Combined treatment modalities using TRAIL and cytotoxic drugs revealed highly additive effects in different tumour cell lines. Little is known about the efficacy and underlying mechanistic effects of a combined therapy using TRAIL and ionising radiation in solid tumour cell systems. Additionally, little is known about the effect of TRAIL combined with radiation on normal tissues.TRAIL (Tumour cell systems derived from breast- (MDA MB231), lung- (NCI H460) colorectal- (Colo 205, HCT-15) and head and neck cancer (FaDu, SCC-4) were treated with a combination of TRAIL and irradiation using two different time schedules. Normal tissue cultures from breast, prostate, renal and bronchial epithelia, small muscle cells, endothelial cells, hepatocytes and fibroblasts were tested accordingly. Apoptosis was determined by fluorescence microscopy and western blot determination of PARP processing. Upregulation of death receptors was quantified by flow cytometry.The combined treatment of TRAIL with irradiation strongly increased apoptosis induction in all treated tumour cell lines compared to treatment with TRAIL or irradiation alone. The synergistic effect was most prominent after sequential application of TRAIL after irradiation. Upregulation of TRAIL receptor DR5 after irradiation was observed in four of six tumour cell lines but did not correlate to tumour cell sensitisation to TRAIL. TRAIL did not show toxicity in normal tissue cell systems. In addition, pre-irradiation did not sensitise all nine tested human normal tissue cell cultures to TRAIL.Based on the in vitro data, TRAIL represents a very promising candidate for combination with radiotherapy. Sequential application of ionising radiation followed by TRAIL is associated with an synergistic induction of cell death in a large panel of solid tumour cell lines. However, TRAIL receptor upregulation may not be the sole mechanism by which sensitation to TRAIL after irradiation is induced. Tumour necrosis factor related apoptosis inducing ligand) is one of the most promising anti-cancer agent being currently under investigation (for review see [TRAIL (view see -5). Initview see ,7. In thview see ,9. Howevview see . This isview see .in vivo. In this regard it has been shown that mice lacking TRAIL display a significantly reduced capacity to eliminate syngenic tumour cells in the liver [One possible explanation for the tumour specifity of TRAIL could lie in its potential role as a mediator of tumour immune surveillance he liver ,13.Tumour necrosis factor) or CD95. Most important in this regard is the fact that at least five, instead of only two resp. one, different TRAIL receptors have been identified: The proapoptotic DR4/Trail-R1 [Although TRAIL is a member of the death receptor ligand family certain differences exist to the well characterised ligands TNF . KeyWith only very few exceptions , apoptosInterestingly, both  pathways are interconnected on several levels. In case of death receptor activation, the propagation of the apoptotic signal is enhanced by caspase-8 mediated activation of Bid . Bid likAlthough TRAIL induces apoptosis when given alone, it has been shown that combination of TRAIL with cytotoxic drugs as 5-fluorouracil, etoposide, paclitaxel, actinomycin C and cisplatin has an even higher apoptotic efficacy -59,6.Whereas abundant data therefore support the combination of TRAIL with cytotoxic drugs, only limited studies of TRAIL combined with ionising radiation have been performed ,33,60-62All biochemicals were obtained from Sigma-Aldrich chemicals (Deisenhofen, Germany) unless otherwise specified. Hoechst 33342 was purchased from Calbiochem and dissolved in distilled water as 1.5 mM stock solution.2. Normal human epithelial cells (HMEC, PrEC, RPTEC, SAEC) and hepatocytes were obtained from Clonetics/Cambrex (Taufkirchen, Germany). Cell culture was performed according to the manufacturer's protocols.The tumour cell lines MDA-MB 231, HCT-15, Colo 205, NCI H460, FaDu DD and SCC-4 cells were purchased from ATCC (Bethesda, MD, USA). HSF6/ HSF7 fibroblasts, HUVEC and SMC were kindly provided from H-P. Rodemann and R. Kehlbach (Tübingen, Germany). Respectively. All cells were grown in RPMI 1640 medium (Gibco Life Technologies, Eggenstein, Germany) and maintained in a humidified incubator at 37°C and 5% COTRAIL induced apoptosis was induced with recombinant human TRAIL/TNFSF10 (R&D Systems, Wiesbaden-Nordenstadt, Germany) in concomitant or sequential application with irradiation.Cells were irradiated with 6 MV Photons using a Siemens Mevatron linear accelerator with a dose rate of 4 Gy per min at room temperature.Apoptosis induction was quantified by counting of cells with a characteristic apoptotic morphology after DNA staining with Hoechst 33342. Cells were stained by incubation with Hoechst 33342 at a final concentration of 1.5 μM for 15 min. Microscopy was performed using a Zeiss Axiovert 200 microscope (Carl Zeiss, Jena, Germany) using an excitation wavelength filter of 380 nm. All apoptotic rates were means of at least three independent experiments. The given error bars represent the standard error of the mean from independent measurements of the same cell batch.6) were lysed for 30 min in a lysis buffer containing 25 mM HEPES, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, 10 mM NaF and 125 mM NaCl on ice. After removing insoluble material by centrifugation for 10 min at 12.000 g, 20 μg lysate was separated by SDS-PAGE. Blotting was performed employing a tank blotting apparatus (Biorad, Munich, Germany) onto Hybond C membranes (Amersham, Braunschweig, Germany). Equal protein loading was confirmed by Ponceau S staining (Sigma). Blots were blocked in PBS buffer containing 0.05 % Tween 20 and 5% bovine serum albumin at 4°C over night. Primary antibodies were detected after repeated washings with PBS/Tween 20 (0.05%) of the membrane, using a secondary antibody (anti IgG-AP 1:10.000, Santa-Cruz-Biotech, Heidelberg, Germany) diluted in PBS/Tween and incubated for 3 hours at room temperature and washed three times with PBS/Tween. Detection of antibody binding was performed employing enhanced chemoluminescence (CSPD®-Solution Tropix, Applied Biosystems, MA, USA). PARP cleavage was tested using a polyclonal antibodies for cleaved and uncleaved PARP from Boehringer (Mannheim, Germany) in a 1: 1000 dilution. Monoclonal antibodies for caspase 8 were a gently gift from Prof. K. Schultze-Osthoff and used in a 1:45 dilution. β-Actin (Santa Cruz, Heidelberg, Germany) antibody was used in a 1: 5000 dilution.Cells (1 × 106) were washed twice with PBS and incubated for 30 min with PE-labeled anti-R1/DR4 or -R2/DR5-antibody (R&D Systems, Wiesbaden-Nordenstadt, Germany) at a dilution of 1: 400 in 0,5% FCS/PBS. FACS analyses of superficial receptor expression was performed according to manufacturer's protocol with the Quantibrite™ kit from BD (Heidelberg, Germany).Cells . No upregulation of DR4/R1 was found in all tested tumour cell lines. Instead, a subtle downregulation of DR4/R1 after irradiation was observed fig. . Fig. 4BAs stated above, hardly any data on normal tissue toxicity after combined treatment are available. We therefore analysed the effect of irradiation plus TRAIL on human hepatocytes, fibroblasts (HSF6 and 7), epithelial cells from prostata (PrEC), kidney (RPTEC), breast (HMEC) and small airways (SAEC), endothelial cells from umbilical cord (HUVEC) and small muscle cells (SMC). Normal tissue cells were treated with 10 Gy and a tenfold higher dosage of TRAIL (1 ng/ml) as used for tumour cells. For evaluation of apoptosis strict morphologic criteria as condensation of chromatin and nuclear fragmentation were used. 48 h after combined treatment no relevant sensitisation of normal tissue cells to TRAIL induced apoptosis could be detected in all cultures. Moreover, even preirradiation did not sensitise normal cells to TRAIL induced apoptosis as observed in tumour cells table .Based on the rationale that radiation and TRAIL induce cell death via distinct but overlapping cell death pathways, tumour cell lines and normal tissue cultures were subjected to either radiation or TRAIL alone or combined with varying application schedules. Our data show that combining radiation with TRAIL induces apoptosis in a significantly higher percentage than either treatment alone. It is important to note that for TRAIL stimulation in our experiments on tumour cells very low concentrations of TRAIL were used (0.1 ng/ml). The pharmacodynamic properties of TRAIL, especially the peak plasma levels in humans, are not known. Since it is likely that toxicity rises with higher doses of TRAIL, the observation of pronounced effects on tumour cell kill at such low doses is particularly intriguing.Any combination of radiation with TRAIL proved to be more effective than either one alone; however depending on dose level and schedule of stimulation less than additive, additive and synergistic effects were detectable. When TRAIL was applied simultaneously with irradiation three of the six cell systems reacted synergistically. In contrast, five of the six cell systems reacted with synergistic effects when TRAIL was given sequentially 12 hours after irradiation. One of the cell lines displayed only less than additive effects.Statistical analysis confirmed, that synergistic effects are more pronounced and occur with greater likelihood after sequential application of TRAIL after irradiation when compared to concomitant treatment schedules.-/- HCT-116 cells and mouse embryonic fibroblasts showing that NF-κB may also be important for a irradiation induced upregulation of death receptors [Possible explanations for the positive interaction of TRAIL and DNA damaging agents including ionising radiation are being disputed. Since the sensitisation was associated with upregulation of the DR5 receptor in some experimental settings ,64,68-70eceptors .Recently, it was shown in overexpression experiments of NF-κB that its subunits c-Rel and RelA regulate expression of cell death molecules in a differential manner. This suggests that RelA, in contrast to c-Rel, acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2 . This prRecently, it has been shown that the interaction of 5-FU with TRAIL is strictly Bax but not Bak dependent in HCT 116 cells . Thus, tHowever, the general mechanism of the positive interaction of TRAIL with irradiation remains unclear. It may ultimately turn out, that manifold mechanisms exist and only some mechanisms will be operative in a single cell system .The second part of our experiments the toxicity of TRAIL combined with radiation on normal tissues. In order to be able to draw relevant conclusions for normal tissue cells , 10 foldOur data suggest that TRAIL has great potential in cancer treatment, especially in sequential combination with radiotherapy. We did not observe any sensitising effect of the sequential treatment on TRAIL sensitivity in normal tissue cells. Xenograft experiments designed to answer the questions regarding the short and long term efficacy of a combination of radiation with either TRAIL or TRAIL specific antibodies are underway in our laboratory.APAF, apoptosis protease activating factor; FADD, Fas-associated death domain protein; NFκB, nuclear factor κB; PARP, poly-ADP-ribosyl polymerase; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand. HMEC: human mammary epithelial cells, PrEC: human prostate epithelial cells, RPTEC: epithelial cells of renal proximal tubule, SAEC: small airways epithelial cells, HUVEC: human epithelial cells of umbilical vein, SMC: smooth muscle cells, HSF 6,7: human fibroblastsThe author(s) declare that they have no competing interests.PM performed FACS-analysis and microscopic evaluation of Hoechst stained cells, analyzed the data, participated in the conception of the trial and participated in the preparation of the manuscript. AS participated in microscopic evaluation of tumour cell apoptosis and accomplished analysis of the resulting data. VJ participated in receptor analysis and microscopic evaluation of Hoechst stained cells. HF carried out Western blotting. PTD participated on the preparation of the manuscript. WB performed isobologramm analysis. CB participated in the conception, design of the study, coordination of the study as well as preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe.As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs.Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species. Microarrays are routinely used for large scale transcriptome analyses and have been widely and successfully employed for simultaneously monitoring the expression of a potentially unlimited number of genes in parallel, thus providing the basis for identifying genes differentially expressed in distinct cell-types, developmental stages, disease states and cells subjected to exogenous reagents . The rapThe advantages of cross-species hybridisation are two-fold. First, cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The advantage of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large so that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. Second, the use of microarrays in studies in mammalian species other than human and rodents, for example nonhuman primates, bovine, sheep and porcine may advance our understanding of human health and disease, for example the use of animal models in drug target validation. However, the inavailability of adequate sequence data and commercial cDNA and oligonucleotide microarrays keeps this technology beyond the reach of investigators working on economically and scientifically important large domestic species such as cattle, pigs and sheep. A potential solution to this problem is the use of cross-species hybridisations, i.e, human sequence-based arrays as tools for undertaking comparative genome expression studies. Such analyses have been performed using ape brain RNA as target on a human oligonucleotide array  and pig,in vitro production of embryos for stem cell derivation and somatic nuclear transfer cloning. Employing the bovine model and sensitive RT-PCR assays, it has been shown that the majority of embryos derived from such sources display distinct mRNA expression patterns in a variety of developmentally important genes compared to their in vivo derived counterparts [in vitro production and/or nuclear transfer of which significant oversize is a predominant feature [A crucial step employing domestic animals in biomedicine is genetic modification which requires extensive embryo and embryo-related technologies, such as terparts . Some of feature . Analysi feature ,11 and aImportant criteria for evaluating any microarray system include the reproducibility of the data generated, the specificity of detection of the targeted gene, and the validity of the results that identify and establish differential gene expression. The experiments described here show the systematic validation of cross-species microarray analysis, with emphasis on the reproducibility and statistical analysis of generated data using standard microarray data analysis tools.Specifically, we investigated the feasibility and reproducibility of cross-species hybridisation of orthologous genes within a defined developmental and metabolic pathway using as a test case and the first description of its kind, human and bovine fetal brain RNA as Cy-dye labelled targets on a human cDNA microarray. The microarray is composed of 349 genes each spotted 20 times to ensure reproducible validation by independent technologies such as semi-quantitative RT-PCR as carried out in this study, or alternatively, Real-Time PCR and Northern blot analysis.HPRT and β-ACTIN (made up of a dilution series of 25-, 50-, 100-, 150- and 200 ng/μl) were employed as endogenous guide dots to enable accurate grid placement prior to image analysis. These spots can be seen as intense yellow signals at the periphery of each block as portrayed in Figure OCT-4 and Arabidopsis cDNAs  were included as negative controls to monitor hybridisation-specificity. The transcription factor OCT-4 is expressed in human embryonic stem cells and primordial germ cells and is down-regulated upon differentiation [The human cDNA microarray used in this study consisted of 349 fully sequenced and annotated cDNAs as described in the Supplemental Table ntiation .Classification of the genes according to Gene Ontology annotations (molecular function) and chromosomal location Figures  and 1C dOur experimental design incorporated dye-swaps and four replicated hybridisations. Three hundred and forty nine cDNAs, each representing a unique gene were spotted 20 times on each microarray. Four independent experiments were performed with human and bovine brain, respectively. Within each experiment the 20 replicates were averaged to yield a reliable signal for the respective probe. In the next step, the replicated signals from the different experiments were averaged to compute overall characteristics. The overall correlation of expression of the human and bovine genes is shown in Figure Additionally, we computed MA-plots  in orderth cycle of a standard PCR reaction. cDNAs were considered as "detected" when their average BG-tag was above 0.9.In order to measure whether a given gene was significantly expressed, we compared its cDNA's signal to a signal distribution derived from negative controls represented by approx. 2,500 empty spot positions on the array. After quantification of each array a low non-zero intensity is assigned to each of these empty spots reflecting the amount of background signal on the array. Since these positions are spread uniformly over the array, the distribution of these signals reflects the distribution for signal noise and is an indicator whether signals are at the background level or reflect reliable expression levels. For each cDNA we counted the relative proportion of empty positions on the array that are smaller than the actual observed intensity (BG-tag). BG-tags from replicated experiments for the same cDNA were averaged. Thus, high values (close to one) indicate that the cDNA is expressed in the respective tissue whereas low values reflect noise. The limit of visual detection of a spot corresponds to a BG-tag level of 0.9, however there is a grey zone around this value. Comparison with RT-PCR analyses showed  that thiWe grouped the 349 genes into three classes of expression levels with respect to their intensity values above background levels (i.e, BG-tag) with class 1 as low (not-detected), class 2 as medium (possibly detected) and class 3 as high level of expression (detected), respectively. Within class 1, are 19 bovine and 17 human genes. Class 2, comprises 26 bovine and 17 human genes and finally class 3 has 304 bovine and 315 human genes, respectively. The genes within expression class 1 have signal intensities below the detection level of our microarray analysis platform and as such these genes can be designated as "Absent"  across the replicated experiments. Histograms are shown in Figure Figure ZNF278, APOARGC, KIAA1609 and MGC12904 – highlighted as diamond shapes in Figure Three statistical tests that judge the significance of differences in the levels of gene expression in human and bovine fetal brains were employed (Student's t-test, Welch-test and, Wilcoxon's rank-sum test) as described previously . Tests wAPOARGC). Thus, Holm's setp-wise correction would start with an adjusted experiment level P-value of p = 0.05/349 = 1.43e-04. Similarly, measuring the false discovery rate by qvalues [Furthermore, taking into account corrections for multiple testing, no gene is differentially expressed at the global experiment significance level of 0.05. For example, the lowest P-value of the Welch test is 1.98e-03, , solute transport- (SLC11A3), protein assembly- (CALNEXIN), anion channel- (VDAC2) and as an endogenous reference, the housekeeping gene, β-ACTIN. Primer sequences were designed to co-amplify the orthologous genes  are expressed at a low level  in human and twenty genes (5.7%) in bovine with variability in expression levels in the four hybridisation experiments. In addition, approximately 305 genes have a CV of less than 0.5 with human brain (87%) compared to 280 with bovine brain (80%). However, this level of variability is low and can be expected due to the fact that the probe set used are of human origin. Moreover, the slight decrease in reproducibility due to species and nucleotide sequence differences can be compensated for by increasing the number of independent repetitions  of the experiments. The unexpectedly low level of variability in gene expression levels between human and bovine fetal brain can also be attributed to the 80 data points examined per gene and the four replicate hybridisations  carried out for each species. Our finding further emphasises the need for sufficient technical and biological replicates in all microarray experiments.CDC27: 0.95, CDC6: 1.36, PCNA: 1.07, respectively, whereas the deduced ratios derived from the semi-quantitative RT-PCR assay were 0.59 for CDC27, 0.35 for CDC6 and 0.87 for PCNA, respectively.In assessing differential gene expression in bovine and human fetal brain, we observed that the expression levels of the individual orthologous genes were roughly in the same broad range. For example, if one considers genes involved in pathways of cell cycle control, the microarray deduced ratios (bovine:human) were for SLC11A3 and CALNEXIN are 0.65 vs. 0.98 and 0.88 vs. 0.97. An illustration of co-amplification with common primers and also the confirmation of the array data for a selection of genes can be seen in Figures Similarly the comparative ratios for The similar expression ratio of the orthologous genes implicated in cell cycle control and initiation of eukaryotic genome replication which is conserved in all eukaryotes highlights the feasibility and importance of our study in examining conserved pathways operative within the fetal brains of human and bovine and of course across other species using human cDNA microarrays. A similar finding has also been confirmed when comparing yeast co-regulated genes against the archaeal and bacterial operons. This implies that the components of the protein translation process are conserved across organisms at the expression level with minor specific differences .We also identified four significantly differentially expressed genes as judged by the Wilcoxon test. The Wilcoxon test is known to be more conservative than the Student's t-test and the Welch-test, however, we have more confidence in this test since it is distribution-free, in particular it does not depend on the partly unrealistic assumption of an underlying Gaussian distribution.ZNF278 and APOARGC were 1.45 – and 1.66 -fold over-expressed in human whereas KIAA1609 and MGC12904 were 1.60 – and 1.37-fold over-expressed in bovine. ZNF278 encodes a zinc finger-containing transcription factor that acts as a transcriptional repressor and also implicated in small round cell tumours [APOARGC encodes a protein with hydrolase activity. MGC12904 and KIAA1609 are uncharacterised ESTs so comments cannot be made with regard to their function.The genes  tumours . The genOrthologous genes between human and mouse and between human and rat both have a mean of approximately 85% sequence identity ,23. In tFurthermore, using this protocol of cross-species hybridisation, we have compared gene expression in bovine unfertilised oocytes and blastocyst using a larger microarray (The Human Ensembl Chip) comprising 15,500 fully sequenced and annotated genes and ESTs. The correlation co-efficient between the RNA samples is 0.26, thus reflecting the diversity between oocyte derived maternal transcripts and embryonic transcripts derived from the blastocyst .In summary, this study highlights the significance and utilisation power of comparative genomics and also demonstrates the feasibility of using human cDNA microarrays to facilitate the identification of differentially expressed genes in human and bovine fetal brain. Our results indicate that cross-species hybridisation is not only a useful short-term solution for studying species for which gene maps, cDNA or oligo microarrays are not yet available, but also possesses tremendous power in enabling the unravelling of common evolutionary evolved mechanisms in different species.For the generation of probes for spotting, cDNA inserts from a single 384-well plate of non-redundant, fully sequenced, annotated human cDNA collection (Human Ensembl set RZPD1.1) were employed-  and a second partial plate consisting of control genes and empty wells to be used for data normalisation were amplified by PCR in a 384-well format. Bacterial clones were transferred to the PCR plates using 384-well replicators (Genetix Ltd, New Hampshire, UK). PCR amplifications were carried out in a total volume of 25 μl consisting of 1x PCR buffer, 1.5 M Betaine (Sigma, Germany), 1U Taq polymerase, 200 mM of each dNTP (Invitek, Germany), 25 pmoles of M13-forward (5'GTAAAACGACGGCCA3') and M13-reverse (5'CAGGAAACAGCTATGAC3') primers respectively. Cycling parameters typically consisted of an initial denaturation step at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, elongation at 72°C for 1 min, then a final elongation step at 72°C for 10 min. All amplified products were analysed by agarose gel electrophoresis and in all cases single bands were obtained per gene.Purification was carried out using isopropanol precipitations. In brief, 24 μl of the PCR product was transferred into 384-well Genetix plates (Genetix Ltd, New Hampshire, UK) previously filled with 18 μl of isopropanol per well. The samples were mixed by gentle vortexing after sealing the plates with transparent tape. After precipitation overnight at -20°C, the plates were spun at 3,500 rpm for 2 hrs at 21°C, the isopropanol decanted off and the plates dried briefly in a SpeedVac without heating. The pellets were resuspended in 6 μl of spotting solution ). Random sampling of cDNAs revealed concentrations ranging between 200 to 300 ng/μl. Finally the samples in each well were spotted twenty times  with four slit pins arranged in a 2x 2y printhead . Prior to every spotting run the performance of all components of the robot is carefully tested – in particular the performance of the washing station in test runs designed to detect possible sample carry over. A modified Genetix Q-array (Genetix Ltd, New Hampshire, UK), controlled by a novel in-house software was used for the arraying of the samples on SuperAmine™ aminosilane-coated microscope slides . Post-processing of the arrays was performed using 1,2-dichloroethane and the acylating catalyst N-methylimidazole following protocols described previously .Human brain RNA was isolated from a medically terminated fetus at 10 weeks gestation. This was provided by the MRC-funded Human Embryonic Tissue Bank maintained at the Institute of Child Health and University College London, England. Bovine fetal brain was obtained from a 3–4 months old bovine fetus at a local abattoir near Mariensee, Germany. Immediately after slaughter of the pregnant female, the tissue was plunged into liquid nitrogen and transported to the laboratory. Brain tissues were homogenised using a Dounce homogeniser, total RNA isolated using TRIzol reagent (Invitrogen) and further purified using phenol/chloroform extractions and precipitation with ethanol after DNase 1 (Promega) treatment. All procedures were as described by the manufacturers. RNA Purity, integrity and concentrations were evaluated on the Agilent 2100 Bioanalyzer. High quality RNAs with A260/A280 ratio over 1.8 with intact ribosomal 28S and 18S RNA bands were utilised for subsequent labelling reactions.°C for 5 min., left at room temperature for 10 min and then cooled on ice for 2 min. The following reagents were then added: 4 μl first-strand buffer, 2 μl 0.1 M DTT, 0.5 μl dNTP mix (25 mM each of dATP, dGTP, dCTP and 10 mM dTTP), 1 μl of 1.0 mM Cy5- or Cy3-dUTP (Amersham Pharmacia) and 1 μl (200 U/μl) Superscript II (Invitrogen). The labelling reaction was carried out at 42°C for 1.5 hrs. The reaction was stopped with 4 μl of 0.5 M EDTA. The input RNA was hydrolysed by the addition of 2 μl of 2.5 M NaOH and incubated at 37°C for 15 mins. followed by neutralization with 10 μl HEPES free acid (2 M, pH 5.5). Labelled cDNAs (four replicates per species which included dye-swaps) were purified from unincorporated Cy-dyes using Microcon YM-30 purification columns (Millipore). All labelled cDNAs were routinely analysed on a Fuji scanner (FL8-8000) and by ethidium bromide staining to ascertain dye incorporation and size-range of synthesized cDNAs. After concentrating cDNAs by evaporation in a SpeedVac, labelled targets were resuspended in 20 μl of hybridisation buffer . After thorough resuspension, the cDNA was denatured by heating at 95°C for 5 mins followed by 20 mins. at 42°C to enable annealing of the blocking reagents to repetitive sequences within the target cDNAs. The hybridisation mixture consisting of Cy3-labelled bovine and Cy5-labelled human RNA and vice versa was placed on the blocked array under a 24 × 40 mm coverslip (Menzel-Glaser, Germany). To maintain humidity inside the chamber, 20 μl of 3x SSC was added to the reservoir wells. The chamber was then tightly sealed and slides incubated at 42°C for 18 hrs in a waterbath. Slides were washed twice in 0.2x SSC / 0.1% SDS and then twice in 0.2x SSC. Washes were carried out at room temperature with 10 min durations per wash. Finally, the slides were dried by centrifugation at 1100 rpm for 10 min.Four independent labelling (including dye-swaps) reactions per species were carried out using 25 μg total RNA from both human and bovine per labelling reaction. Direct incorporation of Cy3 and Cy5 during reverse transcription was carried out in a 20 μl reaction volume using 1 μg of anchored oligo-dT primer. The RNA/primer mix was incubated at 70Fluorescence images were captured using an Affymetrix 428 scanner (Affymetrix, Santa Clara, CA) with appropriate gains on the photomultiplier tube (PMT) to obtain the highest intensity without saturation. A 16 bit TIFF image was generated for each channel for subsequent image analyses. Image analysis was carried out by placing the centre of each spot manually (grid-finding step) using the software AIDA (Raytest-Germany) and then by quantifying in a pre-defined neighbourhood around this spot centre using a two-dimensional Gaussian distribution (quantification step).j the median signal, medj, was computed and a multiplicative factor was calculated according to medref/medj, where medref is the global derived from the average intensity values of the cDNAs across the full batch of experiments. The multiplicative factor was used to adjust the signal of the ith cDNA in the jth experiment, xij, by xij * medref/medj.Data analysis comprised two distinct parts. In the first part, data were normalised to eliminate extrinsic influencing factors and artefacts not attributable to the probe-target interaction. For each tissue (human and bovine brain) under analysis the whole batch of experimental replicates was normalized simultaneously. Normalisation should eliminate multiplicative technical bias between the different experiments and result in the same median signal level for each experiment. In a first step, the local background of each spot was subtracted from the spot's signal intensity. Then, for each experiment In the second part, a number of numerical characteristics were calculated in order to quantify the cross-species comparison. These characteristics were signal detection value, signal reproducibility and statistical significance of differential expression. Signal detection was judged by a number of empty positions that were spread across the array. For each experiment, the proportion of signals of empty positions lower than the actual spot signal was calculated. Then across all repetitions, the average proportion was kept as the signal detection value. Employing this procedure, a signal strength was quantified for each cDNA. Signal reproducibility was judged by calculating the co-efficient of variation (CV) for each cDNA across all experiments.In order to judge differential expression of a gene in human and bovine brain we calculated three statistical tests: Student's t-test, Welch-test and Wilcoxon's rank sum test for each cDNA based on the signal series derived from human and bovine experimental repetitions. The three tests make different assumptions on the signal series. Whereas the first two assume that the series are Gaussian distributed, the Wilcoxon test is distribution-free. Low P-values calculated by these tests indicate significant differences in signal intensity within the human and bovine samples .The list of primers, annealing temperatures and genes under investigation is shown in Table °C for 5 mins and cooled on ice. Thereafter, the following components were added sequentially, 4.0 μl of 5x RT. Buffer (Invitrogen), 2.0 μl of 0.1 M DTT (Invitrogen) 1.0 μl of (10 mM) dNTP (Amersham) and 1.0 μl (200 U/μl) Superscript II (Invitrogen). After pulse spinning, incubation was carried out at 42°C for 1.5 hrs.For primer annealing and reverse transcription, 1.0 μl (2.5μg/μl) of DNase1 treated human and bovine total RNA was added to 2.0 μl (50 μM) Oligo-dT primer plus 9.0 μl of RNAse free water. The mixture was spun briefly and heated to 702, 2.0 μl of the first strand cDNA  and distilled water to 50 μl. All reagents were purchased from Invitrogen. Cycling parameters consisted of an initial denaturation step at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing for 30 sec, elongation at 72°C for 30 sec, then a final elongation step at 72°C for 5 min. Primer sequences, annealing temperatures and predicted size of cDNA products are shown in Table ACTIN was used as endogenous control.Gene-specific PCR amplifications were carried out in a total volume of 50 μl consisting of 5 μl of 10x PCR buffer, 0.2 μl (5 U/μl) Taq Polymerase, 1.0 μl (10 mM) dNTP, 2.5 μl of each (20 μM) primer, 1.5 μl (50 mM) MgClJ.A. conceived the study's idea, designed and optimised the protocols, carried out the hybridisations and was pivotal in developing the analysis plan and writing the manuscript.R.H. developed the analysis plan, analysed the data and was pivotal in writing the manuscript.D.H and M.N designed the cross-species primers and carried out the semi-quantitative RT-PCR.W.W. performed the initial image analysis.A.BK. selected the clones used as probes for creating the Chip and carried out the assignment of orthologous genes.T.B. assisted in the semi-quantitative RT-PCR analysisJ.W.C. was involved in the conceptualisation and writingC.H. was instrumental in the Chip design and production.H.N. is the Head of the Department of Biotechnology at the Institute for Animal Science and was involved in the conceptualisation and writing.H.L. is the Head of the Department of Vertebrate Genomics at the Max Planck Institute for Molecular Genetics.Summary of the expression data and the corresponding gene annotations. The terms used under each column are explained viz;IMAGE ID: Gene specific identifier assigned by the IMAGE consortiumGene: HUGO gene nameChromosome: Chromosomal location of geneDescription: Gene descriptionMolecular Function: Assigned function by the Gene Ontology ConsortiumBG-tag-bovine: Signal detection probability in the bovine samplesBG-tag-human: Signal detection probability in the human samplesCV-bovine: Co-efficient of variation between replicate signal intensitiesCV-human: Co-efficient of variation between replicate signal intensitiesMean-bovine: Mean signal across all hybridisation experiments using bovine RNAlog-Mean-bovine: Logarithm (base 2) of the mean valueMean-human: Mean signal across all hybridisation experiments using human RNAlog-Mean-human: Logarithm (base 2) of the mean valueRatio(bovine vs human): Ratio of the mean valueslog2(Ratio): Log-ratio (base 2) of the mean valuesP-value-Student's-t-test: P-value of Student's t-test using the replicate experimentsP-value-Welch-test: P-value of the Welch test using the replicate experimentsP-value-Wilcoxon-test: P-value of Wilcoxon's test using the replicate experimentsMatch: A BLAST hit with E-value < 1.0e-15 was found (1) or not (0)Homology: Classification of sequence homology between human gene sequence and bovine ESTs. "ortholog" = best match in both directions, "paralog" = bovine EST has its best match with another human sequence.TIGR-BTGI0-51503- Matches: Best results of the BLAST matches using the Ensembl- annotated gene sequence with the TIGR- databaseIdentity: %-identity of best matchOverlap: Overlap in base pairsClick here for file


The epidemiology of travel-associated campylobacteriosis is still largely unclear, and various known risk factors could only explain limited proportions of the recorded cases.Using data from 28,704 notifications of travel-associated campylobacteriosis in Sweden 1997 to 2003 and travel patterns of 16,255 Swedish residents with overnight travel abroad in the same years, we analysed risks for travel-associated campylobacteriosis in 19 regions of the world, and looked into the seasonality of the disease in each of these regions.The highest risk was seen in returning travellers from the Indian subcontinent (1,253/100,000 travellers), and the lowest in travellers from the other Nordic countries (3/100,000 travellers). In Africa, large differences in risk between regions were noted, with 502 /100,000 in travellers from East Africa, compared to 76/100,00 from West Africa and 50/100,000 from Central Africa. A distinct seasonal pattern was seen in all temperate regions with peaks in the summer, while no or less distinct seasonality was seen in tropical regions. In travellers to the tropics, the highest risk was seen in children below the age of six.Data on infections in returning travellers together with good denominator data could provide comparable data on travel risks in various regions of the world. Campylobacter infection is a zoonotic disease, observed in most parts of the world. The disease is caused by Campylobacter jejuni, or less commonly Campylobacter coli. It is estimated to cause 5–14% of diarrhoea, worldwide [Campylobacter infection has emerged to be most important bacterial cause of gastrointestinal infection. Animals (variety of fowl, swine, cattle, sheep, dogs, cats, and rodents) are the major reservoir for the bacteria. Campylobacter does not easily grow in food, but the critical infective dose is low [orldwide . Also ine is low .Unlike salmonellosis with well-known routes of transmission, the epidemiology of campylobacteriosis is still largely unclear, and various known risk factors could only explain limited proportions of the recorded cases . Known rCampylobacter infection is a leading cause together with infections due to enterotoxigenic Escherichia coli (ETEC), salmonellosis, and shigellosis [Approximately 80 million persons from industrialized countries travel every year to places in Africa, Asia, Pacific Islands, Latin America and remote areas of Eastern Europe, and between, 25 and 50 % of travellers to these areas experience travellers' diarrhoea -12. Abougellosis ,13.In this study we have used returning travellers to Sweden as sentinels to estimate the comparative risks for travel-associated campylobacteriosis in 19 regions of the world, and looked into the seasonality of the disease in each of these regions.Campylobacteriosis is a notifiable disease according to the Swedish communicable disease act. Cases are notified in parallel to the Swedish Institute for Infectious Disease Control (SMI) by the clinician having seen the patient  and the laboratory having diagnosed the pathogen (laboratory notification). At the SMI the notifications from the two sources are merged into case records, using a unique personal identification number issued to all Swedes, and used in all health care contacts. The clinical notifications contain epidemiological information of relevance, including country of infection. For this study we retrieved notification information (age, sex, country of infection and month of infection) from the national surveillance database  on all cData on travel patterns were obtained from a commercial database, the Swedish Travel and Tourist Database (TDB) . This daCampylobacter infection (cases) as numerator and extrapolated total numbers of travellers from the TDB as denominator. The actual numbers of interviewed persons (controls) were used for the calculations of 95% confidence intervals (95% CI) for the estimates, using the formula:The age, sex and geographical distribution of the interviewees in the TDB, were standardised against the total population of Sweden to give an extrapolation of the actual number of travellers to each country during the seven years. We then estimated risks per 100,000 travellers (divided on the exposures sex, age and region of travel) using notifications on In risk ± 1.96*√ (1/cases+1/controls)eTo adjust for possible confounding and test for interaction, we also calculated odds ratios (OR) with corresponding 95% CI for the same exposures with a logistic regression model.In an initial crude analysis, odds ratios (ORs) for all exposures (age, sex, and travel destination) on the outcome campylobacteriosis were analysed, with the lowest incidence in each category used as reference. Confounding was then assessed using Mantel-Haenszel stratification. ORs for exposures with significant association with the outcome were included in a logistic regression analysis if they were shown to contribute significantly to the model in a Wald test. The presence of significant interaction was tested with tests for homogeneity. For each region we analysed seasonality separately (OR for disease per month, adjusted for age, sex and number of cases/travellers). All analyses were done using the Stata 6.0 software (Stata Corporation, College Station, Tx, USA).Notification data is regulated by the Swedish Communicable disease act, and contain full personal identification. The TDB contains aggregated data only. The Medical Ethics Committee of the Karolinska Institute, Stockholm, Sweden, approved the study.Of 53,223 persons notified with campylobacteriosis in the period 1997 to 2003, 28,704 (54%) were travel-associated, corresponding to 42.3 cases per 100,000 travellers , East Africa , East Asia , North Africa  and Arab countries/Iran . Adjusting for age, sex, and month in the logistic regression model did not change the rank between the regions  versus 40.8 cases per 100,000 95 % CI 39.7–41.9). After adjusting for destination, age, and month in the multivariate logistic regression model, the risks were reversed with a significantly higher OR in males . However, travel destination was an effect modifier on the association between sex and campylobacteriosis, and this higher risk for males were only significant for travellers returning from a European country   and in small children 0–6 years old . Also the association between age and campylobacteriosis was modified by travel destination, and in travellers from tropical destinations, especially from Africa and Asia/Oceania the highest risk was seen in the youngest children , Western Europe , Eastern Europe , North America , Southern Europe , Northern Africa , Arab countries and Iran , and Australia/New Zealand . In the Eastern Mediterranean the peak risk was seen in the spring , and in Russia and former USSR in late fall . In the tropical regions the seasonality was considerably less distinct. In East Asia the risk peak was in December with nadir in May  and in the Caribbean in February with nadir in September . In the Indian Subcontinent, Sub-Saharan Africa, and Central/South America no distinct, significant seasonal peaks could be identified.In this study we report the risks for travel-associated campylobacteriosis and seasonality of the risks in various parts of the world, based on more than 28,000 notified cases. The large number of cases gives more precise risk estimates for this disease than in previous studies, although the estimates are given for quite large regions in parts of the world with few Swedish travellers. The denominator data from the TDB has previously been used in studies on dengue fever  and rickCampylobacter infections. One should therefore be cautious in drawing conclusions from the magnitude of the figures, and rather focus on the relative risks between the various regions, as estimated by the odds ratios. Since the data are all from the same source, the risk figures from the various regions are directly comparable. However, there may be a tendency of investigating travellers from the tropics more vigorous than travellers from e.g. the other Nordic countries, thus underestimating the risks in nearby countries. However, such selection bias could likely not explain the huge differences between say West Africa and East Africa.Notification data only reflects a small (but unknown) proportion of all travel-related Since we have no comparable data on the length of stay among the cases in our study, we were not able to include length of stay in our logistic regression model. However, the TDB clearly shows a longer median stay among travellers in far-away destinations. For instance the median stay in Spain was 6 nights, while in Thailand it was 14 nights. On the other hand, only cases detected after the return to Sweden are included in the analysis. The disease data therefore mainly reflect infections contracted during the last week of stay at the travel destination. Differences in length of travel are therefore to some extent evened out.It has previously been suggested that the risk of travellers' diarrhoea is higher during the first two weeks in highly endemic areas ,18. The Campylobacter jejuni infection was 10 per 100,000 travellers returning from Spain and Portugal, and 50, 60, and 80 per 100,000 returning travellers from China, Thailand and India, respectively [The differences in risk between various regions were considerable, not only between industrialized and developing countries, but also between different developing countries. The Indian Subcontinent, East Africa, East Asia, and North Africa stood out as special high-risk areas. In a recent Finnish study, the risk of travel associated ectively . The lowectively ,21, but Age, sex and season (month of travel/infection) were identified as possible confounders for the association between travel destination and risk for campylobacteriosis and were thus included in the logistic regression model. All three variables contributed significantly to the model, but the overall effect of these confounders did not alter the rank order between the regions in the logistic regression model compared to the crude analysis.The highest risks were seen in young adults and small children, and especially in the tropics the highest risks were seen in the youngest. This is consistent with previous findings that in developed countries the disease most of hits children below the age of 5 years and young adults, while in developing countries it is most often seen in children below the age of 2 years, with an annual incidence of 40–60 % . The datCampylobacter infection. This pattern is also seen in domestically acquired Swedish campylobacteriosis cases, where 56% of the notified cases in 2003 were males, and in the US where campylobacteriosis is more common in males of all age groups [In travellers returning from Europe, male gender was an independent risk factor for e groups ,9. The he groups .In each of the 19 regions in the study, we looked closely at the seasonality of the disease. As has previously been shown ,6-9, theCampylobacter infection was seen between November and April [In the tropical regions, seasonal peaks of campylobacteriosis have not previously been recognized . Also innd April . With onData on infections in returning travellers together with good denominator data could provide comparable data on travel risks in various regions of the world. This study has revealed large and unexplained regional incidence differences, e.g. between East and Central Africa. The very distinct seasonal pattern seen in all temperate regions could not be discerned in the tropics.The author(s) declare that they have no competing interests.KE raised the original idea of the study, did the statistical analyses, and prepared the first draft of the manuscript. YA contributed with in depth knowledge of campylobacteriosis and revised the draft manuscript. Both authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-).Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry.Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic KSlc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2.Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome. PDS gene SLC26A4, which codes for the protein pendrin [Pendred syndrome is a relatively common autosomal-recessive disorder characterized by deafness and goiter . The syn pendrin . The cau pendrin .-, I-, HCO3 - and formate [Pendrin is an anion exchanger that can transport Cl formate ,7. Expre formate ,3,8. In  formate , mammary formate , uterus  formate , testes  formate  and plac formate . No expr formate ,11.Slc26a4-/- mice, which bear a targeted disruption of the mouse Slc26a4 gene [Slc26a4-/- mice.Expression of pendrin mRNA in the inner ear has been found in several places including the cochlea, the vestibular labyrinth and the endolymphatic sac . The pre6a4 gene . The aim+ concentrations were measured in young adult mice (1–4 month of age) that either have (Slc26a4+/+) or lack (Slc26a4-/-) a functional gene for pendrin [Slc26a4+/+ controls, since Slc26a4-/- mice were propagated in the this strain and generated using a stem cell line derived from 129Sv/Ev. Slc26a4+/+ and Slc26a4-/- were agouti. They did not differ in coat color. Differences in pigmentation were verified using coisogenic age-matched Slc26a4+/+ and Slc26a4-/- that were bred in parallel.The endocochlear potential and the endolymphatic and perilymphatic K pendrin . The mou+ was studied using confocal immunocytochemistry and quantitative RT-PCR. K+ secretion and the generation of the endocochlear potential were measured using electrophysiological techniques. All experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University.Expression of key proteins involved in the generation of the endocochlear potential and the transport of K--free phosphate-buffered Na-gluconate solution containing 4% paraformaldehyde. Temporal bones were removed and cochleae fixed by perilymphatic perfusion, decalcified in EDTA, processed through a sucrose gradient and infiltrated with polyethylene glycol. Mid-modiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were blocked in PBS with 0.2% Triton-X (PBS-TX) and 10% bovine serum albumin. Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 ; rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)]. Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1–3% BSA [donkey anti-rabbit Alexa 488, chicken anti-rat Alexa 594, and chicken anti-goat Alexa 594 (Molecular Probes, Eugene, OR)]. Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave , and viewed by confocal microscopy . Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation.Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl2. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The Slc26a4-/-genotype was verified by the observation of large otoconia in the utricular macula [+ channels KCNJ10, KCNQ1 and KCNQ4 were amplified using gene-specific primers . Kidneys and brain were removed and pulverized in liquid Nr macula . Total Rt) where P is the number of product molecules, F is the fidelity of the reaction and Ct is the cycle at which the number of product molecules reaches a chosen threshold [2 + 0.15 × S3 + 11.38 × 10-3 * S4 + 3.39 × 10-4 × S5. The number of product molecules at threshold (PCt) was determined by amplification of known amounts of 18S rRNA according to PCt = T × F ^Ct. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be <0.02% by omission of the RT step.Template molecules (T) were quantified according to T = P / . Stria vascularis without spiral ligament was obtained by microdissection. Currents generated by the stria marginal epithelium were recorded . A Pt-Ir+] were measured with double-barreled microelectrodes [+-selective electrodes were calibrated in solutions of constant cation (K+ and Na+) concentration of 150 mM. The minor selectivity of the K+ electrodes for Na+ produced a nonlinearity in the calibration curve, which was closely fitted by the Nicolski equation using nonlinear curve-fitting software (OriginLab, Northampton, MA): V = Vi + S × log ([K+] + A × [Na+]), where Vi is an offset term, S is slope, A is selectivity, and [Na+] is Na+ concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K+ concentrations were obtained using a blood analyzer .Animals were anesthetized with inactin . The endocochlear potential and the endolymphatic [Kectrodes . MeasureData are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p < 0.05.In situ hybridization in the cochlea has suggested that pendrin mRNA is expressed in cells that reside immediately beneath the spiral prominence on the lateral wall of the external sulcus [Slc26a4+/+) mice using an established antibody [data not shown). Strong expression of pendrin was observed not only in outer sulcus epithelial cells, as predicted from in situ hybridization data, but also in root cells, in apical membranes of spiral prominence surface epithelial cells and in apical membranes of spindle-shaped cells that are part of stria vascularis  support a role for pendrin in HCO3 - secretion into endolymph.l sulcus . To deteantibody . Staininris Fig. . The pree kidney . HCO3 - te cells ,19. CO2 + channel that is expressed in strial marginal cells, and ZO-1, a tight junction protein that labels basal cells and thereby delineates the boundaries of stria vascularis. Dual labeling experiments demonstrated that pendrin is expressed in spindle-shaped cells, which are surface epithelial cells in stria vascularis adjacent to strial marginal cells near the borders of both the spiral prominence and Reissner's membrane . These observations suggest that a primary event leading to deafness in Slc26a4-/- mice, and potentially in patients suffering from Pendred syndrome, is the loss of the endocochlear potential. Degeneration of hair cells is probably a response to the loss of the endocochlear potential.The onset of pendrin expression during development of the mouse inner ear has been determined to be embryonic day (ED) 13 . Morpholdulthood . Interesdulthood . These osurvival , is estasurvival  and may  hearing . We hypo-/- Fig. . No signtic Fig.  or plasm+ channel KCNJ10 located in intermediate cells [+ from the intrastrial space into endolymph and keep the K+ concentration low adjacent to the KCNJ10 K+ channel [Slc26a4-/- mice, we first determined whether intermediate cells are present in stria vascularis, since a loss of intermediate cells is known to lead to a loss of the endocochlear potential [Slc26a4-/- mice  Slc26a4+/+ and both young and old (~12 month) Slc26a4-/- mice, assessed amounts of isolated tissues by quantification of 18S rRNA, and quantified the expression of KCNJ10 mRNA. Quantities of stria vascularis isolated from these different mice were similar, since no significant differences were found in the numbers of 18S rRNA molecules (log(rRNA) = 9.46 ± 0.08, n = 17, pooled data). In contrast, quantities of spiral ganglia isolated from young and old Slc26a4-/- mice (log(rRNA) = 9.04 ± 0.18, n = 4 and 9.29 ± 0.15, n = 6) were significantly smaller than in Slc26a4+/+ mice (log(rRNA) = 9.48 ± 0.19, n = 7), consistent with morphometric data (see below). Expression of KCNJ10 mRNA was normal in stria vascularis and spiral ganglia of young Slc26a4-/- mice but significantly reduced in old Slc26a4-/- mice . The absence of a change in tissue height is consistent with the presence of intermediate cells [Slc26a4-/- mice was less prominent, spiral ligament thinner and spiral limbus flatter . In addition, we observed an apparent degeneration of stria vascularis, including an increase in pigmentation and an irregular pattern of the tight junctions of marginal cells . KCNQ1 and KCNE1, subunits of the secretory K+ channel, were co-localized in the apical membrane of strial marginal cells; the Na+/2Cl-/K+ cotransporter SLC12A2, which is located in the basolateral membrane of strial marginal cells, was found in stria vascularis; and the gap junction protein GJB2 was found in spiral ligament of Slc26a4+/+ and Slc26a4-/- mice (Fig. Slc26a4-/- mice is consistent with the finding of KCNQ1 mRNA expression (Fig. + secretion by strial marginal cells or a compromise of gap junction mediated K+ cycling.The observation that endolymphatic and perilymphatic Kndolymph ,31. In oecretion ,32,33, a cycling . K+ secrice Fig. . Co-locaice Fig. . Express+ concentrations in spite of an enlargement of this fluid compartment. We found that Slc26a4-/- mice lack the endocochlear potential because they do not express KCNJ10 protein. Intermediate cells protect stria vascularis by converting CO2 to HCO3 - and by detoxifying free radicals (Fig. 2 and free radicals are generated by the large numbers of mitochondria in the metabolically highly active strial marginal cells. Intermediate cells employ carbonic anhydrase to convert CO2 to HCO3 - [3 - into endolymph, results in an accumulation of HCO3 - and an alkalinization of the intrastrial spaces. This extracellular alkalinization may enhance free radical stress, since it may inhibit the uptake of cysteine and thereby limit production of the protective glutathione [+ channels has been shown to be controlled by the cytosolic pH and free radicals, which encode the metabolic state of the cell [+ channel in strial intermediate cells, which is essential for the generation of the endocochlear potential, is probably the direct cause of deafness in Slc26a4-/- mice and patients suffering from Pendred syndrome.We described locations of pendrin expression in the cochlea and vestibular labyrinth and detected normal endolymphatic Kals Fig. . CO2 ando HCO3 - ,19 and co HCO3 - -37. It itathione . Supportthe cell . SuppresNone declared.+ concentration. JHL carried out current measurements on strial marginal cells. SMW, LAE, IER and EDG provided the mice and the pendrin-specific antibody. PW and DCM conceived the study. PW wrote the manuscript. All authors read and approved the final manuscript.PW designed and coordinated the immunocytochemical, morphometrical and molecular biological experiments. EMI and BA carried out confocal immunocytochemistry on cryosections. SJ and SVJ carried out confocal microscopy on whole-mounts of stria vascularis. SVJ and RJM carried out quantitative RT-PCR. DCM designed and coordinated electrophysiological experiments. TW carried out measurements of the endocochlear potential and the endolymphatic KThe pre-publication history for this paper can be accessed here:


Ultrasound contrast agents (UCAs) allow the enhancement of vascular definition, thereby providing more diagnostic information. LK565 is a new second-generation UCA based on synthetic polymers of aspartic acid which is eliminated from the blood stream via phagocytosis. LK565 forms very stable air-filled microspheres and is capable of repeated passage through the pulmonary capillary bed after peripheral intravenous injection. This characteristic allows examination of the cardiac function or extracardiac vessel abnormalities up to 15 minutes.A phase one clinical study was conducted on 15 healthy volunteers to identify the development of an undesirable immune response. Phagocytosis capacity, TNF-α secretion, and MHC class II upregulation of monocytes was monitored, as well as microsphere specific antibody development (IgM, IgG). Furthermore, the kinetics of the activation surface markers CD69, CD25, CD71, and CD11b on leukocytes were analyzed.Due to LK565-metabolism the administration of the UCA led to saturation of phagocytes which was reversible after 24 hrs. Compared to positive controls neither significant TNF-α elevation, neither MHC class II and activation surface markers upregulation, nor specific antibody development was detectable.The administration of LK565 provides a comfortable duration of signal enhancement, esp. in echocardiography, without causing a major activation cascade or triggering an adaptive immune response. To minimize the risk of undesirable adverse events such as anaphylactoid reactions, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent should be encouraged as a safe means to provide additional diagnostic information. Echocardiography allows the analysis of ventricular motion and heart-valve morphology. The discovery that injection of small air bubbles dispensed in saline are capable of causing an opacification of the heart ,2 starteThe particles consist of a synthetic polymer of aspartic acid. Via peptide bonds, the molecules form bold, stable beads filled with air. The average size of LK565 particles is 3 μm in diameter figure . BecauseResults from previous studies indicate that phagocytes are responsible for eliminating the contrast agent from the blood stream . In vitrThe activation of macrophages can lead to different conditions: no activation, normal activation, and overreaction. In the last case, over-expression of tumor necrosis factor alpha (TNF-α) can lead to a septic shock by induction of intravascular blood clots apart from other effects, which may result in disseminated intravascular coagulation ,14. FurtThe experiment was approved by the local ethics committee (registration number 708/98). After medical examination, 15 healthy male volunteers under the age of 30 were enrolled in this phase one clinical study. Each volunteer was exposed to a single dose of the contrast agent LK565 applied intravenously. Four participants were exposed to the contrast agent LK565 for the second time because they had already been involved in a prior study . The 15 Prior to application, the contrast agent LK565 was dissolved in 10 ml physiological sodium chloride solution (37°C) under sterile conditions. To eliminate aggregations, the solution was filtered using a 20 μm mesh just before intravenous injection.Heparinized blood samples (2 ml) were taken before application (t = 0), after 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 1 ml after 10 d, 12 d and 14 d. For routine analysis, additional blood samples were drawn before and 2 hours after intravenous contrast agent injection. The volunteers were under medical care throughout the whole experiment.Phagocytosis capacity was determined in vitro before (t = 0) and after 2 h, 6 h and 24 h with Phagotest™ (Becton Dickinson, Germany) and analyzed via flow cytometry . FITC-labeled E. coli, which were opsonized with antibodies and complement factors, were incubated with leukocytes. After incubation, the increased fluorescence of the phagocytes was due to the uptake of bacteria. The fluorescence intensity is dependent on the amount of phagocytosed bacteria. A sample chilled on ice was used for control.2 atmosphere. After washing, monocytes were surface stained by anti-human CD14 TriColor mAb (Medac, Germany). Red blood cells were lysed using FACS™ Lysing Solution (Becton Dickinson, Germany). Cells were fixed and permeabilized by addition of 250 μl Cytofix/Cytoperm™ (Becton Dickinson, Germany). Finally, the retained intracellular TNF-α was stained by the anti-TNF-α PE mAb. For a positive control, cells were stimulated with 0.25 μg Lipopolysaccharide (LPS), following the same procedure as mentioned above.Intracellular TNF-α production in monocytes and macrophages was analyzed before (t = 0) and after 2 h, 6 h, and 24 h. For detection, an anti-human TNF-α Pycoerythrin (PE) monoclonal antibody (mAb) was applied (Pharmingen, Germany). The specificity of the antibody had been validated in blocking experiments. To 40 μl blood, 145 μl RPMI and 15 μl 40 μM Monensin were added (Monensin prevents cytokine release by blocking the golgi apparatus). Cells were incubated for 2 h at T = 37°C and 5% COSurface activation markers were analyzed before (t = 0) and after 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h. For the detection of activation markers, anti-human CD69 PE (Holzel Diagnostika, Germany), CD25 Fluoresceinisothiocyanat (FITC), CD71 FITC, CD11b PE (Diaclone Research, Germany) and HLA-DR PerCP (Becton Dickinson, Germany) mAbs were used. For a positive control, cells were stimulated with 10 ng/ml Phorbol-12-Myristat-13-Acetat (PMA) and 1 μg/ml Ionomycin for 2 h, while macrophages/monocytes were stimulated with 1.25 μg/ml LPS for 2 h. To distinguish between leukocytes, additional surface markers, anti-human CD45 Allophycocyanine (APC), CD19 APC , CD3 Tri/Color (DAKO, Germany) and CD14 FITC (Diaclone Research, Germany) mAbs were used for detection. Blood samples (40 μl) were stained for 4-color flow cytometry analysis. After staining, red blood cells were lysed, subsequently leukocytes were analyzed. Antibody specificity was validated by isotype controls (Becton Dickinson, Germany).Serum samples were analyzed before (t = 0) and after 24 h, 48 h, 72 h, 96 h, 10 d, 12 d, and 14 d. Specific anti-LK565 antibodies were determined via indirect ELISA. Indirect ELISA was performed in a 96-well plate by LK565 immobilization overnight. Optimal concentrations had previously been determined by common cross-testing. Serum was obtained from blood samples after centrifugation at 400 G. After incubation for 2 h at room temperature, detector antibodies (Biozol Diagnostica GmbH, Germany) were added and incubated at 4°C overnight. Washing was carried out with phosphate buffered saline (PBS) and blocking buffer. For detection, alkaline phosphatase (AP)-linked goat-anti-human IgG and IgM antibodies with para-Nitrophenylphosphate (p-NPP) as substrate (Biozol Diagnostica GmbH, Germany) were used. After development, the enzyme reaction was stopped with 0.5 M NaOH and measured at λ = 405 nm. For the functional (positive) control of the indirect ELISA, total immunoglobuline was captured polyclonally. For a negative control, the serum of a person who had not been exposed to the contrast agent was obtained.We investigated 15 volunteers in a clinical phase one trial over a period of 14 days. The kinetics of activation surface markers CD69, CD25, CD71 and CD11b on leukocytes, the phagocytosis capacity, and TNF-α production in monocytes were analyzed, as was the specific antibody production after LK565 application.The expression of CD69 , CD25 1, CD71, HIndependent of dosage or repeated contact, the contrast agent LK565 was well tolerated by all volunteers. After exposure to LK565 no erythema, no drop in blood pressure, neither significant change of the heart frequency nor fever occurred (data not shown). The volunteers did not complain of relevant clinical symptoms such as chest discomfort or asthenia. It exhibited good opacification of both ventricles a few seconds after application figure . While tThe uptake of LK565 led to the saturation of phagocytes. The uptake capacity of the phagocytes from blood samples 6 h after injection was exhausted figure . NeitherDuring the first 24 h an increase in intracellular TNF-α production was detected in monocytes and macrophages figure . HoweverAn increase in integrin expression CD11b was detected on monocytes and macrophages 6 hours after application. Compared to the positive control, the upregulation of the integrin was only poor. In all cases, the upregulation of the integrin was only of brief duration and returned to normal levels after 24 h figure . Only a Discrete MHC class II (HLA-DR) upregulation on macrophages and lymphocytes was observed figure . We founThe results show that there is only a slight activation of leukocytes esp. of phagocytes after application of LK565, a particular contrast agent based on a polymer of the naturally occurring amino aspartic acid. No specific antibody development against the contrast agent was detected. Thus, the risk of a major immunological activation cascade after repeated dosage can be regarded as minor. Since LK565 does not lead to an adaptive immune activation in this study, the possibility of an overexpression of IgE, which plays a key role in allergic diseases, also becomes more unlikely. Although none of the 15 volunteers enrolled in this phase I study developed an allergic-type reaction, such reactions could, however, occur in further phases of clinical testing or application. An allergic reaction against a pharmaceutical drug is a rare event which is impossible to exclude even in large clinical studies.An increase in CD11b/CD18 is connected with phagocytosis and the diapedesis readiness of phagocytes. LK565 is eliminated from the blood stream via phagocytosis, therefore neutrophils exhibited an increase in CD11b expression and phagocytosis activity. This is also connected to a temporary higher intracellular TNF-α level in monocytes.On lymphocytes, especially T-cells, the cascade of CD69, CD25 and CD71 is closely related to an immune response. After expression of CD69, the interlinkage of the receptor leads to proliferation and secretion of IL-2, IFN-γ and TNF-α. After effective activation, the T-cells secrete IL-2 which activates the development of the high-affinity IL-2 receptor CD25 and therefore launches cell proliferation with an expression of CD71 after 3 – 4 days. During our study after LK565 application no major activation cascade was detected on either T-cells or B-cells.As mentioned above, the best contrast performance for echocardiography was obtained at a LK565 dosage of 0.4 mg/kg. Opacification of both ventricles was not enhanced by a further increase in dosage. Bearing in mind the fact that the contrast agent is phagocytosed and uptake capacity is limited, it is recommended that the daily dosage does not exceed 90 mg, which is equivalent up to three echocardiograms per day.To minimize the risk of an undesirable adverse event such as an anaphylactoid reaction, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent (UCA) with a comfortable duration of signal enhancement esp. in echocardiography should be encouraged as a means to provide additional diagnostic information without causing a major activation cascade or triggering an adaptive immune response. This can be an advantage for the "difficult-to-image" patient with adverse reactions related to other UCAs.H.K. Maerz received salary for 6 months from Dr. F. Koehler Chemie GmbH, Alsbach-Haehnlein, Germany, the developer/manufacturer of LK565. R. Zotz is co-author of one of several patents related to LK565 : Ultrasonic contrast agents, processes for their preparation and the use thereof as diagnostic and therapeutic agents. United States Patent 5.137.928).BF: Prepared and edited the manuscript as well as the figures und performed echocardiographic evaluations; HKM: Drafted the manuscript and participated in the design of the study esp. immunological aspects; SO: Carried out TNF-α evaluations, recruited and coordinated the clinical management of the patients; SP: Carried out expression pattern analysis, recruited and coordinated the clinical management of the patient; IL: Participated in the study design; US: Reviewed the manuscript according to study design and immunological aspects PW and TG: Reviewed the manuscript and edited the figures; RZ: Participated in the development of LK565, the study design and coordination. All authors read and approved the final manuscript.


Control and maintenance of mammalian cell size. BMC Cell Biol 2004, 5:35A response to Cooper S:  BMC Cell Biology [Stephen Cooper was kind enough to send to us an original draft of his paper that now appears in  Biology . AlthougJournal of Biology last year [His paper claims to be an analysis of our paper that was published in ast year . UnfortuOne problem is semantic. He uses the term "cell growth" ambiguously, to mean both cell enlargement and cell number increase, which is confusing when discussing both cell growth and cell proliferation. In the Background, for example, he writes "How do cells maintain a constant cell size and cell size distribution during extended cell growth?".There is more important confusion in the phrase "linear growth". He uses it to mean the addition of an equal amount of mass at each stage of the cell cycle, and he claims that we use it in our paper in the same way. In our paper, however, we clearly defined linear growth to describe our observation that Schwann cells, blocked in S phase with aphidicolin, added a constant amount of volume and mass per cell over time, independent of their size. This confusion leads him to claim erroneously, in his Abstract and elsewhere, that we proposed that mammalian cells grow linearly during the division cycle; we do not believe this, and we did not test it or discuss it in our paper. Much of his paper is based on the premise that we were trying to understand how cell growth changes through the cell cycle. In fact, we have never addressed this question, in the paper or elsewhere. For this reason, much of Cooper's paper is not relevant to ours.Cooper criticizes individual experiments in our paper, but this too is almost always based on unnecessary misunderstanding. He accuses us, for example, of "an egregious error" in studying protein synthesis in Schwann cells that were not synchronized and therefore in all phases of the cell cycle. In fact, however, the cells were all arrested at the start of S phase with aphidicolin, as pointed out in both the text and figure legend.S. pombe) blocked in S phase by a mutation, which grow faster as they enlarge [Despite its length, Cooper's paper never comes to grips with either the two main findings in our paper or the points of the experiments described in it. Unlike yeast cells ( enlarge , we foun enlarge , that if enlarge ,4. By co enlarge . We concCooper also ignores our earlier findings that Schwann cell size at division depends simply on how fast the cells are growing and how fast they progress through the cell cycle and that both of these rates depend on the concentrations of extracellular signals that can regulate the two rates independently . We founCooper's model for how cell growth and cell division can be coordinated is one version of a cell-size checkpoint model, in which progression through the cell cycle is somehow linked to cell size. Such models have been widely accepted in the cell-cycle field to explain how proliferating cells maintain their appropriate size over time . Whereas


Insulin resistance induced by a high fat diet has been associated with alterations in lipid content and composition in skeletal muscle and adipose tissue. Administration of β3-adrenoceptor (β3-AR) agonists was recently reported to prevent insulin resistance induced by a high fat diet, such as the cafeteria diet. The objective of the present study was to determine whether a selective β3-AR agonist (ZD7114) could prevent alterations of the lipid profile of skeletal muscle and adipose tissue lipids induced by a cafeteria diet.Male Sprague-Dawley rats fed a cafeteria diet were treated orally with either the β3-AR agonist ZD7114 (1 mg/kg per day) or the vehicle for 60 days. Rats fed a chow diet were used as a reference group. In addition to the determination of body weight and insulin plasma level, lipid content and fatty acid composition in gastronemius and in epididymal adipose tissue were measured by gas-liquid chromatography, at the end of the study.In addition to higher body weights and plasma insulin concentrations, rats fed a cafeteria diet had greater triacylglycerol (TAG) and diacylglycerol (DAG) accumulation in skeletal muscle, contrary to animals fed a chow diet. As expected, ZD7114 treatment prevented the excessive weight gain and hyperinsulinemia induced by the cafeteria diet. Furthermore, in ZD7114 treated rats, intramyocellular DAG levels were lower and the proportion of polyunsaturated fatty acids, particularly arachidonic acid, in adipose tissue phospholipids was higher than in animals fed a cafeteria diet.These results show that activation of the β3-AR was able to prevent lipid alterations in muscle and adipose tissue associated with insulin resistance induced by the cafeteria diet. These changes in intramyocellular DAG levels and adipose tissue PL composition may contribute to the improved insulin sensitivity associated with β3-AR activation. Dietary fatty acids are known to influence the composition of stored triacylglycerol (TAG) and membrane phospholipids (PL) in adipose tissue . More reChronic activation of the β3-adrenoceptor (β3-AR), which is predominantly expressed in white and brown adipose tissue, by selective agonists exerts both anti-obesity and anti-diabetic effects in rodent models of obesity ,16. ActiThe objective of the present study was to determine whether a selective β3-AR agonist could prevent alterations in the profile of skeletal muscle and adipose tissue lipids induced with the consumption of the cafeteria diet, previously reported to induce weight gain and hyperinsulinemia ,23. The Male Sprague-Dawley rats were purchased from Charles River (L'arbesles, France) at 5–6 weeks of age. Animals were individually housed in temperature-controlled rooms (22°C) with a 12-h light-dark cycle. Ten days before the beginning of the study, 30 rats were provided a normal chow diet  and free access to drinking water. At the end of this period, rats were weighed and pre-selected for their sensitivity to weight gain (i.e. rat presenting at least 35% weight gain after a 10-day selection period). The selected rats (n = 15) were equally randomized into 3 groups. A reference group, consisted of rats fed for 60 days with a standard pellet chow diet containing 28, 57 and 15% E from protein, carbohydrate and fat, respectively (REF group). The remaining rats were fed for 60 days with a cafeteria diet composed of 30 g of a mix containing salami, cookies, cheese, sausage, chips, chocolate and almonds in a proportion of 2:2:2:1:1:1:1 and 30 g of the reference group chow diet. This mixed diet contained 26, 27 and 47% energy as protein, carbohydrate and fat, respectively. At the beginning of the dietary intervention, both groups fed the cafeteria diet received daily, by gavage (0.5 ml/100 g body weight), either the selective β-3AR agonist ZD7114 at 1 mg/kg per day (CAF-ZD group) or water alone (CAF group) until day 60. Rats from the REF group (chow diet) also received a daily gavage of water (0.5 ml/100 g body weight).Body weight and food intake were recorded daily. Rats were fasted for 8 hours before sacrifice, performed under isoflurane anaesthesia. Tissues were immediately collected, weighed, frozen in liquid nitrogen and kept at -80°C until analysed.All procedures in the study were in compliance with the ethical committee of the "Service vétérinaire du canton de Vaud".Estimation of the proportions of the different lipid classes contained in the cafeteria diet (see Discussion) was performed using the USDA Nutrient Database.Fatty acid composition of lipids in adipose tissue was performed by Lipomics Technologies (West Sacramento, USA). Briefly, lipids were extracted from 30 mg of frozen epididymal adipose tissue in the presence of authentic internal standards by the method of Folch et al.  using chThe frozen gastrocnemius muscle was thawed and thoroughly dissected under stereomicroscope to remove extramyocellular adipose tissue. Lipids from lyophilized, finely powdered, dissected muscle (50 mg) were extracted according to the method of Folch . PL  and Crystal Chem Inc. (Downers Grove, USA), respectively. Plasma triglycerides and free fatty acids concentrations were analysed using kits from Roche Diagnostic (Basel, Switzerland) and Wako Chemicals (Richmond, USA), respectively.Comparisons of the means of the dependent variables of each group were performed using a one-way ANOVA.Male Sprague-Dawley rats were fed, during 60 days, either a chow diet used as a reference group (REF group), a cafeteria diet (CAF rats) or a cafeteria diet plus a daily gavage of the β3-AR agonist ZD7114 (CAF-ZD rats).At day 60, the mean body weight of CAF rats was significantly greater than that of REF rats Table . On the Measurement of insulin and glucose concentrations in plasma of fasted animals showed that CAF rats presented a marked hyperinsulinemia with a 4.6 fold increase in insulin concentration as compared to REF animals whereas the glucose level was not changed Table . ZD7114 PL and TAG contents per gram of tissue were measured in epididymal adipose tissue of rats fed with a chow diet or a cafeteria diet treated or not with ZD7114. No significant change was observed in the concentration of PL and TAG in adipose tissue of CAF rats when compared with REF animals Table . FurtherAnalysis of the fatty acid profile shows that cafeteria diet induced an increase in the proportion of monounsaturated fatty acids (MUFA), which was compensated for by a reduction in the percentage of PUFA in both adipose PL and TAG Figure . ChangesZD7114 treatment did not change the proportions of any lipid classes measured in adipose TAG of CAF rats Figure . HoweverPL, diacylglycerol (DAG) and TAG contents were measured in the gastrocnemius of REF animals and CAF rats with or without ZD7114 treatment , but significantly, elevated in DAG of CAF-ZD rats with an increase in the proportion of palmitic and stearic acids Table . Furtherin vitro [The present study demonstrates that feeding the cafeteria diet to rats not only promotes weight gain, hyperinsulinemia and hypertriglyceridemia, but also accumulation of lipids in skeletal muscle. Although TAG levels per gram of adipose tissue was not affected by the cafeteria diet, this dietary intervention strongly enhanced TAG accumulation in skeletal muscle, as previously described ,5. We shin vitro . DAG masin vitro . Howeverin vitro ,5,33, DAin vitro .The fatty acid composition of TAG stored in muscle was previously shown to be affected by dietary lipids . InteresFatty acid compositions of muscle and adipose tissue PL were also modified by the cafeteria diet. More specifically, the proportion of PUFA in PL was decreased and MUFA was increased in both tissues. These differences were more pronounced in PL from adipose tissue than from gastrocnemius, indicating that membrane phospholipids in adipose tissue are more susceptible to variations in dietary composition than skeletal muscle. Interestingly, reductions in the proportion of PUFA in PL from myocytes  and adipAs previously reported with Trecadrine, a selective β3-AR agonist , chronicAlthough the content and the fatty acid composition of TAG stored in adipose tissue was not affected by β3-AR agonist administration, the fatty acid profile of adipose PL was modified by the treatment. The β3-AR agonist was able to restore the proportion of PUFA to a level similar to the REF rats by increasing the percentage of arachidonic acid by 2 fold. As mentioned above, a greater percentage of PUFA in PL has been reported to enhance insulin sensitivity in adipose tissue. The changes in PL composition induced by the β3-AR agonist may be associated with the previously reported improved insulin sensitivity in adipose tissue , and theWhilst the effectiveness of β3-AR agonists is limited in humans, understanding the metabolic changes affected in rodents will provide insights into mechanisms, underlying insulin responsiveness in humans. The present study demonstrates that β3-AR agonist treatment not only limits the hyperinsulinemia induced by a cafeteria diet, but also partially prevents the associated alterations in adipose and muscle lipid composition. Particularly, activation of the β3-AR limits the intramyocellular DAG accumulation and the decrease in the proportion of PUFA in adipose tissue PL. This combined action may contribute to the beneficial effect of β3-AR agonists on insulin sensitivity.CAF rats: Rats fed with a cafeteria diet; DAG: Diacylglycerol; MUFA: Monounsaturated fatty acid; PKC: Protein kinase C; PUFA: Polyunsaturated fatty acid; REF rats: Rats fed with a chow diet; SFA: saturated fatty acid. TAG: Triacylglycerol; ZD-CAF rats: Rats fed with a cafeteria diet and treated with the β3-AR agonist ZD7114.