staining, cells were first reacted with FITC-anti-CD4 or FITC-anti-CD4/APC-anti-CD25 , then washed, fixed and permeabilized following the manufacturer's recommendations. The PE conjugated anti-Foxp3 or anti-IL-17 Mab was subsequently added. The samples were subjected to flow cytometry after multiple washings. Flow cytometry data was analyzed using CellQuest software and the Flowjo analysis program.35–55 immunization. T cells were isolated from the single cell suspensions using MAC pan-T cell beads 35–55 peptide antigen. To determine the biological effect of EPO on antigen specific T cells and their secreted cytokines, the in vitro enriched MOG-specific T cells were washed three times with cold PBS and then incubated with log dilutions of EPO  in culture medium (RPMI1640 supplemented with 10% FCS) for 48–72 hrs. Control cells were incubated with medium alone. MOG44–54/H2-Db and control NP366–374/H2-Db dimers were freshly prepared by adding the truncated peptides to a recombinant soluble dimeric mouse H-2Db:IgG1 fusion protein . Cell culture supernatants were collected from each experimental sample well for cytokine profile analysis (below). Flow cytometry of either EPO treated cells or control cells was performed using dual-color staining with peptide dimer complexes and anti-CD3 mAb to determine the level of MOG specific effector T cells MNCs were obtained from EAE mice spleens 5–7 days post MOGLuminex technology was used to detect multiple cytokines and chemokines simultaneously within the pre-treated EAE T cell culture supernatants (above). Commercial kits  containing multi-cytokine beads  were run with their enclosed buffers and standards, following directions from the manufacturer. To obtain concentration values, mean fluorescence intensities from the bead combinations were analyzed by the MasterPlex QT quantification software.Spinal cords were obtained from sham or EPO treated MOG-EAE animals. Immunohistochemical staining for intracellular Foxp3 was performed using biotinylated Foxp3 antibody  and an avidin-biotin immunoperoxidase detection system on paraffin-embedded spinal cord sections.Data are presented as the mean±SEM. The Mann-Whitney U test was used to determine the significance of the intergroup difference in EAE clinical scores and duration of maximum neurological deficit. The composite data was analyzed by the Kruskal-Wallis one-way analysis of variance. Values of p<0.05 were considered significant.


Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants  and core bacterial determinants . An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect  Legionella pneumophila is a ubiquitous bacterium in natural aquatic environments that can also persist in human-controlled systems containing water, such as air conditioning and plumbing infrastructures. Intracellular growth in protozoa can permit this pathogen to survive chlorination, and the generation of aerosols in these systems contributes to the transmission to humans where infection of alveolar macrophages results in respiratory illness  is the gold standard for diagnosis of Legionnaires' disease but rapid detection of L. pneumophila serogroup 1, the most common subtype associated with human disease, can be achieved with immunofluorescence and serological assays targeting L. pneumophila antigen and L. pneumophila-specific antibodies, respectively  scheme for the molecular subtyping of L. pneumophila strains  or positive immunofluorescence antibody serology (standing titre of ⩾1 : 256 or fourfold seroconversion of ⩾1 : 128).The case definition of Legionnaires' disease was chest X-ray confirmed pneumonia, clinical diagnosis or pathological evidence of pneumonia at autopsy, and living or working within 3 kilometres (1.9 miles) of the outbreak site since a month prior to the outbreak. Each case of Legionnaires' disease was definitively confirmed after a positive culture for Legionella strains were recovered from clinical specimens and environmental samples as described by Legionella spp. from environmental samples were first selected using buffered charcoal yeast agar (BCYE) plates supplemented with polymyxin B, anisomycin and vancomycin (PAV). Colonies resembling Legionella spp. on the BCYE-PAV plates were then differentiated using BCYE, BCYE (lacking cysteine) and blood agar plates. Isolation of Legionella spp. from lung samples was performed using solely BCYE plates. All incubations were performed at 37 °C in candle jars or CO2 incubators. Lung autopsy materials were transported in viral transport media, swabs and water samples were collected in sterile containers, and air samples were collected with a XMX-CV aerosol collection system (Dycor). Direct fluorescent antibody assay (DFA) analysis on clinical specimens were done using monoclonal or polyclonal DFA products directed towards L. pneumophila serogroups . Additionally, total DNA was prepared from 19 lung autopsy samples from 7 patients using the DNeasy tissue extraction kit (Qiagen).Taq polymerase (Invitrogen) per 25 ml reaction, and the following thermocycling conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 30 s ; and a one-round final extension at 68 °C for 5 min. DNA sequencing was performed with the oligonucleotides used to generate the amplicon. SBT using targets designed by the European Working Group for Legionella Infections (EWGLI) and amplified fragment length polymorphism (AFLP) analysis were performed as described by Oligonucleotides designed for molecular detection and typing are described in Table 1Legionella, Pontiac fever. A total of 79 people were hospitalized, of which 54 were residents at the outbreak site. This study describes the molecular detection of L. pneumophila in the lung autopsy material from the first mortalities, and the subtyping of L. pneumophila strains subsequently isolated from clinical and environmental samples.An outbreak of Legionnaires' disease at an extended care facility in Scarborough, Ontario, Canada, from September to October, 2005 resulted in 23 deaths. All mortalities occurred with residents of the care facility  and additional cases were identified in 47 residents, 39 staff and 21 visitors to the outbreak site, as well as in 5 occupants and staff of a neighbouring care facility and residence . A total of 82 people were affected with symptoms resembling Legionnaires' disease and 53 people presented with the milder form of illness cause by L. pneumophila in lung tissue DNA extractions and subtyping of pure cultures was achieved using species-specific oligonucleotides  and with the other publicly available bacterial sequences. Oligonucleotides corresponded to conserved regions amongst the three L. pneumophila genomes, but were unique compared to other genera and/or species (data not shown). The target loci included Legionella-specific virulence determinants  and core bacterial determinants . These core bacterial determinants have been predicted to contain species-specific polymorphisms  and human β-globin locus were used as additional control reactions  using ftsZ, trpS, dnaX and mip L. pneumophila-specific primer sets; control reactions included 293T tissue culture cells spiked with Legionella micdadei ATCC 33218 or spiked with L. pneumophila ATCC 33153 . The two other lung specimens from patient 3 were positive for L. pneumophila and the control PCR reactions for specimen 05-L-025 failed, indicating that DNA extraction was likely unsuccessful for this individual specimen. DFA testing on one lung autopsy specimen from each patient was positive for L. pneumophila serogroup 1, whereas L. pneumophila was cultured from lung specimens for six of the seven patients.PCR-based detection assays for L. pneumophila directly in environmental samples failed, including those that ultimately were cultured for this organism. The amount of L. pneumophila DNA extracted from these samples was likely below the detection threshold for these methods, and therefore these methods might be limited to analysis of pure culture and clinical samples. The only environmental samples that yielded L. pneumophila isolates were associated with the cooling towers at both the outbreak and adjacent sites  demonstrated that isolates from the cooling tower at the outbreak site were related to isolates from lung autopsy specimens, but divergent from isolates recovered from an adjacent building and reference strains. Notably, AFLP analyses distinguished two clones amongst the human and environmental outbreak-associated isolates suggesting increased sensitivity for this method; however, the nature of the observed genetic diversity was undefined. The genetic loci developed in this study for SBT effectively determined the relatedness of clinical and environmental samples, but the established EWGLI SBT protocol was of increased utility because it similarly differentiated the L. pneumophila strains but additionally allowed for comparison to a global collection of SBT data deposited in a web-accessible database.Molecular reagents were developed during this outbreak of Legionnaires' disease to supplement bacterial culture and immunological detection methods for


SILV) gene plays a major role in melanosome development. SILV is a target for studies concerning melanoma diagnostics and therapy in humans as well as on skin and coat color pigmentation in many species ranging from zebra fish to mammals. However, the precise functional cellular mechanisms, in which SILV is involved, are still not completely understood. While there are many studies addressing SILV function upon a eumelaneic pigment background, there is a substantial lack of information regarding the further relevance of SILV, e.g. for phaeomelanosome development.The silver homologue(SILV expression exclusively in pigmented tissues, our experiments provide evidence that the bovine SILV gene is expressed in a variety of tissues independent of pigmentation. Our data show that the bovine SILV gene generates an unexpectedly large number of different transcripts occurring in skin as well as in non-pigmented tissues, e.g. liver or mammary gland. The alternative splice sites are generated by internal splicing and primarily remove complete exons. Alternative splicing predominantly affects the repeat domain of the protein, which has a functional key role in fibril formation during eumelanosome development.In contrast to previous results in other species reporting SILV gene independent of pigmentation suggests SILV functions exceeding melanosome development in cattle. This hypothesis is further supported by transcript variants lacking functional key elements of the SILV protein relevant for eumelanosome development. Thus, the bovine SILV gene can serve as a model for the investigation of the putative additional functions of SILV. Furthermore, the splice variants of the bovine SILV gene represent a comprehensive natural model to refine the knowledge about functional domains in the SILV protein. Our study exemplifies that the extent of alternative splicing is presumably much higher than previously estimated and that alternatively spliced transcripts presumably can generate molecules of deviating function compared to their constitutive counterpart.The expression of the bovine  SILV) gene has been a target for many investigations concerning development of melanosomes, which are the specific pigment carrying compartments within melanophores, e.g. in melanocytes. In humans, SILV plays a major role in studies regarding melanoma diagnosis and therapy, because SILV is a sensitive melanoma marker on transcript and protein level [The silver homologue or phaeomelaneic (red) skin [Dilution locus in the Charolais breed (Dc). The putative role of SILV for various coat color dilution loci was described in a number of species, including mouse, dog, chicken, horse, and cattle [SILV gene in mouse, horses and dogs seems to be restricted to the dilution of eumelaneic pigment. This is underlined by experiments in mice indicating that SILV expression seems to be restricted to melanocytes expressing eumelanin [SILV gene [SILV gene that seems to affect the correct splicing of the gene in merle dogs [SILV gene may be specifically involved in melanosome development. Comprehensive studies in humans revealed that alternative splicing is a frequent mechanism altering spatial expression pattern and function of proteins [Coat color phenotype in mammals is dependent on a series of genes determining distribution of melanocytes , synthe, [MC1R, ), and in, [MATP, ). In cated) skin . Spotteded) skin . Similard cattle -16. Therumelanin . In cattumelanin ,18. FirsILV gene  and a rerle dogs  raise thproteins ,21.SILV gene in pigmented and non-pigmented tissues. Multiple transcript variants affecting functional key domains of the SILV protein indicate that the bovine SILV gene may serve as a model for investigations about alternative splicing to generate molecules of obviously deviating function compared to constitutive transcripts.In the present study, we present a comprehensive description of the complex expression pattern of the bovine SILV gene (GenBank: EF065525). No further promoters were detected. The SILV transcription start obtained in this study adds an additional 7 bp to the previously deposited bovine SILV cDNA sequence [SILV gene comprising 2046 bp organized in 11 exons  skin indicated a sharp peak of transcription start sites (TSS) at position -28 bp to the A of the translation start ATG of the sequence . The cDN01495046 ) indicatSILV cDNA from RNA of eumelaneic (black), non-diluted skin with primers in the 5' and 3' UTR of the SILV gene  of the bovine SILV gene  and exon 7 – 11 (E7F1 – E11R2). In contrast, only single RT-PCR fragments were observed for the primer combinations covering exon 1 – 3, exon 3 – 5, exon 5 – 6, exon 9 – 11 and exon 10 – 11. Notably, all identified fragments were present in all differentially pigmented skin samples investigated (Table SILV gene is not restricted to eumelaneic (black), non-dilute (Dcd/Dcd) skin, but also occurs in skin expressing exclusively phaeomelanin. Furthermore, also white skin sections of spotted individuals with eumelaneic or phaeomelaneic background showed SILV gene expression as well as the crème white skin of a eumelaneic homozygous dilute DcD/DcD individual.A series of PCR amplifications in SILV transcripts additional to the constitutive fragment that had been identified by RT-PCR were isolated from the agarose gel and sequenced to reveal the specific DNA sequence of the different fragments. Alignment of the obtained sequences to the reference constitutive cDNA (GenBank: EF065525) showed that the additional SILV transcripts lacked different parts of the SILV mRNA , three alternative transcripts (according to GenBank: EF065525) were characterized: Δ660–1340 lacking 681 bp corresponding to the entire exon 6, Δ816–1340 lacking 525 bp from the 3' end of exon 6 and Δ1185–1340 lacking 156 bp from the 3' end of exon 6. With primers spanning exon 7 to exon 11 (E7R1 – E11R2), we obtained an alternative transcript Δ1655–1748 lacking 94 bp from the 3' end of exon 9. Furthermore, sequencing of an additional RT-PCR transcript amplified with primers from exon 1 and exon 11 (5UTRF1 – E11R2) detected a SILV cDNA Δ498–1340/Δ1655–1748 lacking exon 5, exon 6 and 94 bp from the 3' end of exon 9  were detected. Sequencing of the respective clones revealed SILV cDNAs starting from the identified transcription start, but devoid of distinct other regions in the remaining SILV mRNA. On the one hand, we received transcripts confirming already detected alternative splice variants e.g., Δ498–1340, Δ660–1340, Δ816–1340, and Δ1185–1340. Additionally, we identified two further internal splice sites generating a transcript Δ105–362 (skipping exons 2 and 3)/Δ1332–1340 , non-dilute (Dcd/Dcd) skin with primers spanning exon 1 to exon 7 (E1F3 – E7R1). Alignment of the generated sequences to the SILV constitutive transcript confirmed previously detected transcripts and yielded an additional new series of transcripts  regardless whether on a eumelaneic or a phaeomelaneic background. Notably, non-pigmented white skin of a spotted, homozygous non-dilute (Dcd/Dcd) individual exhibited this splice site albeit at a lower level than the pigmented counterpart. Skin with a crème white coat color characteristic for individuals homozygous at the dilution locus DcD/DcD also displayed the alternative splicing Δ105-362. It has to be noted that crème white skin has an essentially pigmented background with melanocytes [In order to confirm alternative splice sites in the bovine ts Table . Splice ts Table . In contanocytes , howeverSILV gene in all 17 investigated tissues from an adult individual: pituitary gland, thyroid gland, kidney, adrenal gland, liver, lung, heart, brain, rumen, intestinal fat, subcutaneous fat, perirenal fat, mammary gland, duodenum, colon, jejunum, skeletal muscle. All three SILV primer combinations tested spanning exon 1 to exon 5 (E1F3 – E5R1), exon 5 to exon 7 (E5F1 – E7R1) and exon 9 to exon 11 (E9F1 – E11R2) confirmed this observation  revealed at least two PCR fragments for all tissues with a pattern similar to that obtained for differentially pigmented skin tissues.RT-PCR revealed a specific expression of the bovine While alternative splice variants were rather uniformly distributed across the differentially pigmented skins, substantial variation was seen for the other tissues Table . Only trSILV gene shows a strong structural homology with the respective gene in human and mouse. The bovine mRNA (GenBank: EF065525) is separated into 11 exons and revealed a homology of 87% to the human (NM_006928.3) and 83% to mouse mRNA (NM_021882.4). At protein level, the similarities were 78 or 73%, respectively. The sharp peak of transcription start sites detected in CAP finding RACE experiments and the putative TATA box of the SILV promoter located 30 bp upstream correspond to the classical pattern of TATA box promoter architecture characteristic for genes highly conserved in evolution [Dcd/Dcd) skin yielded essentially an identical transcription start compared to a SILV mRNA obtained by 5'RACE from a homozygous dilute (DcD/DcD), crème-white skin [The bovine volution . Our RACRNA NM_0282.4. At SILV expression in skin is not restricted to areas with eumelanocytes, because we also found SILV transcripts in phaeomelaneic and non-melaneic skin. This result is in contrast to previous reports in mice [SILV expression could only be detected in cells synthesizing eumelanin. Whereas there are many studies about the melanophore-specific expression of SILV in the literature , only oxpressed . This redatabase ). Althouynthesis . But as ynthesis , involvecated by , cattle SILV gene resulting in splicing of cryptic introns predominantly affecting exon 6, but also exons 2, 3, 4, 5 and exon 9. Thus, the splicing pattern comprises the use of single or combined cassette exons and alternative 5' splice sites resulting predominantly in deletions of single or multiple exons, which is in line with other comprehensive studies investigating alternative splicing [SILV cDNA with the nine cattle ESTs homologous to SILV, which are deposited in the Unigene database [SILV transcripts in cattle from our study are in line with previous experiments in humans describing two alternative splice sites in exon 6 and exon 9 of the SILV gene [SILV and indicates that the number of alternative transcripts in humans is also presumably higher than presently considered. Interestingly, humans and cattle are the only species for which alternative transcripts have been experimentally established [RT-PCR with an overlapping panel of primer pairs indicated nine alterative splice sites in the bovine splicing . Sequencdatabase , did notILV gene . The extILV gene ,30 colleablished ,29. No aSILV deletion constructs showed that a protein lacking the Repeat domain (ΔRPT SILV) showed an appropriate intracellular trafficking [SILV gene represent ideal candidates for further study into the potential effects of alternative splicing on function [Comparative analysis of the bovine SILV protein with the respective counterpart in human according  suggestefficking . Howeverfficking  used HMBfficking . The altfunction .SILV sequence that could convincingly be associated with phaeomelanin coat color dilution in a F2 resource population based on the Charolais and the Holstein breed [SILV gene that was associated with coat color dilution and concluded that the mutation might impair correct splicing of the canine SILV transcript. Thus, another as yet undetected mutation in the genomic sequence of the bovine SILV gene could possibly affect the structure of the SILV mRNA, representing the genetic background for phaeomelanin dilution in cattle. Because we could confirm SILV expression in eumelaneic and also phaeomelaneic skin, any genetic variant affecting regulation or coding structure of the gene might theoretically also affect phaeomelanin dilution in cattle, although presently there is no indication, which function SILV may exert on maturation of phaeomelanosomes. Thus, given our expression data the SILV gene could not formally be rejected as the background for phaeomelanin dilution in cattle. However, neither the transcription start, which was conserved between homozygous dilute (DcD/DcD) and non-dilute (Dcd/Dcd) individuals, nor the distribution of splice variants in the differentially pigmented skins, convincingly explained the differences in dilution phenotype. Hence, there is no indication that variation in the primary sequence of the SILV protein either due to variation in the coding sequence or due to alternative splicing is responsible for dilution of phaeomelanin in cattle.In cattle, we did not find any mutation in the coding in breed . In dogsin breed  describeSILV expression to the requirements of the respective cells [Whereas the pattern of transcript variants is rather similar across the panel of differentially pigmented skin, the variability of splice variants detected in other tissues points towards a tissue specific splicing mechanism. This may represent a tool for adapting ve cells .SILV gene is conserved across a variety of species including cattle, its pattern of transcription shows substantial differences regarding alternative splicing between cattle and human on the one side and mice on the other. Our experiments provide evidence for a ubiquitous transcription of the bovine SILV gene not restricted to pigmented cells and show a striking variety of alternative splice sites. These results indicate that potentially SILV may have functions exceeding melanosome development. This would have to be considered in future investigations concerning melanoma diagnostic and therapy and also in studies taking SILV as a model for amyloid formation [SILV as an adequate animal model.Although the structure of the ormation  or for iSILV transcripts are in line with the hypothesis of the new postulated additional SILV functions exceeding synthesis and deposition of melanin. This hypothesis is supported by the detection of SILV transcripts in non-melanophores in our study. However, it has to be considered that our analyses are restricted to the transcription level. Thus, subsequent steps modulating SILV expression due to e.g. nonsense mediated decay [SILV gene. Due to its extensive, up to now unique pattern of alternative splicing, bovine splice variants represent a naturally occurring model for the function of the domains located in the spliced regions of the SILV and for the alternative functions postulated for SILV. Furthermore, factors regulating the processes of alternative splicing in the different tissues can be investigated exemplarily.Whereas alternative splicing is a well established regulatory mechanism for gene expression , the coned decay  and tran2 cattle resource population [Dilution (Dc) responsible for coat color dilution, (ii) Extension (E) responsible for an eumelaneic (black) or a phaeomelaneic (red) phenotype, and (iii) Spotted (S) resulting in a pigmented-white spotting pattern similar to piebald in mice. We collected differentially pigmented neck skin after slaughter: (1) black and white sections of a homozygous non-dilute (Dcd/Dcd), spotted (s/s) eumelaneic (ED/Ee) animal, (2) diluted colored and white sections of an eumelaneic (ED/ED), heterozygous DcD/Dcd, spotted (s/s) individual, (3) diluted colored and white sections of a phaeomelaneic (Ee/Ee), heterozygous DcD/Dcd, spotted (s/s) individual and (4) the crème white skin of a homozygous non-dilute (DcD/DcD), non-spotted, eumelaneic (ED/ED) individual. Hair and subcutaneous tissue was removed and the remaining cutis was snap frozen. Genotypes of the individuals for the SILV c64G>A mutation, presumably responsible for dilution of eumelanin, were determined as described by [Extension locus were determined by sequencing of the MC1R gene. Primers MC1RF1 (5'-TACTACTTTATCTGCTGCCTG-3') and MC1RR1 (5'-GCGTAGAAGATGGAGATGTAG-3') flanking the causal mutations for the alleles ED  and Ee  were used for amplification in genomic DNA of individuals tested and consecutive sequencing of the resulting PCR products. In addition to the differentially pigmented skin samples, 17 tissues  from an adult female Charolais × German Holstein F2 individual were collected at slaughter and immediately snap frozen. Total RNA was isolated with the NucleoSpin® RNAII kit (Macherey and Nagel) except all fat tissues, which were isolated with the RNeasy Lipid tissues kit (Qiagen), essentially as described by the manufacturers.For our study, we included individuals from a Charolais × German Holstein Fpulation . The resribed by . Genotyp(12–18) and a gene specific primer from the 3'UTR of the SILV mRNA  was determined by RT-PCR as described [The isolated RNA was reverse transcribed into cDNA according to  with a p2–18 and escribed .SILV transcripts in differently pigmented skin and non-pigmented body tissues detected more than one RT-PCR fragment for several primer combinations. The respective fragments were excised from the agarose gel, purified using the Nucleospin Extract II kit (Macherey and Nagel) and sequenced on a capillary sequencer  using BigDye® (Applied Biosystems) chemistry. Two prominent RT-PCR fragments > 1000 bp generated with primers spanning exon 1 to exon 11  chemistry.The analysis of SILV gene and to test for alternative promoters, 5' RACE experiments with the GeneRacer Kit (Invitrogen) based on RNA ligase mediated and oligo-capping methods were performed. After CAP selection in a preparation of total RNA from black skin of a eumelaneic (ED/ED), non-dilute (Dcd/Dcd) individual, reverse transcription with oligo dT and re-amplification with the RACE 5' oligo and a SILV specific primer from exon 7 .To determine the transcription start site for the bovine SILV splice variants, SILV cDNA samples were investigated by splice site specific RT-PCR. For this purpose, SILV cDNAs were generated by reverse transcription with the E11R2 primer from total RNA of skins differing in coat color phenotype. Afterwards, the cDNAs were subjected to PCR amplification with one SILV primer corresponding to the sequence of the constitutive SILV mRNA and another primer specific for each splice variant, respectively . Analogous to the RT-PCR in skin tissue, the different RNAs were reversely transcribed and amplified by using primers corresponding to the constitutive SILV cDNA sequence in combinations covering exon 1 to exon 5 (E1F3/E5R1), exon 5 to exon 7 (E5F1/E7R1) and exon 9 to exon 11  was further investigated for alterative splicing by splice variant specific RT-PCR analogous to the procedure described for skin. The respective primer combinations are listed in Table The author(s) declares that there are no competing interests.CK conceived, designed and coordinated the study, carried out PCR analysis of RNAs, participated in sequence alignments and drafted the manuscript. RW participated in coordination of the study, participated in sequence alignments, carried out the RACE and splice variant specific experiments, and helped drafting the manuscript. All authors read and approved the final manuscript.