PRNP) contribute to the genetic determinants of Creutzfeldt-Jakob disease (CJD). Numerous polymorphisms in the promoter regions as well as the open reading frame of PRNP were investigated. Greater than 90% of Korean, Chinese, and Japanese carry the homozygote 129 MM codon. In Korea, polymorphisms have not been comprehensively studied, except codons 129 and 219 in PRNP among Korean CJD cases. Although polymorphisms at codons 129 and 219 play an important role in susceptibility to sporadic CJD, patients with other polymorphisms in PRNP exhibited critical distinctions of clinical symptoms.Polymorphisms of the human prion protein gene (PRNP were carried out among probable CJD patients in comparison with the results from magnetic resonance imaging (MRI) and electroencephalogram (EEG).The genetic analyses of The molecular analyses revealed that three mutations at codons D178N, E200K, and M232R in heterozygosity. Patients with the D178N and M232R mutations had a 129MM codon, whereas the patient with the E200K mutation showed 129MV heterozygosity. They all revealed strong 14-3-3 positive signals. The 67-year-old patient with the D178N-129M mutation showed progressive gait disturbance and dysarthria was in progress. The 58-year-old patient with the E200K mutation coupled to the 129MV codon had gait disturbance, dysarthria, agitation, and ataxic gait, and progressed rapidly to death 3 months from the first onset of symptoms. The 65-year-old patient with the M232R mutation showed rapidly progressive memory decline and gait disturbance, and died within 16 months after onset of symptoms.Despite differences in ethnicity, the clinical and pathological outcomes were similar to the respective mutations around the world, except absence of insomnia in D178N-129M subject. PRNP) on chromosome 20, and is categorized as genetic TSE. These reports have suggested the importance of PRNP mutations in familial CJD (fCJD). PRNP mutations have been discovered from more than 30 sites, and lead to amino-acid substitutions, premature stop codons, or the insertion of additional octapeptide repeats at the N-terminus [PRNP polymorphisms or mutations to predict disease occurrence.Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) fatal familial insomnia (FFI), and Kuru are transmissible spongiform encephalopathies (TSE) diseases in human. TSEs are typically fatal neurodegenerative diseases; 90% of CJD patients die within 1 year of diagnosis which occurs sporadically at an annual incidence of 1 per million populations . Among aterminus ,5. ThesePRNP. Fatal familial insomnia (FFI) seems to be associated with a D178N mutation and methionine at codon 129 of PRNP, whereas the phenotype of sCJD was observed for the D178N mutation with valine at codon 129 [As a rapidly progressing neurodegenerative disorder, the symptoms of CJD are characterized by progressive dementia, ataxia, and myoclonus . FamiliaThe clinical, EEG and neuroimaging features in patients with E200K-129M mutation were similar to sCJD. A typical EEG, with periodic spike and wave (PSW) complexes, was observed in about 75% of all patients . The levThe M232R mutation was reported in eight Japanese patients without any previous family history of neurodegenerative diseases . The cliCJD is clinically diagnosed with specific finding with magnetic resonance imaging (MRI), periodic sharp and wave complexes (PSWCs), and elevated 14-3-3 protein in the CSF -17. The PRNP at codons 178, 200-129, and 232 reported in other countries were first discovered among probable CJD patients in Korea. In addition, the level of 14-3-3 protein released into the CSF was studied and compared with the MRI/EEG results.In this study, the mutations of The clinical findings of probable CJD patients in Korea are summarized in Table A 67-year-old man was admitted to hospital because of progressive gait disturbance and dysarthria. He didn't have family history of similar problems. The subject also had extrapyramidal symptoms such as rigidity and bradykinesia, but no sign was evident of myoclonus, visual symptoms, pyramidal symptoms, or cognitive impairment. The cerebellar function test was conducted by tandem gait, heel-to-shin, finger-to-nose, and rapid alternating movements, and the results were normal. The laboratory findings for electrolytes, antibodies of the thyroid gland, liver function test, immunological test for syphilis, and viral markers were negative. His CSF was clear and colorless without any white blood cells. A few months after onset the patient's health deteriorated gradually into akinetic mutism, which progressed further.A 58-year-old man was hospitalized with gait disturbance and confused mentality, and there was no family history. He had fallen from a bike 2 weeks prior to his visit to hospital. He had stumbled, could not walk in a straight line, and frequently fell down. He presented with a history of chronic alcoholism and hypertension. His symptoms progressed and his condition deteriorated, with dysarthria and agitation. He could not perform the high cortical function test due to reduced attention, and he developed an ataxic gait. Although he looked cautious during the neuropsychological test, he showed frequent disorientation and confusion, and often fell into a stupor. He was not able to be woken from sleep on the 6th day after hospitalization, and on the 12th day developed aspiration pneumonia, which was treated. He later developed myoclonic movements in his limbs. The laboratory findings for electrolytes, antibodies of the thyroid gland, liver function test, immunological test for syphilis, and viral markers were negative. His CSF was clear and colorless without any white blood cells. He died 3 months after the first onset of neurological signs of CJD.A 65-year-old man presented with rapidly progressive memory decline and gait disturbance. He had no known diabetes, cardiac disease, or previous stroke except hypertension. Also, he had no family history of dementia or overseas travel, he was alert. Cranial nerve, sensory, reflex, cerebellar function, and motor power examinations did not revealed any abnormality. But he showed a bilateral swaying gait. The cognitive function test showed frontal lobe dysfunction, and his mini-mental state examination score was 22. His rating score for clinical dementia was 0.5, and his Barthel index was 14. The laboratory findings for electrolytes, antibodies of the thyroid gland, liver function test, immunological test for syphilis, and viral markers were negative. His CSF was clear and colorless without any white blood cells. His symptom progressed further and he died within 16 months after onset of symptoms.PRNP sequences revealed mutations at D178N-129M, E200K-129MV, M232R, 129MV, E219K, E219K-129MV, P102L, and V180I. Among them, further analyses were carried out on three patients with mutations at D178N-129M, E200K-129MV, and M232R under their consents for research only. Control DNA was obtained for analysis from healthy volunteers. This study was approved by the Institutional Review Board (IRB).Blood and CSF samples were collected from 99 probable CJD patients in Korea. Genetic analyses of their ® West Pico Chemiluminescent Substrate . CSF from definite sCJD patient was used as the positive control.A recombinant 14-3-3β plasmid cloned into the expression vector pEF6/V5-His TOPO was transiently transfected into 293T cells using FuGENE 6 . The CSF (32 μl) from probable CJD patients were separated on a 4–12% Bis-Tris gel, then transferred to a PVDF (polyvinylidene fluoride) membrane, followed by 5% dry milk. Polyclonal antibodies specific to 14-3-3β  were used for blotting. The signals were developed using a SuperSignalPRNP. The 30 cycles of PCR were performed as follows: 94°C for 1 min, 60°C for 45 sec, and 72°C for 1 min 30 sec. PCR products for sequencing were purified using a Gel Extraction Kit . DNA sequencing with primers, S13 and T2, was carried out using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI PRISM 3730xl DNA Analyzer . Results of DNA sequencing were analyzed using Sequencing Analysis Software .Genomic DNA of probable CJD patients was extracted from 200 μl blood samples using the QIAamp DNA Blood Mini Kit (Qiagen), following the manufacturer's protocol. Polymerase chain reaction (PCR) was performed with the S13 (5'-AAGCCTGGAGGATGGAACAC-3') and T2 (5'-CCCACTATCAGGAAGATGAG-3') primers, which amplified nucleotide 79–758 of ® 3100 Genetic Analyzer , and the data were analyzed with GeneScan software .SNaPshot was analyzed with the SNaPshot Multiplex Kit according to the ABI protocol, using PCR products amplified under the same conditions as for DNA sequencing . SNaPshot primers were S178 (5'-TTGATTGTGATATTGACGCAGT-3') for the codon 178 variants, S129 (5'-GTGGTGGGGGGCCTTGGCGGCTAC-3') for the codon 129 variants, S200 (5'-AAAAAAAAAACCACCAAGGGGGAGAACTTCACC-3') for the codon 200 variants, and S232 (5'-TATTACCAGAGAGGATCGAGCA-3') for the codon 232 variants. The samples were analyzed on an ABI PRISMEach PCR products amplified using the same condition as for the DNA sequencing for Cases 1 and 2, were digested with the restriction enzymes Tth111 I, Nsp I, and BsmA I (New England Biolabs). The PCR conditions for Case 3 were as follows: 30 cycles of 94°C for 30 sec, 60°C for 1 min, and 72°C for 1 min with the K-7 (5'-GTCACCACAACCACCAAGGG-3') and K-8 (5'-CAGGAAGACCTTCCTCATCC-3') primers. The PCR products for Case 3 were then digested with Nla III . The resulting fragments were separated by electrophoresis on a 10% TBE gel (Bio-Rad Ready Gel), and stained and visualized using EtBr.PRNP were different . Later, this patient was diagnosed as normal. The detected levels of 14-3-3 in CSF from Cases 1, 2, and 3 were similar, although mutated codons in PRNP from genomic DNA of probable CJD patients in Korea, three PRNP mutations were found  were heterozygotes for Met and Val, and the second set of two peaks was from the heterozygotes for Glu and Lys. The heterozygotes at codon 232 for Case 3 represented two peaks of Met and Arg .After amplifying and sequencing d Figure . In Cased Figure , two ove3 Figure . The PCRg Figure . Both CaPRNP PCR products from DNA sequencing and SNaPshot assay verified the three PRNP mutations. In Case 1, the mutation at codon 178 abolished digestion with Tth111 I. Conversion of one allele to Asn presented a band of 680 bp, while RFLP from the healthy control showed two bands of 223 and 456 bp, respectively  images Figure . For EEGIn Case 2, the diffusion-weighted MRI (DWI) showed higher signal intensities in the left side than the right side of basal ganglia or the cerebral cortex Figure . After 5In Case 3, DWI showed high signal intensities mainly from the cortex of the parieto- occipital and temporal lobes, and FLAIR images showed high signal intensity lesion from the cortex of temporal lobe Figure . The EEGThe genotype of the patients seemed to play an important role in the pathogenesis of CJD. The D178N mutation abolished the salt bridge between the conserved residues Asp-178-Arg-164, suggesting that the mutant protein had reduced thermodynamic stability . MoreoveThe D178N mutation with 129MM was the genotype for FFI and sCJD with 129MV or 129VV . JapanesPRNP polymorphisms in the Korean population will be investigated in the future. PRNP polymorphisms among Korean, Chinese, and Japanese populations will provide insights into the associations between polymorphisms, CJD susceptibilities, and their clinical phenotypic variations, especially when 129MM codon has been reported to be a dominant determinant in more than 90% of populations.The D178N-129M, E200K-129MV, and M232R mutations, which were previously reported from other countries, were identified for the first time among probable CJD patients in Korea. Diverse The authors declare that they have no competing interests.nd), SY(3rd) and SS designed, drafted and reviewed the manuscript. SY, SE, YJ, SH and SJ performed the clinical observations and provided MRI and EEG pictures. CK and JS supported the process of examination and reviewed the manuscript. YR overviewed the work. All authors read and approved the final manuscript.BY conducted the Western blotting, genetic analysis, RFLP and drafted the manuscript. SY(2The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/132/prepub


The Galliformes is a well-known and widely distributed Order in Aves. The phylogenetic relationships of galliform birds, especially the turkeys, grouse, chickens, quails, and pheasants, have been studied intensively, likely because of their close association with humans. Despite extensive studies, convergent morphological evolution and rapid radiation have resulted in conflicting hypotheses of phylogenetic relationships. Many internal nodes have remained ambiguous.Arborophila was the sister group of the remaining taxa followed by Polyplectron. This was followed by two major clades:  Francolinus) ) Pavo) and  Lophura) Syrmaticus) Perdix) Pucrasia) )  Tragopan))).We analyzed the complete mitochondrial (mt) genomes from 34 galliform species, including 14 new mt genomes and 20 published mt genomes, and obtained a single, robust tree. Most of the internal branches were relatively short and the terminal branches long suggesting an ancient, rapid radiation. The Megapodiidae formed the sister group to all other galliforms, followed in sequence by the Cracidae, Odontophoridae and Numididae. The remaining clade included the Phasianidae, Tetraonidae and Meleagrididae. The genus The traditional hypothesis of monophyletic lineages of pheasants, partridges, peafowls and tragopans was not supported in this study. Mitogenomic analyses recovered robust phylogenetic relationships and suggested that the Galliformes formed a model group for the study of morphological and behavioral evolution. Gallus gallus), green peacock (Pavo muticus) and turkey , among others. Many galliforms have beautiful ornamentations and they play an important role in hunting and entertainment. Regardless, these birds are best known for their importance in agriculture and as model organisms in scientific studies [The Galliformes, a well-known and widely distributed Order in Aves, contains about 70 genera and more than 250 species including the domestic chicken , Francolinus (Tribe Perdicini) and Bambusicola (Tribe Perdicni) clustered together, suggesting that pheasants and partridges were not monophyletic groups [Mitochondrial DNA analyses revealed that c groups ,12. Subsc groups , combinec groups ,20 and sc groups . Howeverc groups  conflictInternal nodes resolved by previous studies were generally very short. Further, bootstrap support values were very low and many species had unresolved relationships, suggesting a rapid radiation of the Phasianidae ,9. AlthoRecently, mt genomes have been widely used to reconstruct intractable phylogenies -33. In gThe general characteristics of mt genomes of 34 galliforms and six anserforms are summarized in Additional file P > 0.05). Only Alectoris chukar for CoxI and ND5, Alectoris lathami for ND5, and Bonasa bonasia for CoxII, ND2 and ND4 differed marginally significantly. However, all five outgroup taxa failed the stationarity tests for some data partitions as follows: Anas platyrhynchos for CoxI, CR, ND2 and ND5; Anser albifrons for CR, ND2 and ND5; Anseranas semipalmata for CR; Aythya americana for CoxI, CR, ND2 and ND5; Branta canadensis for CoxI, CR and ND5; and Cygnus columbianus for CR .Tests for stationarity of base composition among the taxa for each mt gene revealed that the Galliformes did not differ significantly in their base content  revealed a single, robust tree using MP and ML, and BI produced a summary of numerous trees  were 2,744 aligned sites in length. The tRNA dataset (combined 22 tRNA genes) contained 1,612 bp of aligned sites. The individual partitions  showed very limited power for phylogenetic inference, leaving many unresolved nodes  obtained less resolution compared to protein-coding genes  to form a single dataset with a length of 16,508 aligned sites for a genomic-level phylogeny . The BI owl Fig. .Regarding the phylogenetic positions of the Cracidae and Odontophoridae, the BI trees based on 10,502 nucleotide positions  and four nuclear introns , were similar in some respects, but have many differences. For example, within Gallus, they resolved the species relationships as  G. gallus) G. sonneratti) and our ML and BI analyses resolved them as  ). Whereas no nodal support was obtained in the former set of relationships, our ML and BI trees were strongly supported at each node  Coturnix and (2) gallopheasants, tragopans, turkey and grouse. In contrast, our analyses did not place Gallus near the base of the Phasianidae, but rather clustered it with Coturnix. This pair then clustered with Pavo, which then became the sister group of gallopheasants, tragopans, turkey and grouse  retrotransposable elements ,36. ThesMuscle or feather tissue was obtained from 14 species. Further, 26 additional complete mt genomes were obtained from GenBank for the Galliformes and Anserformes , and approximately 10 ng LA-PCR products. PCR amplifications were carried out using the following parameters: 95°C 4 min, 20 cycles of denaturation at 94°C for 1 min, annealing at 60-50°C , extension at 72°C for 1 min, and finally 15 cycles of 94°C 1 min, 50°C 1 min, 72°C 1 min. PCR products were cleaned using Watson RCR Purification Kits .FJ752423-FJ752436).PCR products were sequenced at least three times in both directions on an ABI 3730 Sequencer  using the ABI PRISM BigDye Terminator v3.0 sequencing kit. DNA sequences were edited using DNAstar Seqman software . The newly determined genomes were deposited in GenBank  implemented in PAUP* 4.0b10 . MP heur6 generations and sampled every 100 generations. When the log-likelihood scores were found to stabilize, a consensus tree was calculated after omitting the first 25% trees as burn-in.Bayesian inference (BI) was performed using MrBayes 3.1.2 . The BayIn order to detect the possible bias of substitution saturation, we plotted transitions and transversions against the pairwise sequence divergence using 12 mitochondrial protein-coding genes in DAMBE .Two additional weighting strategies were applied in the analysis of combined 12 protein-coding genes to avoid possible bias of nucleotide composition and saturation: (1) excluding the 3rd codon positions, and (2) recoding the 3rd codon position nucleotides to two-state categories, R (purine) and Y (pyrimidine), i.e., RY-coding. RY-coding can greatly improve consistency in phylogenetic resolution by reducing bias from differences in nucleotide composition ,52.CR) partitions were each used to reconstruct the phylogeny using BI.To examine the performance of individual genes, a partitioned Bremer support (PBS) analyses  were perCR) was also used to reconstruct the phylogeny using BI, ML and BP methods.To maximize the amount of phylogenetic information, the entire mt genome  All codon sites; (B) 1st codon position; (C) 2nd codon position; (D) 3rd codon position.Click here for fileBayesian tree based on 12 mitochondrial protein-coding genes. In order to emphasize the topology of Galliformes, we did not include the outgroup on the tree. Numbers are maximum likelihood bootstrap support and Bayesian posterior probabilities. Branches are drawn proportionally to the average number of expected DNA substitutions per site among all trees sampled after a burn-in period, as indicated by the scale at the bottom left. Species belonging to the Tribe Phasianini are marked in purple, and to Tribe Perdicini in green.Click here for fileSubstitution saturation of 12 mitochondrial protein-coding genes. Transitions and transversions plotted against the pairwise sequence divergence for 12 mitochondrial protein-coding genes. (A) 1st codon position; (B) 2nd codon position; (C) 3rd codon position; (D) all codon sites.Click here for filerd codon positionBayesian phylogenetic analyses of RY-coding and exclusion the 3. Bayesian phylogenetic analyses of two weighting strategies in the combined 12 protein-coding gene sets. Bayesian posterior probabilities >70% are indicated on the branches. (A) Recoding the 3rd codon position nucleotides as to two-state categories, R (purine) and Y (pyrimidine), (RY-coding); (B) Excluding the 3rd codon position.Click here for fileBayesian tree for 22 tRNA genes. Bayesian inference consensus tree for the Galliformes based on combined data from mitochondrial 22 tRNA genes. Anseriformes forms the outgroup. Bayesian posterior probabilities >70%, and maximum likelihood bootstrap proportions >50% are indicated on the branches.Click here for file12S rRNA and 16S rRNA genesBayesian tree based on combined data from . Bayesian inference consensus tree for Galliformes based on combined data from mitochondrial 12S rRNA and 16S rRNA genes. Anseriformes forms the outgroup. The Bayesian posterior probabilities >70%, and maximum likelihood bootstrap proportions >50% are indicated on the branches.Click here for fileBayesian analyses of individual mt genes. Bayesian inference analyses of individual mt genes and control region (CR). Each run was conducted with 5,000,000 generations and sampled every 100 generations. Bayesian Posterior Probabilities >70% are indicated on the branches. (A) ND1, 972 aligned sites; (B) ND2, 1,038 aligned sites; (C) ND3, 348 aligned sites; (D) ND4L, 291 aligned sites; (E) ND4, 1,377 aligned sites; (F) ND5, 1,818 aligned sites; (G) ND6, 519 aligned sites; (H) CoxI, 1,548 aligned sites; (I) CoxII, 681 aligned sites; (J) CoxIII, 783 aligned sites; (K) ATP6, 681 aligned sites; (L) ATP8, 165 aligned sites; (M) CytB, 1,137 aligned sites; (N) CR, 1,294 aligned sites; (O) 12S, 1,047 aligned sites; (P) 16S, 1,695 aligned sites.Click here for fileBayesian trees based on combined datasets of the Cracidae and Odontophoridae. Bayesian inference (BI) consensus trees based on combined datasets of the Cracidae and Odontophoridae. Anseriformes forms the outgroup. Bayesian posterior probabilities > 70% are indicated on the branches. (A) The BI tree for the dataset of 10,502 nucleotide positions for 11 species in the Cracidae (marked in red) and 40 galliform/anseriform birds that have complete mt genomes; (B) The BI tree for the dataset of 3,262 aligned nucleotide positions for 11 species in the Cracidae (marked in red), four in the Odontophoridae (marked in blue) and 40 galliform/anseriform birds.Click here for fileSource of sequence data for the Cracidae and Odontophoridae. No complete mitochondrial genomes were available for the Cracidae and Odontophoridae in GenBank. Thus, we mined GenBank and created a dataset with as many genes as possible for them.Click here for filePrimers for amplifying the complete mitogenomes. List of primers used in this mitogenomic study of the Galliformes.Click here for fileParameters of evolutionary models. Evolutionary models, log-likelihood values (-ln L), and settings identified by Modeltest for different DNA sequence datasets from the Galiformes.Click here for file


While Rb2 downregulation was observed in several malignant tumours including endometrial cancer, the role of p130 in breast carcinomas is still unknown. We investigated Rb2 protein expression in tumour tissue from 68 mammary and 41 endometrial carcinomas, 4 mammary cell lines, and normal tissue samples. Therefore, we performed Western blot experiments for Rb2, Rb, and the oestrogen and progesterone receptors . Weak or absent Rb2 expression was more often found in endometrial (59%) than in mammary carcinomas (24%). We found significant positive correlations of Rb2 expression with Rb, ER, and PR-B in breast cancer samples, and of Rb2 with Rb, PR-A, PR-B, and younger age in endometrial carcinomas. No significant associations with histological grading, stage, nodal involvement, or Ki67 staining were detected. Rb2 mRNA expression was studied by semi-quantitative RT-PCR in 56 endometrial or mammary tissue samples and correlated significantly with Western blot results. Our results indicate that loss of Rb2 expression, mostly by transcriptional down-regulation, may be associated with the development and dedifferentiation of most endometrial and a subset of mammary carcinomas. © 2001 Cancer Research Campaign


Drosophila and mice. This studies aim was to characterise the evolution of akirin gene structure and protein function in the eukaryotes.Akirins are nuclear proteins that form part of an innate immune response pathway conserved in akirin genes are present throughout the metazoa and arose before the separation of animal, plant and fungi lineages. Using comprehensive phylogenetic analysis, coupled with comparisons of conserved synteny and genomic organisation, we show that the intron-exon structure of metazoan akirin genes was established prior to the bilateria and that a single proto-orthologue duplicated in the vertebrates, before the gnathostome-agnathan separation, producing akirin1 and akirin2. Phylogenetic analyses of seven vertebrate gene families with members in chromosomal proximity to both akirin1 and akirin2 were compatible with a common duplication event affecting the genomic neighbourhood of the akirin proto-orthologue. A further duplication of akirins occurred in the teleost lineage and was followed by lineage-specific patterns of paralogue loss. Remarkably, akirins have been independently characterised by five research groups under different aliases and a comparison of the available literature revealed diverse functions, generally in regulating gene expression. For example, akirin was characterised in arthropods as subolesin, an important growth factor and in Drosophila as bhringi, which has an essential myogenic role. In vertebrates, akirin1 was named mighty in mice and was shown to regulate myogenesis, whereas akirin2 was characterised as FBI1 in rats and promoted carcinogenesis, acting as a transcriptional repressor when bound to a 14-3-3 protein. Both vertebrate Akirins have evolved under comparably strict constraints of purifying selection, although a likelihood ratio test predicted that functional divergence has occurred between paralogues. Bayesian and maximum likelihood tests identified amino-acid positions where the rate of evolution had shifted significantly between paralogues. Interestingly, the highest scoring position was within a conserved, validated binding-site for 14-3-3 proteins.akirins and provides insight into their multifaceted and conserved biochemical functions.This work offers an evolutionary framework to facilitate future studies of eukaryotic  Drosophila melanogaster immune deficiency (Imd) pathway, which responds to gram-negative bacterial infection  and suped . Whatevakirin duplication, Akirin1 evolved at a faster rate than Akirin2. This result was confirmed by significant relative rate test results for several vertebrate lineages (result not shown). To examine whether this difference in evolutionary rate was accompanied by altered selective constraints, we examined pairwise rates of synonymous (dS) and non-synonymous (dN) substitutions between Akirin1 and 2 for several vertebrate lineages. Two approaches were implemented: firstly, the likelihood method of Goldman and Yang [It is known that Akirin1 and Akirin2 differ in at least one function [and Yang  and secoand Yang . Both reakirin function'). Thus, there are many potential residues of functional significance in Akirin (i.e. within putative binding sites) that may have evolved at different rates between Akirin1 and 2. Furthermore, binding sites for 14-3-3 proteins have been biochemically confirmed in Akirin2 [θ) between Akirin1 and 2 clusters in the phylogenetic tree topology provided  and ide Akirin2 ,29). Ini Akirin2  to test  Akirin2 ). The mo Akirin2 . To summnificant ,33). NexThe extreme N-terminus (first 30 residues) and C-terminus (last ~70 residues) of Akirin proteins are clearly under strong purifying selection based on the near absence of fast-evolving sites 9). As exAlmost all of the highest scoring candidate positions for functional divergence between Akirin paralogues are found in the middle region of the protein , which also has numerous sites that evolved at a significantly higher rate in both Akirin1 and 2 compared to the average of all positions . Therefore, the number of potential 14-3-3 binding sites in Akirin proteins increased rapidly at the base of metazoan evolution. However, sites are absent or greatly reduced in certain metazoan lineages, including D. melanogaster (0 sites), Anopheles gambiae (0 sites), Lumbricus rubellus (0 sites) and Caenorhabditis elegans (1 site) (not shown). The preferred binding motifs of 14-3-3 proteins are Arg-Ser-x-Ser-x-Pro and Arg-x-x-x-Ser-x-Pro, although functional variations in these motifs are tolerated [Of the five 14-3-3 protein-binding sites identified in rat Akirin2 [olerated . Almost akirin genes in order to provide novel insight into their biochemical functions. We hope that this will prompt the sharing of akirin literature between researchers from different fields and open up new avenues of investigation.In this section, we combine the findings of this study with available literature on the known roles of akirin and akirin2 in flies and mice respectively [akirin in the embryos of the nematode C. elegans by RNAi knockdown was also lethal ( search term: E01A2.6). Further, RNAi knockdown of akirin in ticks  dramatigen 4D8',. These fmammals:  and broa; ticks: ; zebrafi; ticks: ; mammalsterature ,31.akirin1 is not essential for embryonic development, and has even been lost in the class Aves. Thus, relative to Akirin and Akirin2, Akirin1 has diverged in at least one essential function  has a role in regulating vertebrate myogenesis, as it was identified in mice from a suppression subtraction hybridization cDNA library produced using myostatin-null mice as the 'tester' material [akirin1 was reportedly upregulated in the muscles of myostatin-/- mice [akirin1 proximal promoter [akirin1 also functions in myogenesis in flies. Specifically, Akirin (as Bhringi) bound the bHLH factor Twist and this interaction was necessary for the normal expression of Twist target proteins [akirin had considerable defects in muscle mass and morphology [akirin1 in mdx mice, where muscle mass, fibre size and structural integrity was markedly increased [akirin1 in regulating muscle growth may be conserved from the akirin proto-orthologue. If the function of akirin1 in amniote muscle growth is essential, then its absence in birds, where muscle physiology is strongly conserved with mammals, particularly in terms of the functions of key genes (e.g. myostatin), could only be fulfilled by akirin2.In vertebrates, material . Myostatmaterial . akirin1-/- mice . Mstn prpromoter . Interesproteins , represerphology . This isncreased . Thus, takirin2 (as FBI1) was also shown to promote carcinogenesis by interacting with the phosphoserine-threonine-binding protein 14-3-3β [akirin2 was also upregulated in tumour cell lines and its mRNA downregulation reduced tumour metastasis by inducing the expression of MAP kinase phosphotase 1 (MKP1), which reduced the activation of the extracellular-signal regulated kinases (ERKs), ERK1 and ERK2 [akirin2-14-3-3β complex functioned as a transcriptional repressor of the MKP-1 promoter [akirin1 cannot be excluded. However, distinct evolutionary rates in positions within, or adjacent to 14-3-3 binding sites in Akirin1 and Akirin2 are probably important explanatory variables underlying their functional divergence . The 14-3-3β  acting t 14-3-3β . akirin2nce fig. . Interes pathway ,44. akirakirin2 functions at a level close to, or downstream of NF-κB to selectively regulate some of its target genes [IL-6 and RANTES, presumably disrupting the interaction of p65 and chromatin [IL-6, RANTES) were strongly repressed in akirin2 knockout mice following TLR, IL-1β and TNFα treatment [akirin2 knockout mice is accounted for by altered 14-3-3-protein activity. In addition to a predicted interaction with 14-3-3 proteins to regulate chromatin, fly Akirin (as Bhringi) was shown to bind Bap60 [In vertebrate immune response signalling pathways, et genes . Since aet genes . 14-3-3 et genes ,40. Impoet genes . IκB is et genes  and furtet genes . TNFα tret genes . Furtherhromatin . It was hromatin . Interesnd Bap60 , a DNA bnd Bap60  which isnd Bap60  and withnd Bap60 , , Arthropods , nematodes (C. elegans) and Fungi (Saccharomyces cerevisiae).BLASTp searches of the NCBI akirin1/akirin2 orthologues in a broader range of metazoans, directed tBLASTn searches of NCBI nucleotide and EST databases were performed for the following taxa: Acoelomorpha, Annelida, Arthropoda, Brachiopoda, Bryozoa, Chaeognatha, Chordata , Cnidaria, Ctenophora, Echinodermata, Entoprocta, Hemichordata, Mollusca, Nematoda, Nematomorpha, Nemertea, Onychophora, Placozoa, Platyhelminthes, Porifera, Rotifera, Tardigrada and Xenoturbellida. Non-metazoan eukaryotes were also screened by the same approach, including the following taxa: Amoebozoa, Choanoflagellata, Chromalveolata, Fungi, and Plantae. Finally, genome databases at the DOE Joint Genome Institute , Welcome Trust Sanger Institute , Arabidopsis Genome Initiative  and TIGR Rice Genome Annotation  were BLAST screened for akirin orthologues for the following taxa: Amoebozoa , Archea , Bacteria , Choanozoa (Monosiga brevicollis), Chromalveolata , Excavata , Fungi , Placozoa (T. adhaerens) and Plantae .To identify akirin1 and akirin2 were constructed using data manually obtained from release 50–52 Ensembl genome assemblies for H. sapiens, M. musculus, G. gallus, X. tropicalis, D. rerio and G. aculeatus. The genomic neighbourhoods surrounding H. sapiens akirin1/akirin2 were used as a starting reference. The intron-exon organisation of eukaryotic akirin orthologues was established by loading genomic and corresponding cDNA sequences into Spidey [Synteny maps for the genomic neighbourhoods surrounding o Spidey . PSORTIIo Spidey  was usedakirin1/akirin2 in Urochordates (H. roretzi), Cephalochordates (B. floridae), Hemichordates  and Echinoderms (Strongylocentrotus purpuratus). Sequence alignment was performed using PROMALS [. The first output was improved by removing indels and low scoring regions of the alignment as well as manual checking of alignment quality. ML was performed using PhyML [. The JTT substitution model was utilised with concurrent estimation of the gamma distribution parameter. 1000 bootstrap replicates were sampled to obtain a measure of branch confidence. The Bayesian approach was implemented in MrBayes3.12 [27 full coding amino acid sequences of Akirin were used for phylogenetic analysis. This included Akirin1/Akirin2 sequences spanning broad vertebrate taxa as well as deuterostome outgroups representing the single invertebrate gene related to both vertebrate  PROMALS  at . Theng PhyML  at . Theayes3.12  with estayes3.12 , in eachayes3.12  using thakirin1 and akirin2 containing chromosomal tracts in at least two vertebrate classes  and non-synonymous (dN) substitution rates for Akirin1 and Akirin2 orthologues from mammals, birds, amphibians and teleost fishes as performed by two methods (see main text). Also shown are the accompanying dN/dS ratios.Click here for fileAmino acid alignment of 27 Akirin sequences showing the full results of the rate-shift likelihood ratio test. This file shows an output from the rate-shift analysis server  where the LRT (ref [P. marinus), H.s (H. sapiens), X.t , D.r (D. rerio), T.r (Takifugu rubripes), G.g , G.a (G. aculeatus), O.l (Oryzias latipes), P.p (P. promelas), S.a (Sparus aurata).LRT (ref  in the mClick here for file


Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation.Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear β-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors . CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors.These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. In recent years, substantial experimental evidence has been generated in support of the role of a small population of self-renewing cells that could sustain malignant growth lowCSCs were identified utilizing flow-cytometry-based cell sorting and NOD/SCID mice xenografting. CSCs express tissue-specific cell surface markers: e.g., breast CSCs express CD44+/CD24Flow-cytometry-based cell-sorting which enables the isolation of a “side population” (SP) with enriched cancer stem cell activity was described by Goodell et al Due to their high drug resistance and tumorigenicity, CSCs are thought to be responsible for tumor regeneration after chemotherapy In the present study drug surviving cells (DSCs) were isolated from human cancer cell lines treated with cisplatin, doxorubicin, or etoposide. Isolated DSCs exhibited high clonogenic capacities, enrichment with SP cells, expression of CSC cell surface and embryonic stem cell markers, a capacity for self-renewal, the generation of differentiated progeny, and high tumorigenic potential following SCID mice transplantation. We concluded that these DSCs were CSCs.It has also been suggested that CSCs have high metastatic potential It continues to be unclear what properties of CSCs confer increased tumorigenicity and metastatic potential. We hypothesized that the tumorigenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors, which stimulate tumor cell proliferation as well as promote formation of the tumor vascular system in order to provide oxygen and nutrients for local tumor growth or distant growth after dissemination of tumor cells into different anatomical locations. Thus, the highly efficient production of growth and angiogenic factors is a fundamental property of tumor-initiating cells. VEGF is a potent angiogenic factor in vitro on cultured cells and in vivo utilizing the human tumor xenografted model in SCID mice. Human tumors growing in SCID mice consist of human tumor cells and murine stroma. Sonicated extracts of the xenografted human tumors contained cytokines produced by human tumor cells as well as by murine stromal cells. Concentrations of each type of cytokines can be analyzed using multiplex kits developed specifically for the detection of human or murine cytokines. This could provide information about the cytokine network produced by CSCs and their ability to stimulate stroma formation.Therefore, in the present study, we performed a comprehensive analysis of various cytokines, chemokines, and growth factors produced by parental H460 tumor cells and isolated CSCs using multiplex xMAP technology , which allows simultaneous analysis of numerous soluble factors. This analysis was performed Our studies demonstrate that drug surviving lung tumor cells have the characteristics of CSCs, and produce elevated levels of multiple cytokines, chemokines, growth and angiogenic factors and their receptors. These findings bring new insight to our understanding of the mechanisms responsible for high tumorigenic and metastatic potential of lung CSCs and their ability to survive chemotherapy.Human cultured cancer cell lines, OVCAR-3 (ovarian), MCF-7 (breast), and H460 (lung), were obtained from the American Type Culture Collection . Cells were grown in culture media, as recommended by ATCC, supplemented with 20% FBS .Cisplatin, doxorubicin, etoposide, and Hoechst 33342 were from Sigma-Aldrich . Fluorochrome-conjugated antibodies against human CD24, CD34, CD44, CD24, CD117, CD90, VLA-4, and VLA-5 were from Beckman Coulter . Antibodies against FGFR2, VEGFR1, VEGFR2, and CXCR1, 2, 4 were from R&D Systems INC. . The antibody against VLA-6 was obtained from AbD Serotec . Antibodies against CD 133 and cytokeratins 8/18 were from Abcam Inc. . Alexa Fluor®-488 conjugated mouse antihuman TRA-1-60, TRA-1-81, SSEA-1-4, and the antibody against human β-catenin were purchased from BD Biosciences Inc. . The antibody against phosphor-β-catenin was from Cell Signaling Technology Inc. . An embryonic stem (ES) marker sample kit, designed for detection of SSEA-1, 3, 4, TRA-1-60, TRA-1-81, and Oct-4, was obtained from Chemicon International . Alexa Fluor®-488 phalloidin and secondary Abs conjugated with Alexa 488, 546, and 680 were from Molecular Probes .2 in 100 mm2 Petri dishes or at a density of 0.5 cells/well in 96-well plates, and cultured for 14 days. For colony counting, cells were fixed and stained with Coomassie brilliant blue.Cells were plated at a density of 10–50 cells/cmFor side population (SP) analysis of cells we used standard protocol Cells were fluorescently stained as described To detect intracellular proteins, cells were fixed, permeabilazed, and incubated with primary antibodies against embryonic stem cells markers, β-catenin, and cytokeratins 8/18 for 1 h and with secondary antibodies conjugated with Alexa 488, 546, or 680 fluorochromes (Molecular Probes/Invitrogen) for 1 h. For actin cytoskeleton staining, cells were incubated with Alexa Fluor 488 Phalloidin for 1 h. Cell nuclei were then stained with Hoechst 33342 at 2 µg/ml for 20 min to identify individual cells and to optimize focusing.To stain tumor spheres, all manipulations were done under the microscopic control: tumor spheres were gently placed into individual wells of ultra low adherent 96 well plates, and incubation with primary and secondary antibodies was the same as described above for the adherent cultures.All incubation and fixation procedures were performed at room temperature.The Cellomics ArrayScan HCS Reader  was utilized to collect information on distribution of fluorescently labeled components in the stained cells. The ArrayScan HCS system scans multiple fields in individual wells, acquiring and analyzing each of the cell images according to defined algorithms. The scanner is equipped with emission and excitation filters  for selectively imaging fluorescent signals. Data were captured, extracted, and analyzed with ArrayScan II Data Acquisition and Data Viewer version 3.0 (Cellomics), Quattro Pro version 10.0.0 , and MS Excel 2002 .Suspension growth was assessed in methyl cellulose-based (MC-based) medium as described 4 cells/mL on 96-well plates precoated with Collagen IV (BD Biosciences) in culture media supplemented with 10% FBS without growth factors and transferred into new plates when cultures reached confluence. To test the self-renewing potential of differentiated cells, cells were transferred into semisolid serum-free media supplemented with EGF, FGF, and insulin and their ability to form tumor spheres was evaluated as described above. To perform phenotypic characterization of cells from spheres and cells after differentiation, cells were seeded in 96-well plates (5×103 cells/well) and stained with various antibodies as described above.Cells dissociated from spheres (third generation) were plated at 1×10H460 parental cells, cells obtained from lung cancer spheres, and three weeks after CSCs differentiation cells were plated into 96-well plates precoated with Collagen IV (BD Biosciences) and cultured in RPMI 1640 media supplemented with 10% FBS. After 24 h doxorubicin and cisplatin were added at the final concentrations . After 72 h of treatment, cells were stained with Hoechst 33342, and the number of cells per well were counted with Cellomics Array Scan VTI , as described The migration and invasion activity of tumor cells in response to human recombinant IL-8 (100 ng/ml) was measured in BD BioCoat Matrigel Invasion Chambers  according to manufacturer protocol.Experiments were carried out in accordance with guidelines provided by the Pittsburgh University Institutional Animal Care and Use Committee (IACUC) and the National Institute of Health Guide for the Care and Use of Laboratory Animals. SCID mice, 7−8 weeks old , were maintained in the animal facility of the University of Pittsburgh Cancer Institute (UPCI).3–5×105 cells per mouse (5 mice per group) without Matrigel. Tumors were measured twice a week. Mice were sacrificed when their tumors measured approximately 2 cm in diameter.To compare tumorigenic properties, H460 cells and DSCs were harvested and injected (in 200 µl of PBS) subcutaneously (s.c.) into SCID mice at concentrations of 5×104 cells were inoculated intravenously (i.v.) into the tail vein of SCID mice. The SCID mice were T, B, and NKT cell-deficient but had active NK cells that could eliminate human tumor cells circulating in the blood stream. To deplete NK cells, SCID mice were inoculated intraperitoneally (i.p.) with 0.2 ml of anti-asialo GM1 IgG (diluted 1:20) 24 h before i.v. inoculation of tumor cells To analyze the ability of H460 cells and CSCs to form metastases, 5×10Tumors grown s.c. in SCID mice were removed and snap-frozen in liquid nitrogen. Frozen tumor tissues were cut, sonicated for 20 s, and centrifuged at 15,000 g for 10 min to remove cell debris. Tumor extracts were stored at −80 °C.www.upci.upmc.edu/facilities/luminex). Mouse cytokines were analyzed using 19-plex kit for IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, MIG, GM-CSF, MIP-1α, IL-12p40/p70, KC, TNFα, MCP-1, VEGF, and bFGF (Invitrogen/Biosource). Analyses of tumor supernatants and sonicated tumor extracts were performed in 96-well micro plate format according to manufacturers' protocols as previously described Analysis of human cytokines and growth factors in cell culture medium and in sonicated tumor lysates was performed using multiplexing xMAP technology . Multiplex kits for detection of 49 human cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-13, IL-15, IL-17, GM-CSF, IFN-α, TNFα, MCP-1, MCP-2, MCP-3, IP-10, MIP-1α, MIP-1β, RANTES, VEGF, bFGF, G-CSF, EOTAXIN, HGF, MIG, GROα, sIL-2R, sVCAM-1, CTACK, LIF, M-CSF, NGF, PDGF-BB SCF, SCGF-β, SDF-1α, TNF β, TRAIL IFN-γ, EGF, TNFRI, TNFRII, DR5, IL-1Rα, and sIL-6R were purchased from BIO-RAD Laboratories . Multiplex kit for detection of sFas, sFasL, TGFα, Fractalkine, sCD40L, TRAP, CS154, MIF, sVCAM-1, sICAM-1, MPO, Adiponectin, MMP-9, and tPAI-1 were purchased from Linco/Millipore . The kit for detection of MMP-2 and MMP-3 was purchased from R&D Research . The multiplex kit for detection cancer antigens CEA, CA-125, CA 19-9, CA 15-3, CA72-4, AFP as well as mesothelin, IGFBP-1, kallikrein 10, EGFR, ErbB2, and Cyfra 21-1 were custom-made at the UPCI Luminex Core Facility  H460 cells were treated with cisplatin (1–5 µM), etoposide (1–5 µM), or doxorubicin (0.067–0.125 µg/ml) for 3 days. A vast majority of the cells died. Over the next 7 days some surviving cells resembled senescent cells with enlarged and flattened morphology The clonogenic capacity of parental H460, OVCAR3, and MCF7 cells and DSC populations was tested as described in Analysis of SP fractions revealed that tested parental cell lines differed in the proportion of SP fraction, ranging from 0.6% in OVCAR3, 0.5% in MCF7, to 5.2% in H460 cells . SP cellThe Cellomics Array Scan HCS Reader (Cellomics/ ThermoFisher) was used for imaging and analysis of expression of CSCs and embryonic stem cell markers in DSCs. This approach is based on a combination of microscopy and flow cytometry methods in a 96-well format. The advantages of the approach include: 10 times less cells are needed than for flow cytometry analysis, multi-spectral fluorescence micro-imaging is automated, and images are stored, visualized and analyzed using powerful software applications.The analysis revealed that the DSC population from MCF-7 cells were CD44 positive with low levels of CD24 expression (data not presented), which corresponds to the previously identified phenotype of breast CSCs To date, human lung CSCs are poorly characterized Lung cancer cells are known to express type I CK18 and type II CK8 cytokeratins, known differentiation markers in these cells Wnt signaling proteins have been shown to play a role in controlling stem cell self-renewal. β-catenin is a key player in the Wnt pathway, transmitting Wnt signals to the nucleus and playing a crucial role in tumorigenesis in vitro migration and invasion assay using the IL-8 as the chemoattractant. Whereas, only 13.7% of parental H460 cells invaded through the Matrigel, 87.5% of DSCs were invasive.Our morphological analysis of DSCs and parental H460 cells revealed differences in cell shape and adhesion properties, suggesting potential differences in their cytoskeletal organization. To test this hypothesis, we used Alexa 488 phalloidin to visualize the F-actin. As shown in Adhesion molecules, including integrins, facilitate cell survival signaling and are involved in motility, intercellular adhesion, chemotaxis, and metastasis in vitro assay of self-renewal potential The ability to self-renewal and generate differentiated progeny are fundamental properties of CSCs. Tumor sphere generation is an The differentiation potential of cells from third generation lung cancer spheres was evaluated. Cells dissociated from spheres were cultured in RPMI 1640 medium supplemented with 10 % FBS in plates precoated with Collagen IV to improve cell adhesion. After 3 weeks of culture under adherent conditions, the cells acquired the typical morphologic features of parental H460 cells with increased expression of the differentiation marker cytokeratins 8/18 and loss of expression of CSC marker CD133 .Next, we analyzed the self-renewal potential of differentiated cells. After 3 weeks of culture under adherent conditions, cells were transferred onto low-adherent plates and cultured in serum free stem cell medium. Tumor sphere formation was evaluated. Cells maintained under differentiating conditions for 3 weeks demonstrated a substantial reduction in their ability to form spheres, indicating a reduction in their self-renewal potential .We considered the question of whether chemotherapeutic drugs could select cells with self-renewing characteristics from already differentiated tumor cells. Cells that were differentiated for 3 weeks under adherent condition were treated with cisplatin (1 µM) for two days and high proportion of these tumor cells were killed by drug. The ability of cisplatin surviving cells to form tumor spheres in serum free medium supplemented with growth factors was analyzed. Untreated differentiated tumor cells displayed a low ability to form spheres, while cells that survived cisplatin treatment showed a high capacity for sphere formation and self-renewal . These dTaken together, all these data strongly indicate that DSCs express markers conventional for CSCs (CD133), ESC markers , low levels of differentiation markers CK8/18, and demonstrate a capacity for self-renewal and differentiation.As shown above  parental3–5×105 cells without Matrigel which provides artificial environment, stimulates production of various cytokine, and angiogenesis. As shown in 3–5×105 DSC cells, whereas no tumor growth was observed after inoculation with 5×103 H460 cells. H460 cells grew in four out of five SCID mice inoculated with 5×104 cells, and it required an injection of 5×105 H460 tumor cells to develop tumors in 100% of SCID mice. Thus, DSCs demonstrated a substantially higher tumorigenic ability than H460 cells. Moreover, all tumors that developed from DSCs grew faster than those developed from parental cells as assessed by the time required for mice to bear tumors of 2000 mm3. All mice bearing DSC-derived tumors were sacrificed 2 wk earlier than animals inoculated with parental H460 cells. Tumor samples were frozen and used subsequently for cytokine analysis.To compare the tumorigenic potential of drug-isolated CSCs in comparison with H460 cells, SCID mice were inoculated s.c. with 5×104 H460 cells and 5×104 DSCs were inoculated i.v. into SCID mice. Sixty days after inoculation, metastatic nodules were found only in the lungs. It was also observed that parental H460 cells and DSCs differed dramatically in their capacity to develop lung metastases in SCID mice  in CSC-derived tumor lysates as compared to H460-derived tumor lysates. In addition, increased levels of stroma-derived factor-1α (SDF-1α) and stem cell factor (SCF) were found in lysates of CSC-derived tumors . CSCs alTaken together, these data demonstrate that high tumorigenic and metastatic potentials of CSCs correlate with superior production of angiogenic and growth factors involved in cell proliferation and angiogenesis. Increased levels of SCGF-β, SDF-1α, and SCF in tumors from CSCs are indicative of their stem cell origin.in vitro also showed differences in cytokine secretion. Lung CSCs produced twenty-fold more bFGF than H460 cells  as well as CA 125 and CA 72-4 when compared with parental cells.Many cancer-associated antigens are encoded by genes normally expressed in germ cells, trophoblasts, and embryonic cells To measure cytokines produced by host stroma, multiplex kits for detection of 19 murine cytokines were used. Most factors were present in mouse tumor stroma at low or undetectable concentrations; however, levels of mouse proangiogenetic cytokines VEGF, bFGF, MCP-1, and MIP-1α in CSC-derived tumor samples were significantly higher than those in parental H460 extracts . These rin vivo and in vitro by CSCs and H460 cells show multiple differences in the range and amount of cytokines, thus highlighting the advantage of CSCs in proinflammatory niche formation and metastatic ability. Cytokines and growth factors exert their functions by binding to their respective receptors. Therefore, next we compared expression of growth and angiogenetic factors receptors in parental H460 cells and lung CSCs growing in adherent condition and in tumor spheres.Taken together, the comparative analysis of human factors produced Lung CSCs produced three-fold increased levels of VEGF , a potenWe used ArrayScan®VTI HCS Reader (Cellomics Inc) to identify VEGFR1 and VEGFR2 receptor expression in parental H460 cells and lung CSCs cultured under adherent conditions for 8 h. Both H460 parental tumor cells and CSCs expressed VEGFR1 . Howeverin vivo and vitro  is well known multifunctional prosurvival and proinflammatory chemokine involved in tumorigenesis, angiogenesis, and metastasis formation in vivo  and in vin vitro . We alsoin vitro , 10 as win vitro .Stromal derived factor-1  and its receptor, CXCR4, were significantly elevated in lung CSCs when compared with parental H460 cells .Our results clearly show that lung CSCs are characterized by higher production of growth and angiogenetic factors as well as higher expression of their receptors. These upregulations in cytokine network could serve as the basis for the enhanced tumorigenic and metastatic potentials of lung CSCs.Our findings demonstrate that CSCs are resistant to chemotherapeutic drugs and can be selected from the parental population of human H460 lung tumor cells by treatment with drugs. Cells, which survived drug treatment, have all the properties of CSCs. Indeed, DSCs had high clonogenic efficiency, were enriched in SP phenotype, and expressed markers associated with CSCs, such as CD133. Expression of CD133 was found in CSCs of several different cancers, including brain, prostate, and pancreatic Self-renewal and the ability to generate differentiated progenitors are considered fundamental properties of CSCs Finding that lung CSCs are resistant to different drugs  suggests the involvement of the complex mechanisms in their drug resistance that require further investigation. It is intriguing that changes during differentiation lead to the drug sensitivity of their differentiated progenitors.in vitro for many years put forth the idea that CSCs may be important for the maintenance of the tumor cell population not only in vivo but also in vitro. Elimination of drug sensitive differentiated tumor cells and enrichment of CSCs following drug treatment observed in our in vitro model of human lung cancer suggest that similar selection of drug resistant CSCs could be observed in clinical practice during chemotherapy. Recently it was reported that breast tumors from patients treated with chemotherapy contained higher proportion of CD44+, CD24low cells with CSC properties than breast tumors from untreated patients The presence of CSCs in tumor cell lines propagated 4) of tumor cells into SCID mice, DSCs formed numerous metastatic tumors in the lungs, whereas parental H460 cells failed to form any metastases in the majority of mice.High tumorigenic potential is a hallmark of CSCs. We found that DSCs, in comparison to parental H460 cells, have higher tumorigenic potential following s.c. inoculation into SCID mice. It has also been suggested that CSCs have high metastatic potential and can initiate the formation of distant metastases in vitro and in vivo experiments demonstrate that DSCs possess all known characteristics of CSCs. What remains unknown; however, are the precise mechanisms by which CSCs are highly tumorigenic and metastatic. We found increased levels of adhesion molecules  as well as MMP2 and MMP3 that could be contributing factors to the metastatic potential of CSCs. Recently it was shown that the CD133+ CXCR4+ pancreatic CSC population is the only cell population responsible for tumor metastasis in pancreatic cancer Thus, our The high tumorigenic and metastatic properties of CSCs could be based on their increased ability to produce cytokines, chemokines, and angiogenic and growth factors, which, through autocrine and paracrine mechanisms, stimulate proliferation of tumor cells and migration and proliferation of stromal cells to form of a network of blood vessels that support tumor growth. We performed a comprehensive analysis of these factors in sonicated lysates of xenografted tumors derived from parental H460 cells and CSCs. We found that CSCs, in comparison with H460 tumor cells, produced up to two to threefold higher level of angiogenic and growth factors . Moreover, lung CSCs showed higher levels of VEGFR1 and FGFR2 receptors. FGFb is a known fundamental growth factor, supporting the maintenance of embryonic stem cell and CSC populations CSC-derived tumors contain increased levels of proinflammatory cytokine IL-6 and chemokines IL-8 (CXCL8), MCP-1 (CCL2), and MIP-1α (CCL15), which are potent stimulators of angiogenesis and tumor cell proliferation and play an important role in tumor progression and metastasis in a variety of human cancers, including lung cancers Human tumors growing in SCID mice consist of human cells and murine stroma. Multiplex analysis of murine cytokines and growth factors revealed an increased level of murine angiogenic factors VEGF, bFGF, as well as MIP-1α and MCP-1 in CSCs-derived tumors in comparison to parental H460 tumors growing in SCID mice, indicating that CSCs more efficiently stimulate murine proangiogenic factors and blood vessel formation to support the growth of xenografted human tumor.Drug resistance of CSCs represents a serious obstacle for current chemotherapy-based treatment strategies, for which alternative approaches must be considered Clinically, chemotherapy is typically administered in several cycles separated by three-week intervals in order to allow the body to restore hematopoietic and other normal cells damaged by drugs. However, some reports indicate that during this resting period tumor cells can aggressively repopulate the tumor and restore pre-treatment tumor size Further characterization of lung CSCs and the mechanisms of their drug resistance, as well as an investigation of their complex cytokine network, may provide key information on relevant pathways to be targeted to increase the therapeutic response.


Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with a dismal prognosis and no effective conservative therapeutic strategies. Although it is demonstrated that histone deacetylases (HDACs), especially the class I HDACs HDAC1, 2 and 3 are highly expressed in this disease, little is known about HDAC isoenzyme specific functions.Depletion of HDAC2, but not HDAC1, in the pancreatic cancer cell lines MiaPaCa2 and Panc1 resulted in a marked sensitization towards the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Correspondingly, the more class I selective HDAC inhibitor (HDACI) valproic acid (VPA) synergized with TRAIL to induce apoptosis of MiaPaCa2 and Panc1 cells. At the molecular level, an increased expression of the TRAIL receptor 1 (DR5), accelerated processing of caspase 8, pronounced cleavage of the BH3-only protein Bid, and increased effector caspase activation was observed in HDAC2-depleted and TRAIL-treated MiaPaCa2 cells.Our data characterize a novel HDAC2 function in PDAC cells and point to a strategy to overcome TRAIL resistance of PDAC cells, a prerequisite to succeed with a TRAIL targeted therapy in clinical settings. In addiIn this study we show that specific depletion of HDAC2, but not HDAC1, sensitizes PDAC cells towards tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, suggesting a new therapeutic strategy.We recently observed the HDAC2 mediated control of the DNA-damage response in PDAC cells . To inveSince valproic acid (VPA) is a more class I specific HDACI and known to deplete HDAC2 via a proteasomal pathway ,15, we vSince HDAC-dependent non-redundant functions are ill defined, we tested whether HDAC1-depletion also sensitizes for TRAIL-induced apoptotsis. Transfection of HDAC1 siRNA into MiaPaCa2 and Panc1 cells resulted in a specific knockdown of HDAC1, whereas HDAC2 expression was not affected figure . NeverthRecently we observed that depletion of HDAC2 results in an increased expression of the pro-apoptotic BH3-only protein NOXA in PDAC cells . To inveXL was observed in HDAC2-depleted MiaPaCa2 cells  or the mitochondrium were described in PDAC cells ,19. Howeescribed ,34-43. Ubutyrate . The faipoptosis . Here, apoptosis . Consistpoptosis . Alike, poptosis  and thatpoptosis  or a p65poptosis  resultedHDACI sensitize cancer cells towards TRAIL and several mechanisms, like upregulation of TRAIL and TRAIL receptors, downregulation of c-Flip, XIAP or members of the anti-apoptotic bcl2 family members as well as the modulation of NF-κB activity were shown to contribute ,49. As oIn summary, we have described a novel non-redundant HDAC2 function in PDAC cells that provides the rationale for further preclinical evaluation of a HDACI (HDAC2 isoenzyme-specific) and TRAIL combination therapy. Furthermore, our experiments point to a way to overcome TRAIL resistance of PDAC cells, needed to succeed with a TRAIL targeted therapy in clinical settings.The pancreatic cancer cell lines MiaPaCa2 and Panc1 were cultivated as recently described . ValproiAll data were obtained from at least three independent experiments performed in triplicate, and the results are presented as mean and standard error of the mean (S.E.M.). To demonstrate statistical significance a two-tailed Student's t-test was used. p-values are indicated and * denotes a p-value of at least < 0.05.Total RNA was isolated from pancreatic carcinoma cell lines using the RNeasy kit  following the manufacturer's instructions. Quantitative mRNA analyses were performed as previously described using real-time PCR analysis  ,53. ExprXL ; NOXA, mcl1 ; c-Flip, cleaved PARP, caspase 8 ; HDAC1 ; β-actin . One representative western blot out of at least three independent experiments is shown. Western blots were quantified using Odyssey Infrared Imaging System , assuring measurements in the linear range.Whole cell lysates were prepared and western blots were done as recently described ,53. The Viability of the cells was measured using MTT-assays performed according to the manufacturer's protocol . Caspase 3/7 activity was determined using Promega's Caspase-Glo 3/7 assay according to the manufacturer's instructions .Cells were transfected as indicated. After 48 h cells were trypsinized, washed once with PBS and suspended in PBS containing 2 μg/ml primary antibody  or control IgG. Cells were stained on ice for 60 min, then washed with 3 ml cold PBS and incubated with the secondary antibody (1:100 dilution PE-conjugated goat anti-mouse F(ab')2 anti-IgG+IgM ) for 60 min, on ice, in the dark. After an additional wash with 3 ml cold PBS, cells were suspended in 1 ml cold FACS buffer and 10.000 cells per sample were analyzed using fluorescence flow cytometry  and results were analysed with the FLOMAX software (Dako).The authors declare that they have no competing interests.SS, PF, SD, AA, DS, and GS designed and conducted the experiments. SS, PF, SD, AA, DS, and GS analyzed and interpreted data. SS, PF, SD, AA, DS, RMS and GS drafted the manuscript and revised it critically for important intellectual content. All authors read and approved the final manuscript.


Fruits and vegetables are important sources of fiber and nutrients with a recognized antioxidant capacity, which could have beneficial effects on the proinflammatory status as well as some metabolic syndrome and cardiovascular disease features. The current study assessed the potential relationships of fruit and vegetable consumption with the plasma concentrations and mRNA expression values of some proinflammatory markers in young adults.2) were enrolled. Experimental determinations included anthropometry, blood pressure and lifestyle features as well as blood biochemical and inflammatory measurements. The mRNA was isolated from peripheral blood mononuclear cells (PBMC) and the gene expression concerning selected inflammatory markers was assessed by quantitative real-time PCR. Nutritional intakes were estimated by a validated semi-quantitative food-frequency questionnaire.One-hundred and twenty healthy subjects , independently of gender, age, energy intake, physical activity, smoking, body mass index, systolic blood pressure and circulating non-esterified fatty acids. In addition, plasma CRP, homocysteine and TNFα concentrations and ICAM1, TNFα and NFκB1 gene expression in PBMC showed a descending trend as increased fiber intake (>19.5 g/d) from fruits and vegetables (P for trend < 0.05). Furthermore, the participants within the higher tertile (>11.8 mmol/d) of dietary total antioxidant capacity showed lower plasma CRP and mRNA values of ICAM1, IL1R1, IL6, TNFα and NFκB1 genes (P for trend < 0.05). The inverse association between fruit and vegetable consumption and study proinflammatory markers followed the same trend and remained statistically significant, after the inclusion of other foods/nutrients in the linear regression models.The highest tertile of energy-adjusted fruit and vegetable consumption (>660 g/d) was associated with lower plasma concentrations of C-reactive protein (CRP) and homocysteine and with lower A higher fruit and vegetable consumption was independently associated not only with reduced CRP and homocysteine concentrations but also with a lower mRNA expression in PBMC of some relevant proinflammatory markers in healthy young adults. A low-chronic inflammatory status is a recognized link between excessive adiposity and metabolic syndrome features, diabetes, and atherosclerosis ,2. In faIn turn, fruits and vegetables contain components such as plant proteins, potassium, magnesium, fiber, and others compounds with antioxidant capacity, whose consumption may reduce the risk of suffering metabolic syndrome manifestations -8. Thus,In this context, nutrigenomic studies have demonstrated the healthy effect of specific nutrients and calorie-restriction on PBMC proinflammatory gene expression ,14. HoweICAM1), interleukin-1 receptor-type 1 (IL1R1), interleukin-6 (IL6), tumor necrosis factor-alpha (TNFα), subunit-1 or p50 (NFκB1) and subunit-3 or p65 (RELA) of NFκB in PBMC, from young adults.Overall, the present study assessed the potential association of fruit and vegetable consumption with plasma concentrations of CRP, homocysteine, IL6, and TNFα as well as gene expression profiles, which were assessed through messenger RNA (mRNA) levels of genes encoding intercellular adhesion molecule-1 , in accordance with the principles of the Helsinki Declaration.One hundred twenty (50 men/70 women) subjects with a mean age of 20.8 ± 2.6 (range 18-30) years old and mean body mass index of 22.3 ± 2.8 (range 18.5-30.5) kg/mAssessment and information about dietary intake were obtained by the questionnaire of the Seguimiento Universidad de Navarra (SUN) Study . This sead hoc computer program specifically developed for this aim /22.5 /22.5 . Plasma g, 30 min, at 20°C) by using PMN medium , and total RNA from PBMC was extracted and subsequently purified by DNase treatment  as previously described [ICAM1, IL1R1, IL6, TNFα, NFκB1, RELA) were also supplied by Applied Biosystems . Gene expression levels were assessed and normalized by using 18s rRNA as internal control following previously described protocols [-ddCt) in the target genes, normalized to 18S and relative to the lowest expression profile, was calculated for each sample according to the manufacturer's guidelines .PBMC were isolated by differential centrifugation  and physical activity practice (Yes/No and volume of activity). To quantify the volume of activity, an activity metabolic equivalent (MET) index was computed by assigning a multiple of resting metabolic rate (MET score) to each activity . Metabol2), daily energy intake , physical activity during leisure time (METs-hour per week), smoking , systolic blood pressure (mmHg), and non-esterified fatty acid concentration (mmol/l). The same procedure was performed in order to analyze the relationships of dietary TAC and dietary fiber (from fruit and vegetable intake) with proinflammatory markers. In addition, we categorized the participants by quintiles of fruit and vegetable consumption, and the ratios between the mean values of selected proinflammatory marker concentrations , computed as the quotient between fifth and first quintiles of this food-group consumption were calculated.Results are shown as mean ± standard deviations (SD). The Shapiro-Wilk test was used to determine variable distribution. Comparisons between three groups were performed by ANOVA one-factor tests, while the post-hoc Tukey test was used concerning multiple comparisons. Dietary intakes were adjusted for the daily energy intake by the residuals method, applying separate models among women and men . To asseP-value < 0.05 was considered as statistically significant.Furthermore, we used stepwise multiple regression  to identP < 0.05). The circulating levels of other proinflammatory markers followed a decreasing trend, when analyzed with such criteria (Table P < 0.05) reached for homocysteine and TNFα concentrations.Anthropometrical, clinical, and metabolic characteristics were examined by tertiles of fruit and vegetable consumption Table . Thus, sia Table . In addiia Table , with stR2 = 0.45 and 0.42, respectively). Other important contributing food items to the dietary intake are listed in the Table Moreover, those participants who were included in the third tertile also presented higher intake values for vegetable protein, potassium, magnesium, and vitamin C, as compared with subjects in the lowest tertile of fruit and vegetable consumption. Interestingly, fiber and dietary TAC also were statistically higher in those individuals included in the highest tertile of fruit and vegetable consumption Table . MoreoveP for trend < 0.05) values to plasma concentration of CRP and homocysteine (Table P < 0.05) across tertiles of fiber intake from fruits and vegetables, while levels of CRP were also lower across tertiles of increasing dietary TAC values  values to mRNA expression of ICAM1, IL1R1, IL6, TNFα, and NFκB1(p50) in PBMC, independent from assumed confounding factors  gene expressions were significantly lower in the subjects included in the highest tertile of dietary TAC  genes were markedly reduced in the third tertile of dietary fiber from fruit and vegetable consumption  lower in those subjects included in the third tertile of energy-adjusted fruit juice consumption (> 100 ml/d), adjusting for gender, age, BMI, energy intake, smoking habit, physical activity counts, NEFA concentration, and systolic blood pressure.Furthermore, other analyses were performed in order to evaluate the association of fruit and vegetable consumption and proinflammatory markers. Since fruit and vegetable consumption correlated with other foods/nutrients, the association between fruit and vegetable consumption (as independent variable) and proinflammatory markers (as outcomes) were analyzed, using linear regression models, additionally adjusted for vegetable protein, potassium, magnesium, total fat, polyunsaturated fatty acid or saturated fatty acid intake as well as for dietary TAC and fiber, as continuous variables. The inverse association between fruit and vegetable intake and proinflammatory markers remained statistically significant (data not shown). Moreover, we performed additional analyses including fruit juices in the categories of fruit and vegetable consumption, which did not change the interpretations. In this context, we separately analyzed the fruit juice consumption . The CRP and homocysteine concentrations as well as mRNA expression of In addition, the separate regression analyses of the influence of the smoking status  or including the smoking habit categorized by number of cigarettes/day (1) 0-5 or ≥ 5 cigarettes/d; 2) 0-10 or ≥ 10 cigarettes/d) as covariates, produced the same outcome concerning the inflammatory markers. Finally, when we performed linear regression analysis, including the waist circumference as independent variable, the results followed the same trends and remained statistically significant (data not shown).ICAM1, IL1R1, IL6, TNFα, and NFκB1(p50) gene expression in PBMC.A high intake of fruits and vegetables has been inversely associated with metabolic syndrome features ,6, cardiILs, TNFα, ICAM1 genes and of their respective receptors in adipose tissue as well as in PBMC has been implicated in the higher risk of suffering metabolic syndrome and cardiovascular disease [An altered expression of  disease ,4,35. In disease . In this disease . TherefoIn this study, we also found a statistically significant inverse association of fruit and vegetable consumption with CRP and homocysteine concentrations. These results are consistent with some earlier cross-sectional studies carried out on children and adolescents ,39 as weICAM1 and VCAM1 genes [in vitro studies have evidenced an anti-inflammatory effect of flavonoids and carotenoids, by an inhibition of NFκB activity, through suppressing the activation-related phosphorylation, and inhibiting the nuclear translocation [The anti-inflammatory mechanisms related to fruit and vegetable consumption are still unclear. Fruits and vegetables are sources of essential nutrients, which could be implicated in inflammation and oxidative stress reductions ,41. For M1 genes , althougM1 genes ,38,47. Ilocation -50.In this study, we also found inverse associations of dietary fiber and TAC with plasma concentrations and the gene expression of certain proinflammatory markers, suggesting a participation of these specific dietary factors in the beneficial effect of fruit and vegetable consumption. The gene expression of investigated proinflammatory markers had statistically significant lower values across tertiles of fruit and vegetable consumption, independently of dietary fiber and dietary TAC. In addition, the inverse associations of fruit and vegetable consumption with proinflammatory gene expression maintained the trend and the statistical significance after including others specific dietary factors such as plant protein, potassium, magnesium, total fat, polyunsaturated fatty acid and saturated fatty acid. In this context, our findings suggest that additive and synergistic effects of bioactive compounds provided by fruits and vegetables as responsible for the antioxidant and anti-inflammatory activities of these foods.Interestingly, we found lower values to BMI, waist circumference, and blood pressure in those participants who were in the highest tertile of fruit and vegetable consumption. These results are in accordance with findings obtained in the SUN Study ,51 and iIn this translational study, the fruit and vegetable consumption was inversely associated with mRNA expression of certain proinflammatory markers from PBMC in healthy young adults, suggesting a beneficial effect of high fruit and vegetable consumption on decreasing proinflammatory status and providing new light for the nutrigenomic involved-mechanisms as well as new tools for the assessment of nutrient-gene interactions.BMI: Body mass index; CRP: C-reactive protein; DASH: The Dietary Approaches to Stop Hypertension; ICAM1: intercellular adhesion molecule-1; IL1R1: interleukin-1 receptor-1; IL: interleukin; MET: activity metabolic equivalent; mRNA: messenger RNA; NEFA: non-esterified fatty acid; NFκB1: nuclear factor-nuclear-kappa-B (p50); PBMC: peripheral blood mononuclear cells; RELA: nuclear factor-nuclear-kappa-B (p65); SUN: Seguimiento Universidad de Navarra; TAC: total antioxidant capacity; TNFα: tumor necrosis factor-alpha.The authors declare that they have no competing interests.HHMH: Design, field work, data collection, analysis, and writing of the manuscript. BP: Design, field work, and data collection. MZ: project co-leader, design, financial management, and editing the manuscript. JAM: project leader, general coordination, design, financial management, and editing of the manuscript. All authors read and approved the final manuscript.


Infusing arginine vasopressin (AVP) in vasodilatory shock usually decreases cardiac output and thus systemic oxygen transport. It is still a matter of debate whether this vasoconstriction impedes visceral organ blood flow and thereby causes organ dysfunction and injury. Therefore, we tested the hypothesis whether low-dose AVP is safe with respect to liver, kidney, and heart function and organ injury during resuscitated septic shock.13CO2/12CO2 enrichment during 1,2,3,4,5,6-13C6-glucose infusion), visceral organ function , nitric oxide  and cytokine production (interleukin-6 and tumor necrosis factor-α blood levels), and myocardial function (left ventricular dp/dtmax and dp/dtmin) and injury (troponin I blood levels) were measured before and 12, 18, and 24 hours after peritonitis induction. Immediate post mortem liver and kidney biopsies were analysed for histomorphology (hematoxylin eosin staining) and apoptosis (TUNEL staining).After intraperitoneal inoculation of autologous feces, 24 anesthetized, mechanically ventilated, and instrumented pigs were randomly assigned to noradrenaline alone  or AVP  to treat sepsis-associated hypotension. Parameters of systemic and regional hemodynamics , oxygen transport, metabolism (endogenous glucose production and whole body glucose oxidation derived from blood glucose isotope and expiratory AVP decreased heart rate and cardiac output without otherwise affecting heart function and significantly decreased troponin I blood levels. AVP increased the rate of direct, aerobic glucose oxidation and reduced hyperlactatemia, which coincided with less severe kidney dysfunction and liver injury, attenuated systemic inflammation, and decreased kidney tubular apoptosis.per se and/or to the decreased exogenous catecholamine requirements.During well-resuscitated septic shock low-dose AVP appears to be safe with respect to myocardial function and heart injury and reduces kidney and liver damage. It remains to be elucidated whether this is due to the treatment  It is still a matter of debate whether this vasoconstriction impedes visceral organ blood flow and thereby causes organ dysfunction [Infusing arginine vasopressin (AVP) in vasodilatory septic shock is usually accompanied by a decrease in cardiac output and systemic oxygen  is safe with respect to liver, kidney, and heart function in a clinically relevant porcine model of fecal peritonitis-induced septic shock . AVP was2 uptake and carbon dioxide (CO2) production, arterial, portal, hepatic, and mixed venous blood gases and oximetry), intrathoracic blood volume, extravascular lung water and indocyanine-green plasma disappearance rate , blood glucose, lactate, pyruvate, bilirubin, creatinine, troponin I, nitrate+nitrite , TNFα, and IL-6 concentrations, as well as the alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities were determined as described previously [2-+NO3- concentrations and the ALAT and ASAT activities are normalized per gram of plasma protein to correct for dilution by intravenous fluids [13C6-glucose and the mixed expiratory 13CO2, respectively, during continuous intravenous isotope infusion, after gas chromatography-mass spectrometry assessment of plasma and non-dispersive infrared spectrometry measurement of expiratory gas isotope enrichment [®, Millar Instruments, Houston, TX, USA) that allowed measuring maximal systolic contraction (dp/dtmax) and diastolic relaxation (dp/dtmin), as well as the frequency-independent relaxation time (τ).The study protocol was approved by the University Animal Care Committee and the Federal Authorities for Animal Research . Anesthesia, surgical instrumentation, measurements have been described in detail previously . Systemieviously . The bils fluids . Endogenrichment . Left veImmediate postmortem liver, kidney, and heart biopsies were evaluated for histomorphologic changes (H&E staining) and the number of apoptotic nuclei  staining) . Evidenc2O, inspiratory-to-expiratory (I/E) ratio 1:1.5, respiratory rate adjusted to partial pressure of arterial carbon dioxide (PaCO2) 35 to 45 mmHg (but maximum 40 mmHg/min), peak airway pressure less than 40 cmH2O, fraction of inspired oxygen (FiO2) 0.3 . If partial pressure of arterial oxygen (PaO2)/FiO2 less than 300 mmHg or less than 200 mmHg, I/E ratio was increased to 1:1 and PEEP to 12 or 15 cmH2O, respectively. Lactated Ringer's solution was infused as maintenance fluid (7.5 mL/kg/h), and normoglycemia (4 to 6 mmol/L) was achieved with continuous intravenous glucose as needed. Following instrumentation, an eight-hour recovery period, and baseline data collection, peritonitis was induced by intraperitoneal instillation of 1.0 g/kg autologous feces incubated in 100 mL 0.9% saline for 12 hours at 38°C [Body temperature was kept between 37 and 39°C, that is ± 1°C of the pre-peritonitis value, with heating pads or cooling. Ventilator settings were : tidal v at 38°C . Hydroxy at 38°C ) allowed at 38°C  we aimedData are presented as median (quartiles) unless otherwise stated. After exclusion of normal distribution using the Kolmogorov-Smirnoff-test, differences within groups were analyzed using a Friedmann analysis of variance on ranks and a subsequent Dunn's test with Bonferroni correction. As our primary hypothesis had been that AVP was safe with respect to liver and heart function in our model, intergroup differences for blood ASAT and ALAT activities as well as bilirubin and troponin I levels were tested using a Mann-Whitney rank sum test with Bonferroni adjustment for multiple comparisons. Because of the multiple statistical testing of the numerous variables measured, all other intergroup comparisons have to be interpreted in a secondary, exploratory, and hypotheses-generating, rather than confirmatory, manner.One animal in the control group died following data collection at 18 hours, and thus statistical analysis at 24 hours comprises 23 animals. Colloid resuscitation was identical in the two groups (controls: 15 (14 to 15), AVP: 14 (13 to 14) mL/kg/h). AVP-treated animals did not require any additional noradrenaline during the first 12 hours of the experiment, and, consequently, the median duration and rate of the noradrenaline infusion were significantly lower (duration: 111 (0 to 282) versus 752 (531 to 935) minutes; infusion rate: 0.06 (0.00 to 0.10) versus 0.61 (0.33 to 0.72) μg/kg/min).2 exchange, acid-base status, and metabolism . Although the rate of whole body glucose oxidation increased comparably, the progressive rise of endogenous glucose production rate was less pronounced in the AVP animals . Consequently, the directly oxidized fraction of the glucose released was significantly higher in the AVP group, which coincided with significantly lower arterial lactate levels at 18 and 24 hours.Tables sm Table . None of2 kinetics, acid-base status, and function. Except for a lower portal venous flow (P = 0.053 at 24 hours), liver hemodynamics and O2 exchange did not significantly differ between the two groups. Nevertheless, AVP attenuated the portal and hepatic venous acidosis .Table P = 0.061).Histomorphologic evaluation showed some non-specific subcapsular inflammatory cell infiltration and a few biliary tract concrements in the liver, and tubular swelling in the kidney; however, this was without any intergroup difference, and no pathologic findings at all in the myocardium. Although TUNEL-positive nuclei were absent or rare (without intergroup difference) in the heart and the liver, respectively, AVP-treated animals showed less TUNEL-positive renal tubular nuclei (3 (3 to 9) versus 11 (5 to 15), respectively, The aim of the present study was to test the hypothesis whether low-dose AVP infusion is safe for heart and visceral organ function in a clinically relevant, resuscitated, and hyperdynamic porcine model of fecal peritonitis-induced septic shock. AVP supplemented with noradrenaline was compared with noradrenaline alone, which were titrated to maintain comparable blood pressure. The key findings were that: AVP decreased heart rate and cardiac output without affecting myocardial relaxation, and significantly decreased troponin I blood levels; increased the rate of direct, aerobic glucose oxidation, and reduced hyperlactatemia; attenuated kidney dysfunction as well as liver injury, which coincided with less severe systemic inflammatory response.max was significantly lower in the AVP group, whereas dp/dtmin remained unchanged. Thus our experiment seems to confirm negative inotrope properties of AVP in isolated hearts [max and dp/dtmin crucially depend on heart rate. In the mentioned studies, however, heart rate was not affected at all [2 consumption, was well maintained under constant flow conditions [In our experiment, left ventricular dp/dtd hearts ,24 and ed hearts . As firsd at all ,24 or ded at all . Furtherd at all  or AVP dd at all ,24. Clead at all . These ad at all . It remanditions . Finallynditions . In the nditions  and redunditions .2 exchange and, moreover, was associated with less severe hepatic venous metabolic acidosis and attenuated liver injury. Furthermore, AVP infusion resulted in significantly less severe kidney dysfunction. Controversial effects were reported on the effects of AVP infusion on visceral organ blood flow and function during large animal sepsis and septic shock: although AVP decreased mesenteric arterial and portal venous flow during porcine and ovine bacterial sepsis [Despite the lower portal venous flow infusing AVP did not have any detrimental effect on liver Ol sepsis ,15,16 orl sepsis ,10, othel sepsis ,10,19. Tl sepsis ,16, and l sepsis  renal bll sepsis ,10 and/ol sepsis . In endol sepsis .+ excretion was significantly increased. Therefore, it could be argued that AVP deteriorated or, at best, did not influence kidney function [+ excretion probably mirrors the physiologic response to AVP [+ elimination both under healthy [+ excretion: noradrenaline per se was demonstrated to reduce Na+ elimination [AVP did not affect creatinine clearance, and fractional Nafunction , which wfunction ,13,35 anfunction ,36. It sfunction . Moreovee to AVP  rather t healthy ,40 and p healthy . Finallymination ,43.per se, the lower noradrenaline doses may have attenuated the inflammatory response: catecholamines may mimick [2 consumption is higher for glycolysis than for β-oxidation, because reduced nicotineamide adenine dinucleotide (NADH) as an electron donor provides three coupling sites rather than two only provided by reduced flavine adenine dinucleotide (FADH2) [per se and/or the reduced catecholamine requirements: Noradrenaline increases endogenous glucose release [Several mechanisms may explain the AVP-related less severe organ dysfunction and tissue injury. First, AVP was associated with significantly lower IL-6 levels, that is, an attenuated systemic inflammatory response, which is in good agreement with the anti-inflammatory properties of AVP reported in endotoxic mice . In addiy mimick  and/or ey mimick ,48 the i (FADH2) . Again,  release , and Reg release , whereas release .2 delivery was always well maintained and portal drained viscera O2 extraction, hepatic O2 uptake, regional venous O2 saturation, and lactate/pyruvate ratios were identical.Mean blood pressure was significantly lower in the control group during the last six hours of the experiment due to the resuscitation protocol imposing a maximum noradrenaline infusion rate at heart rates of 160 beats/min or higher. Hence, any beneficial effect of AVP on organ function and/or damage could be referred to a higher perfusion pressure . We thinWe used hydroxyethyl-starch for fluid resuscitation, because in swine this colloid caused less pulmonary dysfunction than Ringer's lactate  and atteFinally, we investigated young and otherwise healthy pigs during the first 24 hours of sepsis, which precludes any conclusion on the safety of AVP infusion with respect to organ injury during prolonged administration and/or with underlying ischemic heart disease, congestive heart failure, or peripheral vascular disease.max in young animals without underlying heart disease, the use of AVP should be cautioned in patients with heart failure and/or cardiac ischemia, such as in the recent VASST [per se and/or to the reduced noradrenaline requirements needed to achieve the hemodynamic targets.In our clinically relevant model of fecal peritonitis-induced septic shock, low-dose AVP infusion supplemented with noradrenaline proved to be safe with respect to myocardial and visceral organ function and tissue integrity. Nevertheless, as we observed a reduced dp/dtnt VASST . It rema• Low-dose AVP appears to be safe with respect to myocardial function and heart injury and even attenuates kidney and liver dysfunction and tissue damage during well-resuscitated porcine septic shock.• An increased aerobic glucose oxidation and reduced hyperlactatemia suggests improved cellular energy metabolism, which coincides with less severe systemic inflammation.per se and/or to the decreased exogenous catecholamine requirements.• It remains to be elucidated whether this is due to the treatment 2: carbon dioxide; dp/dtmax: maximal systolic contraction; dp/dtmin: maximal diastolic relaxation; FADH2: reduced flavine adenine dinucleotide; FiO2: fraction of inspired oxygen; H&E: hematoxylin and eosin; I/E: inspiratory-to-expiratory; IL-6: interleukin-6; NADH: reduced nicotineamide adenine dinucleotide; NO2-+NO3-: nitrate+nitrite; O2: oxygen; PaO2: partial pressure of arterial oxygen; PaCO2: partial pressure of arterial carbon dioxide; PEEP: positive end-expiratory pressure; τ: diastolic relaxation time constant; TNFα: tumor necrosis factor-α; TUNEL: terminal deoxynucleotidyltransferase-mediated nick-end labeling assay; VASST: vasopressin and septic shock trial.ALAT: alanine aminotransferase; ASAT: asparatate aminotransferase; AVP: arginine vasopressin; CORL is a full-time salaried employee of Ferring Research Institute Inc., San Diego, CA, USA. PA, PR, and EC received a research grant from Ferring Research Institute Inc., San Diego, CA, USA. PR and PA received consultant fees from Ferring Pharmaceutical A/S, København, Denmark, for help with designing preclinical experiments. The other authors declare that they have no competing interests.PA, RL, PR, and EC played a pivotal role in planning and designing the experimental protocol. FS, MG, and FP carried out the anesthesia, surgical instrumentation as well as the on-line data collection. RG, BH, and MG were responsible for the data analysis. AS and PM provided the histomorphology and immunohistochemistry findings and the analysis of these data. JV and UW were responsible for the isotope data acquisition, analysis, and interpretation. MG, PR, and BH wrote the manuscript.