ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these “silent” variations can have a significant impact on protein expression and function and should no longer be considered “silent”. Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of  A Disintegrin-like and Metalloprotease with Thrombospondin type-1 repeats, member-13) plays an integral role in vascular hemostasis by cleaving von Willebrand Factor (VWF) within intact blood vessels under shear stress Thrombotic Thrombocytopenic Purpura (TTP [OMIM 274150] - http://www.omim.org/), a life threatening hematological disease ADAMTS13 (Single nucleotide polymorphisms (SNPs), originally defined as single site codon substitutions that occur in >1% of the population, are prevalent and are found across the entire human genome coding sequence, with few exceptions. Approximately 962,258 unique SNPs have been reported in the coding sequence of the human genome, although frequency data are not available for all of these SNPs. Therefore, SNPs are now classified as genomic variants and it is no longer possible to distinguish between SNPs and mutations based on their frequency ADAMTS13 variants – six synonymous variants and six non-synonymous variants (the latter defined as single site codon substitutions that do change the encoded amino acid) and originally listed in the coding region of the gene in the NCBI dbSNP . Some of these variants have been previously investigated using in vitro methods by other researchers At the inception of this project, we chose to investigate twelve ex vivo, in cells. In addition, we have employed a variety of computational methods to analyze the potential effects of these variants on ADAMTS13 mRNA splicing, change in mRNA structure, codon usage and amino acid conservation as well as the relationships between the location of these variants in the encoded polypeptide chain and the wild-type (WT) ADAMTS13 (predicted) protein structure.Here, we have used a transient expression system to study the effects of the twelve variants mentioned above on mRNA and protein expression levels, protein activity and conformation ADAMTS13 are not neutral. Furthermore, we demonstrate that in silico analysis may serve as a tool to identify variants that may potentially have an effect on the protein bearing them, altering its expression levels and/or activity. In silico variables with high correlation to ex vivo results  may become important for the characterization of potential TTP patients carrying genetic variants. These in silico variables may also be used in the future for developing safer and more effective therapeutic recombinant proteins. This may be achieved by taking into account the predicted effects of variants (and even haplotypes) on ADAMTS13 or any other therapeutic recombinant protein characteristics.Substantial differences in protein expression levels, activity and conformation were found between WT ADAMTS13 and ADAMTS13 variants, suggesting that both non-synonymous and synonymous variants in e.g. only KineFold is able to predict pseudoknots . Nevertheless, synonymous variant 354, independently of the length of the RNA fragment analyzed, was predicted (using both programs) to affect RNA stability to a similar extent , while synonymous variant 420 showed considerably different ΔΔG values and trends associated with RNA fragments of different lengths . Overall, the highest ΔΔG calculated by mFold was observed for synonymous variant 4221 (for a fragment of 75 nucleotides in length) and the lowest ΔΔG was observed for synonymous variant 420 (for a fragment of 151 nucleotides in length)  . AlthougADAMTS13 mRNA expression levels in transfected cells. mRNA levels were monitored 24 hours (h.) post-transfection. Significant differences in ADAMTS13 mRNA levels between cells transfected with a plasmid harboring WT ADAMTS13 gene and those transfected with ADAMTS13 variants were observed for variants 1451, 1716, and 2699   . On averctively)  for the ctively)  and theictively) .Materials and Methods for details). In brief, the differences between a variant’s codon usage and the corresponding WT codon usage were calculated using either the Relative Synonymous Codon Usage (RSCU) values ADAMTS13, and are shown in ADAMTS13  to ensure that final measurements were done within the region of linear response  was used to determine the effect of each variant on the expression of the mutant proteins as compared to the WT ADAMTS13. Both intra- and extra-cellular ADAMTS13 expression levels were determined. Transient transfections with WT response . Based oresponse . It shout test) . The non-synonymous variant 1852 showed a significant decrease, resulting in less than a third  of the extracellular WT expression level. As both intracellular and secreted proteins represent the active forms of ADAMTS13 t test, respectively), while non-synonymous variants 1342  and 1423 ADAMTS13 variants  showed increased  intracellular expression compared to WT. In lieu of an increase in extracellular expression above WT, the ADAMTS13 construct harboring synonymous variant 354 also revealed a 146±21%  increase in intracellular expression above WT.Cells expressing ADAMTS13 non-synonymous variant 3097 revealed the highest increase in extracellular ADAMTS13 expression of 179±35% of WT  . ADAMTS1ADAMTS13 variants did not show any significant correlation between intra- and extracellular expression . Mutants highly expressed in the intracellular compartment were not necessarily highly secreted and vice versa. A representative example, in which three independently transfected ADAMTS13 constructs  were simultaneously analyzed, is shown in ADAMTS13 mRNA levels and either extracellular protein expression  or intracellular protein expression levels .We found that changes in intracellular ADAMTS13 expression levels correlated strongly  with corresponding changes in RSCU values calculated using human codon frequencies. In addition, changes in mRNA minimum free energy values ) unexpectedly correlated with the corresponding changes in ADAMTS13 extracellular expression levels. We found no correlation between t test, respectively). At the same time non-synonymous variants 1451 and 1423 were found to have the lowest specific activity . We further chose to investigate the non-synonymous 2699 variant  because the ADAMTS13 protein bearing this variant revealed the lowest intracellular expression level compared to WT protein, with no change in extracellular protein expression levels compared to WT. This suggests possible conformation changes within the mutant protein that perhaps might lead to enhanced intracellular degradation of non-synonymous variant 2699 species that do not pass quality control mechanisms in the ER. However this protein might also travel faster through the secretory pathway and thus the total amount of the extracellular protein may remain unchanged. In order to detect any minor changes in conformation between 2699 and WT, a more detailed kinetic analysis was performed in the case of non-synonymous variant 2699 and WT (max (RFU min−1) and KM (µM) for WT ADAMTS13 and non-synonymous variant 2699 mediated cleavage of the fluorogenic substrate FRETS-VWF73 are shown in m and Vmax values between the intracellular forms of the non-synonymous variant 2699 and WT proteins, respectively. Thus, our analysis of the specific activity of 12 ADAMTS13 variants and the detailed kinetic analysis of non-synonymous variant 2699 clearly demonstrated differences in specific activity as compared to WT.Activity of each ADAMTS13 variant was determined (and compared to that of the WT protein) using the FRETS-VWF73 fluorogenic substrate and normalized to extracellular ADAMTS13 expression levels to yield the specific activity . UnexpecTo probe and compare the conformations of the variant ADAMTS13 proteins, we subjected them to limited proteolysis with trypsin. We specifically chose to investigate non-synonymous variant 1852, because this ADAMTS13 variant revealed the lowest extracellular expression levels and reduced specific activity as compared to the WT protein. We hypothesized that the differences in expression and function may result from conformational changes within the protein. Increased resistance to limited proteolysis with trypsin was revealed for non-synonymous variant 1852, as compared to WT .e.g. domain linkers) were suggested to elicit more substantial effects on protein folding in comparison with other non-synonymous or synonymous variants in silico predictions). Similar to in silico findings, 4 out 5 variants in the ADAMTS13 (287–682) X-ray fragment encode residues located in beta-structures (Pro475 (ADAMTS13 variant 1423), Arg484 (ADAMTS13 variant 1451), Thr572 (ADAMTS13 variant 1716) and Pro618 (ADAMTS13 variant 1852), respectively) with 2 (ADAMTS13 variants 1451 and 1852) located at the very edges of the secondary structures  and by employing the ConSeq server An interesting question to consider is whether ADAMTS13 variants under investigation are found at codons that encode in WT ADAMTS13 evolutionary conserved amino acid residues. One would expect that non-synonymous variants will be found at less conserved residues, as variants at conserved residues are expected to severely affect protein function, while synonymous variants, should, in principal, be subjected to less evolutionary pressure. To answer this question, we have determined the conservation of the ADAMTS13 amino acid residues using sequence alignments of 50 homologues proteins from Swissprot database  et al. also reported the extracellular expression of non-synonymous variant 1342 to be 95% of WT; similarly Kokame et al. reported similar expression of non-synonymous variant 1342 as compared to WT  et al., observed that non-synonymous variant 1423 showed no change in expression as compared to WT In sum, we have demonstrated that non-synonymous and synonymous variants can significantly alter both intracellular and extracellular protein expression as well as protein activity. Currently, there are only a handful of ADAMTS13 variants studied here alone or in concert with additional variants, however their results cannot be directly compared to the results reported here due to differences in the expression systems and methods used; yet, some valuable information can be extracted. In a different system which examines the expression and function 72 h. post-transduction, Tao et al. used a synonymous 3097 ADAMTS13 variant as a positive control and observed equal extracellular expression and VWF-cleavage activity compared to WT ADAMTS13 in HeLa cells in vivo. In addition to these ex vivo studies, Schettert et al. reports an association between the non-synonymous variant 2699 and increased risk of death by an adverse cardiovascular event and lower cholesterol levels, while no such association was found for the 1852 variant Other reports have investigated several of the in vivo protein folding ADAMTS13 variants may result from the changes in codon usage and corresponding ribosomal translation speeds. This change in translation speed prompts a divergence from the WT folding pathway and therefore might create a new protein conformation. Statistical analysis revealed no inherent bias in our codon usage tools (RSCU and log ratio) resulting from our choice of twelve variants, compared to the RSCU and log ratio of all variants within ADAMTS13. No correlation was found between the ΔRSCU using genomic codon usages and the specific activity of ADAMTS13 using the FRETS-VWF73 substrate. Extracellular protein expression may be significantly influenced by variant-induced conformational changes and this may explain an additional lack of correlations between specific activity and in silico analyses. This indeed suggests that codon usage plays an important role in the protein activity implying that codon usage affects the local rates of translation and thus protein folding and specific activity.So, how do variants under study, particularly synonymous variants, exert their effects  without altering protein composition? One of the elements that might influence protein folding is codon-usage bias. A co-translational, sequential folding model which incorporates a folding funnel characterized by conformation intermediates and an eventual free energy minima ground-state conformation has been suggested as a plausible hypothesis for In the last few years, the general belief that synonymous variants are silent has been reevaluated due to the accumulation of data suggesting that they can interfere with the mRNA structure, codon usage and/or conformation of the resultant protein, thereby possibly affecting its function. Moreover, many recombinant proteins carry variants or common haplotypes with currently little attention being paid to which haplotype is used in their production. A set of experimental approaches and tests used here are precise enough to distinguish between slight changes in protein expression and activity levels. Therefore, we believe that these tests and type of analysis may serve as useful tools to better understand the effects of different variants in any target protein and may be also used during the design and evaluation of therapeutic recombinant proteins.2. 1.2×106 cells were plated 24 hours (h.) before transfection in 2 mL growth medium in 6-well plates. Prior to transfection, DMEM was replaced with 1.6 mL Opti-MEM reduced Serum Medium . Cells were transfected with 1 µg of plasmid DNA using 6 µL of Lipofectamine 2000 (Invitrogen) in a total of 400 µL Opti-MEM (Invitrogen). Independent transfections using the variant and WT constructs were performed in triplicate for each variant. A ratio of 1∶9 and 1∶5 eGFP: ADAMTS13 was used with the same transfection conditions for co-transfection of eGFP and ADAMTS13 in order to confirm similar transfection efficiencies of the WT ADAMTS13 and each of the variants (data not shown).Human embryonic kidney cells  were grown in Dulbecco’s Modified Eagle Medium (DMEM)  with 1% glutamine , 1% penicillin- streptomycin  and 10% fetal bovine serum  at 37°C under humid conditions in 5% COpcDNA4-ADAMTS13 , which carried a full-length ADAMTS13 (NM_139025) cDNA conjugated to V5 and poly (His) tags was used as a backbone for all constructs. Site directed mutagenesis (SDM) was performed using the QuikChange Site Directed Mutagenesis Kit  to change this sequence to WT. This sequence was then used as the WT reference standard and as the backbone for all twelve variants containing constructs produced via SDM. Bi-directional sequencing was used to confirm that variants were successfully introduced. An empty vector was used for mock-transfection control studies. All ADAMTS13 plasmids were purified using cesium chloride gradient  to obtain high quality supercoiled plasmids. Plasmid expressing enhanced green fluorescence protein  was used for co-transfection assays. All the variants investigated in this study are described in To detect minor changes in ADAMTS13 expression or function, we chose to harvest cells 24 h. post–transfection because ADAMTS13 expression and function are in a linear response range. Cells and media were collected, cell lysates were prepared and cell media was concentrated, as previously described Quantitative real time PCR (qPCR) was done using the LightCycler RNA master SYBR Green Kit and LightCycler 480 , using 1 ng of total RNA with an initial melting cycle of 30 seconds (s) at 95°C followed by 40 cycles of 8 s at 95°C, 12 s at 58°C and 10 s at 72°C. ADAMTS13 was probed with 5′-tcacagccaacctcacctcg’3′ (Forward (F)) and 5′-ccgcacctgccggttac-3′ (Reverse (R)) primers using GAPDH as a reference gene with primers 5′-tcgtggagtccactggcgtctt-3′ (F) and 5′-tggcagtgatggcatggactgt-3′ (R). Crossing Point (Cp) values were obtained and presented in the ΔΔCp method using WT as the comparative control for each variant as previously described RNA was isolated (24 h. post-transfection) using RNeasy Plus Mini Kit  following manufacturer’s instructions. Reverse Transcription was carried out as previously described http://rsbweb.nih.gov/ij/) or the 4000 MM Pro Image Station  with CareStream software. The following controls, in addition to those specified under cell harvest: cell lysate and media preparation subsection, were applied in order to assure reproducibility between different transfection experiments and exclude results due to method variations: (i) samples for Western blotting were loaded per total protein concentrations, (ii) a loading control to verify equal total protein loading using anti-β-actin  or anti-GAPDH  were used in lysate samples, (iii) purified recombinant ADAMTS13  was used as a control, (iv) immunoblot analysis of three independent transfections using the linear V5 epitope, was used to determine the internal variation of the variants on the level of ADAMTS13. eGFP co-transfections were performed and harvested 24 h. post-transfection as described above. Cells were washed twice with PBS and eGFP was detected in FL-1 to determine transfection efficiencies between ADAMTS13 plasmid constructs that were shown to be similar .Samples were sonicated twice for 5 min., boiled in SDS reducing buffer for 10 min. and then resolved by electrophoresis in NuPAGE 7% Tris-Acetate gels (Invitrogen). Mouse anti-V5 primary antibody (Invitrogen) and goat anti-mouse HRP secondary antibody (Thermo Scientific) were used to detect the transfected ADAMTS13. Antibody staining was detected using Amersham ECL Western Blotting Detection Reagents  on Hyblot CL Autoradiography film . Densitometry analysis of band intensities was performed using ImageJ 1.42 q software (ADAMTS13-cell lysates (1852 vs. WT) were diluted using 100 mM Tris-HCl  and subjected to trypsin digestion for 5 minutes (min.) at 37°C with increasing concentrations of freshly made enzyme  at the following concentrations ranges 0–0.0005 mg/mL. The reaction was stopped with 1 mg/mL Trypsin soybean inhibitor (Sigma-Aldrich) and analyzed by immunoblotting as described below. Half life of the protein was calculated using GraphPad Prism software.Fluorogenic FRETS-VWF73 substrate  was prepared and assayed according to manufacturer’s instructions and as previously described ADAMTS13 as described by Sharp and Liij is the number of occurrences of the jth codon for the ith amino acid, which is encoded by ni synonymous codons ADAMTS13 cDNA as well as the codon frequency within the entire human genome using the Codon Usage Database  ADAMTS13 constructs used here are under transcriptional control of a CMV promoter, which was reported to have a 2,500% increase in activity when compared to a human BL-3 promoter ADAMTS13. Interestingly, the CG content of the coding region of WT ADAMTS13 is 64.8%, while in the average, protein coding sequence it is only about 46% ADAMTS13 cDNA codon usages to mimic the strain of such over-expression. RSCU values were calculated for each variant using the variant codon. At all variant locations, the ΔRSCU value was computed between the variant and WT sequence.Relative Synonymous Codon Usage (RSCU) was calculated for all variants in the coding region of ADAMTS13 cDNA codon frequencies (ii). Derived from the human Codon Usage Database et al.The frequency of the given codon divided by the frequency of all synonymous codons for the encoded amino acid was calculated for each variant. A log ratio of this frequency analysis was performed in order to assess differences between variant and WT. At each variant location, the log ratio of the frequency of the individual codon was calculated. Two different frequencies were used in these calculations: (i). Derived from human WT http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) as well as the KineFold online server (http://kinefold.curie.fr/cgi-bin/form.pl); variants were centered within these sequences Using default parameters, short fragments of ADAMTS13 ORF  were analyzed by mFold’s online server  was used to compare different model structures and the structures with the lowest E-value were chosen for further analysis http://conseq.tau.ac.il/ver1.1/index.html), the conservation of each individual variant was calculated http://www.uniprot.org/, last accessed 7 June 2011).Using the ConSeq server  to each parameter. ΔRSCU and Δlog ratio for all variants within the coding region of ADAMTS13 were calculated based on data given the NCBI dbSNP database . Since the distribution of ΔRSCU and Δlog ratio for all variants within the coding region of ADAMTS13 cannot simply assumed to be normal, we used the MAD score as a more robust estimate of outliers. All ΔRSCU and Δlog ratio scores for our set of variants had |MAD score| <3 i.e. they fall within the range of median ±3MAD of the distribution of all variants within ADAMTS13.The mean in silico data  and ex vivo data in order to determine if highly correlating sets of computational data could possibly provide explanation for ex vivo data. We also looked at correlation of ex vivo data between themselves. Additionally, a linear regression analysis was performed for all the above cases to ensure that obvious outliers that would skew the correlation coefficient were not present.Further, Spearman’s correlation matrix was computed for Table S1Changes in predicted binding motif of splicing site regulators between variant and WT using SPmap web server.(DOC)Click here for additional data file.Table S2Summary of in vitro data for synonymous and nonsynonymous variants.(DOC)Click here for additional data file.


Arabidopsis genome contains a large number of gene pairs that encode sense and antisense transcripts with overlapping 3′ regions, indicative for a potential role of natural antisense transcription in regulating sense gene expression or transcript processing. When we mapped poly(A) transcripts of three plant gene pairs with long overlapping antisense transcripts, we identified an unusual transcript composition for two of the three gene pairs. Both genes pairs encoded a class of long sense transcripts and a class of short sense transcripts that terminate within the same polyadenylation region as the antisense transcripts encoded by the opposite strand. We find that the presence of the short sense transcript was not dependent on the expression of an antisense transcript. This argues against the assumption that the common termination region for sense and antisense poly(A) transcripts is the result of antisense-specific regulation. We speculate that for some genes evolution may have especially favoured alternative polyadenylation events that shorten transcript length for gene pairs with overlapping sense/antisense transcription, if this reduces the likelihood for dsRNA formation and transcript degradation.The  Animal and plant genomes contain a surprisingly large number of partly overlapping convergent gene pairs representing approximately 7.5% of all protein-encoding genes in Arabidopsis HBA2) α-globin gene, which induced methylation and silencing of the HBA2 promoter Bidirectional transcription of two yeast genes causes transcriptional interference between the two RNAII polymerase complexes affecting transcript elongation and termination Other regulatory effects based on antisense transcription include selective transcript editing by dsRNA-dependent adenosine deaminases (ADARs) and retention of hyperedited RNAs in the nucleus Arabidopsis, we examined three gene pairs that encode convergent transcripts with long overlapping 3′ region. Surprisingly, we detected in two gene pairs the same arrangement of two classes of polyadenylated sense transcripts, one of which shared a polyadenylation region with the antisense transcripts. Contrary to the expectation that antisense transcription or processing regulates alternative polyadenylation of the sense gene, we find that the two alternative sense transcript classes are independent of antisense transcription. The common presence of shortened transcripts in two sense/antisense gene pairs may be the result of evolutionary selection, if this reduces potentially negative effects from dsRNA formation.To examine potential sense/antisense effects on polyadenylation sites in http://mpss.udel.edu/at/mpss_index.php) The Arabidopsis genome contains 956 pairs of coding genes that overlap at their 3′ ends Microarray data We assumed that the efficiency of any RNA interference effects between sense and antisense transcripts should be reduced if transcription of one of the genes is down-regulated. Equally, it should be enhanced if transcription of one of the genes is up-regulated. We therefore examined if T-DNA insertion lines for the six genes showed antagonistic changes in sense and antisense transcript levels. Depending on the activity of T-DNA promoter and enhancer elements, insertion lines can reduce or increase transcript levels downstream of the T-DNA integration site. The SAIL line contain a pCSA110 vector with a 1′2′ dual promoter near the left border, the GK line contain a pAC161 vector with a 35S promoter at its right border, and the SALK lines contain a pROK2 vector with a 35S promoter near the left border. These and other T-DNA-specific promoter fragments have the potential to induce read-through transcription vice versa, we mapped poly(A) sites for the six genes  gene that encodes a putative ubiquitin E3 ligase and overlaps with the KOKOPELLI (KPL) gene. In sperm cells only, the overlapping transcripts generate a nat-siRNA pair, which regulates depletion of ARI14 transcripts, a prerequisite for double fertilisation Natural antisense transcription has been proposed to comprise a second tier of gene expression in eukaryotes due to its influence on sense transcript synthesis, stability or processing Non-quantitative effects of natural antisense transcripts have not been described for plant genes but there are examples of antisense transcripts in animals regulating RNA editing cis-acting elements that control RNA localisation and/or translation Total RNA preparations from floral tissue contain one type of long At5g02370 sense transcripts with an unusually long 3′ UTR. These transcripts are not detectable in polysomal fractions, which contain two types of long transcripts with shortened 3′ UTRs due to a combination of early polyadenylation and splicing. The 3′ UTRs of many mRNAs contain ∼40 times lower levels  and At5g67310 (SALK_106055), in the promoter sequence of At5g02380 (SALK_144899) and At5g16940 (SALK_067777) and the SAIL insertion line with T-DNA insertion in the coding sequence of At5g16930 (SAIL_229-H04) were obtained from the Nottingham Arabidopsis Stock Centre. The GK_820-E01 line with a T-DNA insertion in the promoter sequence of At5g67310 was obtained from the Gabi-Kat seeds collection .EcoRI and the EcoRI fragment was inserted into the EcoRI site of vector pGreen0029.The At5g67300/At5g67310 sense/antisense construct was prepared in two steps. A 5788bp fragment containing both the sense and antisense gene and their promoter regions was amplified using Fhusion High-Fidelity DNA Polymerase  with primers 2288 and 2236. PCR conditions were 98°C for 30 sec, and 30 cycles at 98°C for 10 sec, 58°C for 30 sec and 72°C for 3 min, followed by an elongation step at 72°C for 10 min. The PCR product was cloned into the pCR-Blunt vector (Invitrogen). The resulting construct was digested with EcoRI and XmnI, and was inserted into pGreen0029 that had been digested by EcoRI and EcoRV. The resulting construct lacks 198 bp of the 5′ region of the antisense gene and the ∼300 bp antisense promoter region.For the preparation of the sense construct, a 5472 bp fragment was amplified using primers 2236 and 2130 at the same conditions described above, and the fragment was cloned into the pCR-Blunt vector (Invitrogen). The fragment, containing the At5g67300 sense promoter and At5g67300 sense gene was cut out with Plasmid DNA, isolated with the QIAGEN plasmid Midi Kit, was used in transient assays  according to the manufacture's instruction and were sequenced.Protoplasts were transformed by PEG-mediated transfer Polysomes were isolated from flowers


T regulatory cells (Treg) play an important role in the induction and maintenance of immunological tolerance. Recent findings in experimental transplant models combined with the development of functional reporter mice have opened new avenues to study Treg biology and their therapeutic potential. In particular, recent advances in understanding Treg function and lineage stability revealed unexpected plasticity of this lineage. Nevertheless, pre-clinical and pilot clinical trials using Treg cells as cellular therapies have been initiated suggesting the safety and feasibility of such treatment. In the transplant setting, numerous findings during the last 25 years have demonstrated the importance and therapeutic potential of Treg in the active control of rejection responses. Although the existence of various cell populations with regulatory/suppressive activity, such as IL-10 secreting Tr-1 cells, CD28−CD8+ cells or B regulatory cells, has been demonstrated, in this review we will focus on classical CD25+CD4+ Treg cells.The appropriate balance between effector and T regulatory cells (Treg) is indispensable for the maintenance of self-tolerance and of functional immune responses in vivo.Treg mediate suppressive effects by several mechanisms including anti-inflammatory cytokines , direct cytotoxic effect , metabolic disruption (adenosine production and IL-2 deprivation) and modulation of dendritic cell function  as discussed in [+CD4+ cells (nTreg) [−CD4+ nonregulatory cells or expanded from CD25+CD4+ cells in response to the antigen [Treg can be divided into two populations: thymic-derived naturally occurring CD25 antigen . These cIn this review, we explore recent advances in understanding Treg function and lineage stability and its impact on tolerance induction protocols. Next, we focus on the current attempts to expand human Treg for clinical application as a cellular therapy in organ and cell transplantation. Finally, we discuss the arising or potential issues relating to the therapeutic application of Treg and proposed solutions.Expression of the transcription factor FoxP3 is essential for the development and function of Treg . It was in vitro expansion of Treg, CpG methylation increased correlating with loss of FoxP3 expression and emergence of pro-inflammatory cytokines [+FoxP3+ naïve Treg showed no increase in CpG methylation after 3-week culture, whereas CD45RA−FoxP3+ memory-like Treg from the same donors lost CpG demethylation status and converted into non-Treg cells. Recent advances in our understanding of the complex regulation of FoxP3 expression have led to new methods of analysing Treg based on quantitative DNA methylation analysis of FoxP3 locus [ex vivo manipulated Treg cells.Several epigenetic markers, such as histone acetylation and methylation, and cytosine residue methylation in CpG dinucleotides, have been reported at the Foxp3 locus •. In parytokines •. IntereP3 locus •, which et al. examined the stability of Treg cells by tracing cells that induced and downregulated FoxP3 during their life span [+ Treg cells and with conventional T cells suggesting that they originated from both nTreg and iTreg. These ‘ex-Treg’ had an activated-memory phenotype and produced pro-inflammatory cytokines. Notably, an autoimmune microenvironment favoured loss of FoxP3, and ‘ex-Treg’ cells from diabetic mice were able to transfer diabetes [+CD4+ cells lose their FoxP3 expression and start producing IFNγ and IL-17 after transfer to a lymphopenic host [FoxP3 epigenetic analysis and the development of functional reporter mice questioned the dogma of natural Treg lineage stability. An elegant study by Zhou ife span ••. The adiabetes ••. Notabnic host •.in vivo or ex vivo generation and/or expansion of Treg. The most common in vivo approaches are based on the fact that exposure to antigen increases Treg frequency and/or potency by either expanding naturally occurring Treg or inducing the generation of adaptive Treg from cells that do not originally possess regulatory activity [+CD4+ cells able to prevent skin graft rejection [in vitro culture of mouse CD4+ or CD25−CD4+cells in the presence of alloantigen and anti-CD4 antibody results in the enrichment of CD62L+CD25+ cells effective in controlling graft survival [+ cells in the presence of interferon-γ (IFN-γ) and immature DC can also generate FoxP3+ cells that are able to protect both skin and islet transplants from rejection [in vivo tolerised mice demonstrate increased levels of IFN-γ production transiently after antigen-specific reactivation through T cell receptor [In vivo, IFNγ produced locally where the Treg are present, the draining lymph nodes and the graft [Many strategies exist for the activity •. Generaejection . Moreovesurvival . Interesejection •,20. Notreceptor •. In vivhe graft •, createhe graft •.in vivo is to create Treg-favouring conditions. In the transplantation setting, patients are treated with diverse immunosuppressive drug combinations, which may have a different impact on Treg. It was demonstrated that calcineurin inhibitors (CNI), especially cyclosporine A, are detrimental to Treg, whereas the mTOR inhibitor rapamycin was shown to be beneficial for Treg both in terms of in vivo generation and function in mouse models [in vitro cultures of human Treg [et al. who in vivo expanded Treg by injecting mice with IL-2/anti-IL-2 monoclonal antibody complexes [Another approach to enrich Treg e models  and in iman Treg . It was man Treg . Interesman Treg •,27•. Noomplexes •.+FOXP3+CD4+ will be genuine Treg and therefore isolation strategies based on CD25hi/+CD4+ are likely to be imperfect. Other markers are therefore needed to enrich Treg from human peripheral blood mononuclear cells. Recently, it has been demonstrated that CD127loCD25+CD4+ T cells are characterised by a higher percentage of FoxP3+ cells with a more pronounced suppressive capacity [loCD25+CD4+ cells resulted in high yield of regulatory cells which maintained high FoxP3 expression [ex vivo expanded CD25hiCD4+ and CD127loCD25+CD4+ Treg cells have been very effective in inhibiting vasculopathy, with CD127loCD25+CD4+ cells being five times more efficient than conventional Treg [+CD45RA+FoxP3lo and activated CD25hiCD45RA−FoxP3hi regulatory cells. A third population of FoxP3+ cells phenotyped as CD25+CD45RA−FoxP3lo was demonstrated to consist of cytokine-secreting, non-suppressive cells [in vitro, only resting Treg were able to proliferate in vitro and in vivo, converting into activated CD45RA negative Treg.Human Treg are currently less well characterised and understood than mouse Treg, so a thorough understanding of their biology is vital before clinical applications can be initiated. It is also important to highlight that there are substantial differences between human and mouse Treg; most notably the differences in FoxP3 expression between mouse and human. In human, FoxP3 is also expressed by activated nonregulatory T cells as well as by Treg, and activated nonregulatory cells also upregulate CD25 expression. Thus not all CD25capacity . Expansipression ••. Impornal Treg ••. Interve cells •. NotablA number of different strategies for the isolation/enrichment of human Treg have been described in the literature, but to date there is no consensus as to which strategy produces the optimal population for use in cell therapy applications. The critical steps and the questions awaiting answers in the process of developing clinically approved Treg cellular therapy are outlined in ex vivo multiplication of the cells prior to their use in vivo. The most commonly used expansion protocol at present is based on stimulation by anti-CD3/anti-CD28 beads in the presence of high doses of recombinant IL-2, supplemented in some protocols with rapamycin. This protocol results in the efficient expansion of polyclonal Treg, generating sufficient numbers of cells for cellular therapy [in vitro than polyclonaly-driven cells and, surprisingly, expanded at a similar rate as polyclonaly-stimulated cells [One of the obstacles in the implementation of clinical protocols using Treg is their low frequency in the peripheral blood leading to the need for  therapy •. Howeveed cells .et al., 51st ASH Annual Meeting and Exposition, New Orleans, LA, December 2009). Another clinical trial in the University of Minnesota, USA, led by Bruce Blazar and co-workers, used ex vivo expanded bead-enriched, third-party, partially HLA-matched Treg cells from umbilical cord blood (UCB). These UCB Treg cells were administered into myeloablated or nonmyeloablated recipients of two unrelated UCB units [There are several ongoing clinical trials with the application of Treg cellular therapy. To date regulatory cells have been adoptively transferred into haematopoietic stem cell transplant (HSCT) recipients in Germany, USA and Italy •. In oneCB units •.et al. [+CD127lo Treg. One of the patients, with chronic GVHD, responded to the therapy with alleviation of the symptoms and reduction of immunosuppression. However, in the case of the second patient with acute, grade IV GVHD, only transient improvement was observed. Notably, in the case of the patient treated successfully with cellular Treg therapy, only 1 × 105 Treg cells/kg body weight was sufficient to achieve clinical improvement. Published reports and unpublished results from other ongoing studies indicate that Treg cellular therapy is proving to be effective in clinical situations. Currently, further clinical studies are being planned to apply Treg therapy in solid organ transplantation.Whereas aforementioned studies utilised Treg to prevent GVHD, Trzonkowski et al. • have rein vitro expanded Treg to convert into cytokine producing, nonregulatory cells upon prolonged TCR stimulation [ex vivo Treg expansion protocols. Importantly, it was demonstrated that a proportion of circulating Treg have the capacity to secrete IL-17 and express RORγt [+ Treg are as equally suppressive as CCR6− Treg [+IL-17+ clones after strong TCR stimulation in the presence of APC [in vivo are still debatable, the possibility that they may elicit unwanted responses when transferred to patients cannot be excluded. However, several markers of ‘true’ Treg have been described such as IL-1R2, LAP and GARP that may allow additional post-expansion purification steps to be introduced into clinical protocols to reduce the risk of introducing cells that do not have regulatory function [+CD45RA+CD127lo) as a starting population for expansion [The ability of mulation • has ledss RORγt . These IR6− Treg , wherease of APC . Althougfunction •,41•. Alex vivo culture. Recently, retinoic acid (RA) has been described as inhibiting IL-17 polarisation and to promote FoxP3 expression [ex vivo expanded human Treg, especially when isolated with magnetic beads [Another way to prevent conversion into Th17 cells may be to use pharmacologic intervention during pression . Anotheric beads .in vitro Treg cultures that maintain FoxP3 expression in ex vivo manipulated cells appears to be an attractive way of ensuring their effectiveness and safety for the patient. Additionally, obtaining cells with higher per cell activity would potentially allow a significant reduction in the number of Treg required for each clinical application.Introduction of additional factors to the in vivo ablation of Treg was recently demonstrated to lead to accelerated fatal infection during mucosal herpes simplex virus infection, suggesting that in some situations Treg facilitate early protective responses to local viral infection [in vivo induced Treg which were able to induce tolerance in primary and secondary allograft recipients did not affect anti-influenza response even when bystander regulation was deliberately induced [One of the concerns regarding the application of Treg in transplant recipients is the possibility of inhibition of anti-tumour and antiviral immunity. Theoretically, infusion with large numbers of potent suppressor cells may present a serious obstacle to the induction of effective immune responses towards infectious pathogens and a reduction in immune surveillance against tumour cells. As is often the case with the immune system, reality may not fit with theoretical predictions. For example, nfection . Notably induced .Numerous studies suggest that the accumulation of Treg at tumour sites may affect anti-tumour immunity, therefore infusion of substantial numbers of Treg may particularly influence the response towards already existing early tumours. Notably, high numbers of Treg in the blood have been recently associated with increased risk of new tumour development in kidney transplant recipients with non-malignant squamous cell carcinoma . On the Recent progress in understanding Treg biology and the development of experimental mouse models has highlighted potential avenues in the translation of research-based knowledge to the clinic. Insights into the biological role of FoxP3, the effects of immunosuppression on Treg and new protocols to expand or induce Treg provide a knowledge base for developing clinical strategies to achieve long-term graft survival without life-long immunosuppression.Authors declare no conflict of interest.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection  within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway. Salmonella infection, the number of people infected with Salmonellae each year has not changed appreciably in the United States over the last two decades Salmonella gastroenteritis worldwide, resulting in 155,000 deaths Salmonella (NTS) infection is recognized as a major health problem and is the leading cause of pediatric bacteremia + T cell help, which is required to prevent disseminated infection, may be diminished or absent.Despite extensive efforts to understand and prevent Salmonella infection, because they develop diarrhea with clinical and pathologic features similar to those in people Salmonella-induced enteritis in both humans and cattle. These naturally occurring clinical and pathological similarities between human and bovine infection make the calf an ideal animal to model the clinical signs of salmonellae infection in humans Calves are ideal models for human Salmonella enterica Serovar Typhimurium (S. Typhimurium) is one of the top Salmonella serotypes that cause disease in humans and cattle. Invasion of the intestinal epithelium is critical for the establishment of infection and occurs within the first 15 minutes of bacterial contact S. Typhimurium invades the non-phagocytic enterocytes of the host intestinal epithelium through manipulation of the host actin cytoskeleton by Salmonella effector proteins secreted through the Type III secretion system (T3SS-1) encoded at Salmonella Pathogenicity Island I Salmonella effector proteins bind to the actin cytoskeleton and induce the formation of cell membrane ruffles that facilitate Salmonella internalization Salmonella containing vacuole (SCV); SopE2 activates actin cytoskeleton rearrangements through Rho family GTPases; and SopD participates in invasion and has a role in membrane fission and macropinosome formation S. Typhimurium. Loss of all five genes together  significantly diminishes fluid secretion, PMN influx, and lesions in the intestine Salmonella induced bovine and human diarrhea, however, the host cellular pathways and pathway mechanistic genes critical to fluid secretion, PMN transmigration, enteritis and diarrhea are less well defined. We hypothesized that comparison of the response of bovine intestinal mucosa to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. In addition, we also hypothesized that this comparison would lead to the discovery of specific pathway mechanistic genes. To test our hypothesis, we challenged bovine ligated ileal loops containing Peyer's patch with three conditions; wild type Salmonella Typhimurium, an isogenic SipA, SopA, SopB, SopD and SopE2 deletion mutant, or media as a negative control. We measured the host mucosal gene expression profile sequentially for 12 hours post-inoculation. We then compared the data from the host response to the three conditions by conventional and dynamic Bayesian analytical methods, confirming known cellular pathways associated with salmonellosis and identifying additional cellular pathways. Known and newly discovered host cellular pathways were merged to establish functional models of the host∶pathogen interface and to identify pathway mechanistic genes for future study. In this study, we report the comparison of host responses to wild type Salmonella as compared to the SipA, SopABDE2 mutant demonstrating significantly different patterns and perturbations of host cellular pathways at early time points of infection and disease expression relative to the inflammatory response and diarrhea.Beyond invasion, the T3SS-1 delivered effector proteins are required for PMN migration and fluid secretion in the intestine. Previous studies have shown that PMN migration and fluid secretion in bovine intestinal loop model are the result of the coordinated actions of five effector proteins secreted by T3SS-1: SipA, SopA, SopB, SopD, and SopE2 Salmonella enterica serotype Typhimurium strain IR715 and ZA21, were used in this study. IR715, a derivative of ATCC 14028, is a spontaneous nalidixic acid resistant derivative referred to hereafter as “wild type” (WT) and ZA21 is a previously described ΔsipA sopABDE2 mutant (MUT) g and re-suspended in fresh LB broth to obtain a final concentration of approximately 0.75×109 colony-forming units/ml (CFU/ml). The bacterial concentration of the inoculum was confirmed by spreading serial 10-fold dilutions on agar plates containing the appropriate antibiotics.Derivatives of ad libitum. To minimize the possibility of interference from other enteropathogens on the host gene expression profile, calves were tested twice for fecal excretion of Salmonella spp. and Eimeria spp. oocysts and only animals that tested negative for these bacteria and parasites were used. Fecal specimens and rectal swabs were collected from calves two weeks prior to and immediately before the experiment and cultured for the presence of Salmonella serotypes. Swabs were placed in tetrathionate broth  overnight and subsequently plated onto XLT-4 agar plates (BBL). Fecal flotation with a hypertonic saline solution was used for detection of Eimeria spp. oocysts. All four calves were clinically healthy before the experiment and were culture negative for fecal excretion of Salmonella. We elected to use four calves because previously published work in our lab indicated that four calves would be sufficient to detect significant differences between the treatments used in this study Four male Holstein calves, 4–6 weeks of age and weighing 45–55 kg, were used in each experiment under an approved animal use protocol in accordance with animal use policy under the supervision of the Texas A & M University Institutional Animal Care and Use Committee (IACUC). All animal work was approved under Animal Use Protocols 2004-029 and 2004-037. Calves were obtained from a local, commercial source and were unrelated. The calves were fed antibiotic-free milk replacer twice daily and water S. Typhimurium strain IR715 (WT) or ΔsipA sopABDE2 mutant (MUT) in LB broth containing approximately 0.75×109 CFU/ml. Loops injected with sterile LB broth (3 ml) served as uninfected, negative controls (UI). Loops were alternately inoculated with LB broth, WT, or MUT starting at the loop closest to the ileocecal junction and were subsequently excised in the same order as inoculated. Three loops were removed at each time point, one loop for each condition. Each loop was inoculated with only one bacterial strain or the negative control. The loops were replaced into the abdominal cavity until collection by excision. At each of seven time points , three loops  were excised. From each of these loops, samples were collected for histopathology, bacteriology, electron microscopy, frozen sections, and RNA extraction.The bovine ligated ileal loop surgical procedure has been described previously For morphologic analysis by light microscopy, full cross-sections of each loop (including the Peyer's patch) were fixed in formalin, processed according to the standard procedures for paraffin embedding, sectioned at 5 µm thickness, stained with hematoxylin and eosin, and examined by light microscopy. All tissue sections collected contained Peyer's patch, which is continuous for approximately 1.2 meter proximal to the ileocecal junction in the calf. Previous work has demonstrated that ligated ileal loops constructed in the manner described have virtually identical morphology and cell composition Salmonella as a consequence of leakage of the loops, cross contamination during material processing, or Salmonella migration via lymphatic or blood vessels. To determine the number of viable CFU of S. Typhimurium in tissues, lysates were serially diluted and cultured on LB agar with nalidixic acid (50 mg/l). Bacterial counts represent tissue-associated bacteria and do not distinguish between intracellular and extracellular bacteria. For RNA isolation, only the intestinal mucosa and submucosa were collected, but for bacteriology, the tissues were full thickness intestinal tissue containing the mucosa, submucosa, and smooth muscle layers.Two-6 mm biopsy punches (0.1 g) from the anti-mesenteric side of the intestinal mucosa to maximize Peyer's patch collection were excised from every loop, washed three times in PBS to remove extracellular bacteria, homogenized, and diluted in 1 ml of PBS. Similar procedures were followed for the control loops to identify the presence of viable t test.For analysis of invasion percentages, the data were transformed logarithmically. The geometric means were determined, and the statistical significance of differences was calculated using two-tailed Student's Total RNA was extracted after dissection of the ileal loops obtained at 15 min and 30 min and at 1, 2, 4, 8, and 12 hours post-infection. A 6 mm biopsy punch was used to collect 8 tissue pieces of Peyer's patch from the excised loop. The mucosa and submucosa of the samples were immediately dissected, the tissue pieces finely minced with a sterile scalpel blade, and two biopsy punches (0.1 mg of tissue) transferred to 1 ml of Trizol reagent . Tissues were immediately further disrupted with an RNase, DNase free plastic disposable pestle. The RNA extraction was performed using the recommended protocol from the manufacturer. The resultant RNA pellet was re-suspended in DEPC-treated water . Genomic DNA was removed by RNase-free DNase I treatment  according to the manufacturer's instructions, and samples were stored at −80°C until used.The quality of the RNA was initially assessed by measurement of the λ260/280 ratio and agarose gel electrophoresis. RNA concentration was quantified by measuring absorbance at λ260 nm using a NanoDrop® ND-1000 , and the RNA quality was determined using a Nano-Chip® on an Agilent 2100 Bioanalyzer . Reference RNAs  were included on each of the four chips used for analysis. The RNA analyzed in all samples was of good to excellent quality (results not shown). The average RNA integrity number (RIN) was 7.42 and the median was 7.7 . Samples were characterized by distinct 18S and 28S rRNA peaks and RNA size distribution. Ten micrograms of RNA was used as starting material for each array.Salmonella but also to other pathogens, including Brucella melitensis, Mycobacterium avium subsp. paratuberculosis. Each of these pathogens cause significant disease in ruminants and potentially humans. Comparison of intestinal host response to these pathogens was a first step in building a model of intestinal host pathway interactions with the intent of building an in silico interactome model. These studies and description of the interactome model will be described elsewhere. A reference RNA was chosen over genomic DNA as reference RNA is subjected to the same procedures, such as reverse transcription, as sample RNA thereby providing control for RNA or dye degradation during processing. Total RNA was isolated from Madin-Darby bovine kidney (MDBK) and bovine B lymphocyte (BL-3) cell lines  and fresh bovine cerebral cortex and cerebellum collected from surgery calves following euthanasia at the conclusion of each experiment. RNA from this combination of tissues was experimentally shown to produce cDNA that would hybridize to the majority of the open reading frames (ORFs) represented on the microarray 2 cell culture flasks with Eagle's minimum essential medium (E-MEM) (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS). Tissues were homogenized in ice-cold Tri-reagent® (Ambion). RNA concentration and quality from each sample was determined before and after pooling the samples. Total RNA isolated from three samples was pooled together in equal amounts, aliquoted and stored at −80°C until needed.A reference RNA was used to normalize signal intensity across all arrays A custom, bovine cDNA array consisting of 13,257 unique 70-mer oligonucleotides representing 12,220 cattle ORFs was designed from normalized and subtracted cattle cDNA libraries. The oligos were printed in 150 mM phosphate buffer at 20 µM concentration in duplicate on aminosilane-coated glass slides at the W. M. Keck Center (University of Illinois at Urbana-Champaign). The oligos were annotated based on the GenBank accession number, when available. Further description of the microarray and its development has been previously published Microarrays were used to examine the transcriptional profiles of bovine intestinal epithelia (control and wild type or mutant infected) across seven time points . Experiments were performed in quadruplicate , generating a total of 84 slides with the array printed in duplicate on each slide. The labeling and hybridization procedures have been described previously 2 value of 0.70 or greater. This R2 value may have resulted from poor RNA quality in a small number of the arrays (predominantly arrays generated from the 12 hour time point). Pairwise comparisons and Student's t test with Benjamini-Hochberg correction were performed using GeneSifter , and Spotfire DecisionSite 9.0 . Genes were considered to be significantly altered if the average fold-change was at least 1.5, the adjusted p value was less than 0.05, the alteration was reproducible across replicates, and the magnitude of difference between conditions was greater than 50% of the fold-change obtained for any two replicate controls. Replicate spots, representing a single gene were also expected to “agree.”Immediately after washing, the slides were scanned using a commercial laser scanner . The spots representing genes on the arrays were adjusted for background and normalized to internal controls using image analysis software . Spots with fluorescent signal values below background were disregarded in all analyses. Samples were normalized against the bovine reference RNA signals across slides and within each slide (across duplicate spots). Regression analysis was performed to verify reproducibility of replicate arrays, all of which had an Rz-score method employing a Bayesian estimate of variance z-scores and fold-change data are provided in Comparative pathogenicity analysis and modeling was performed using an integrated platform termed the BioSignature Discovery System (BioSignatureDS™) . This approach for genomic data analysis and modeling at the system biology level offers an integrated view of biological mechanisms and networks of interactions. Specifically for the analysis reported herein, the tools were used to: 1) determine significant gene modulations via a statistical inference http://www.ncbi.nlm.nih.gov/geo/), Accession #GSE13847.All microarray data is MIAME compliant and the original images and extracted data were deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (T) were averaged among each condition and normalized to the CT of the bovine GAPDH, from the same cDNA samples before calculating the fold-change using the ΔΔCT method Ten selected bovine genes with differential expression by microarray results were analyzed by quantitative RT-PCR (qRT-PCR). Two micrograms of RNA samples were reverse-transcribed using TaqMan® Reverse Transcription Reagents (Applied Biosystems). For relative quantification of target cDNA, samples were either run in individual tubes in SmartCycler II  using one SmartMix bead (Cepheid) for 2–25 µl PCR reaction along with 20 ng of cDNA, 0.2X SYBR Green I dye (Invitrogen) or run using 20 ng of cDNA in Sybr Green PCR Master Mix (Applied Biosystems) on an ABI 7500 Real-time PCR System according to the manufacturer's instructions. In either case, Primer Express Software v2.0 (Applied Biosystems) was used to design forward and reverse primers that were used at a concentration of 0.3 µM . For eacSalmonella culture negative calves underwent ligated ileal loop surgery. Loops in each calf were inoculated with wild type S. Typhimurium (WT), a ΔsipA, sopABDE2 mutant (MUT), or sterile culture medium as a negative control (UI). Individual loops from each treatment were collected at seven time points  post inoculation. Each intestinal loop contained Peyer's patch which in the calf extends approximately 1.2 meters cranial from the ileocecal junction. The morphologic description of the ligated ileal loops and Salmonella infection in the calf intestine has been previously described Four male, unrelated, RNA was isolated from the mucosa and submucosa of the Peyer's patch of each intestinal loop. There were a total of 84 RNA samples that were used to generate indirectly labeled cDNA. The cDNAs were co-hybridized on a custom bovine microarray with indirectly labeled cDNA generated from a bovine reference RNA. The same bovine reference RNA was used throughout the experiment and was used for normalization of the microarrays. Gene expression changes associated with infection by WT and the MUT were compared with gene expression in the uninfected loops (UI) and with each other.t test with Benjamini & Hochberg (B&H) multiple hypothesis testing and a statistical inference z-score method employing a Bayesian estimate of variance t test employed a B&H-corrected p value of <0.05 (95% confidence) combined with a |fold-change| of 1.5 as the cutoff values for significance testing. Likewise, the Bayesian statistical inference method employed a |z-score| >1.96 (95% confidence) combined with a |fold-change| >2 as the threshold values for inference testing. Whereas significance testing is a case of post-hoc analysis, Bayesian inference generates probabilistic statements about hypotheses based on the microarray data (a priori estimation) t test alone Two methods of microarray data analysis were employed to determine gene expression changes: Student's Temporally, WT infection induced a biphasic host response with a period of intense gene expression activity at one hour followed by a second phase at later time points of progressively increased activity over time. In MUT loops there was no biphasic response. The number of significantly affected genes increased progressively over time with a pattern of primarily increased gene expression at later time points . While tSalmonella infection in the bovine intestine CCL2, CCL8, CXCL6 (GCP2), and IL8 (CXCL8), and their receptors including CCR1 and CCR3 was seen in both WT and MUT inoculated loops at 4, 8, and 12 hours (z-score| ≥2.24 at any one time and reflects 97.5% confidence in the data (note that no fold-change cutoff was applied). The magnitude of expression of these genes was greater in WT than in MUT, but there was no significant difference between these conditions. Expression of IL8, IL8RB, CXCL6, and CCL8 differed between WT and MUT at 8 hours between these conditions  method described more fully in File S1. The DBGGA method is similar to Bayesian network modeling methods employed by other investigators Salmonella infection including Regulation of Actin Cytoskeleton, Apoptosis Salmonella-induced diarrhea.There were 193 pathways that were significantly altered between WT and MUT infected loops (WT vs MUT) at various time points throughout the experiment . PathwayHelicobacter pylori  and late time points  were explored in greater depth  and 3. Tr pylori . At eighr pylori . PathwayAKT2, JUN, MAPK1, NFKB1, NFKB2, PIK3C2G, PIK3C3, PIK3R2, PRKCG, RAC1, and RHOA. Based on Bayesian z-score, only AKT2, RHOA, and NFKB1 at 1 hour were appreciably regulated (|Bayesian z-score| >2.24). None of these genes were significantly modulated at later time points, however small alterations in expression of these key genes can have significant effects on other genes as reflected in the dynamic Bayesian model.All 14 of the pathways exhibited activation in the MUT loops at 2 hours, 4 hours, and 12 hours. This was not the case in WT inoculated loops where the pattern was more complex. At 15 minutes post-infection, Phosphatidylinositol Signaling System, Regulation of Actin Cytoskeleton, Tight Junction, Wnt Signaling, TGF-β Signaling, BRC Signaling, and CCR3 Signaling in Eosinophils pathways were down-regulated. By one hour, CCR3 Signaling, Regulation of Actin Cytoskeleton, TGF-β Signaling, and Tight Junctions were all activated. These differences between WT and MUT are consistent with differential regulation of host response through the five deleted effector proteins. These seven pathways are connected through regulation of the actin cytoskeleton and phosphatidylinositol signaling and despite early down-regulation, by one hour all but BRC Signaling, Wnt Signaling and Phosphatidylinositol Signaling System were activated. Within the 14 selected pathways, there were 103 genes that were common key mechanistic genes within the pathways. Of these genes, 11 were identified as key intersection points having mechanistic functions in more than five of the 14 pathways . These mSalmonella infection is complex. SopB is a phosphoinositol phosphate phosphatase required for activation of Akt, early trafficking of the Salmonella-containing vacuoles (SCV), and activates an SH3-containing guanine nucleotide exchange factor (SGEF) which is an exchange factor for RhoG Salmonella infection explain the presence and absence of a biphasic response in gene expression in WT and MUT infection respectively. Rac1 and MAPK1, link multiple pathways including the Adherens Junction, BRC signaling, CCR3 Signaling in Eosinophils, MAPK Signaling, Regulation of Actin Cytoskeleton, TGF-β Signaling, Toll-like Receptor Signaling, mTOR Signaling and Wnt Signaling pathways. With the addition of Akt2, Apoptosis, ErbB Signaling, and Tight Junction pathways can be added to the model. Calcium Signaling can be added through the effects of NFKB1 and NFKB2. The Phosphatidylinositol Signaling System cannot be directly linked through these genes but SopB can hydrolyze inositol phosphates and phosphoinositides in vitro and is required for formation of phosphatidylinositol 3- phosphate on SCVs PIK3C2G, PIK3C3, and PRKCG may represent attractive targets for future studies.Although it is not possible to separate the effects of SipA, SopA, SopB, SopD and SopE2 in the data presented here, the known direct and indirect effects of SopE2 and SopB provide crucial links between these 11 mechanistic genes and 14 pathways and the differences between the early phase of WT and MUT infection. The role of SopB during Salmonella infection or the role of SipA, SopA, SopB, SopD or SopE2. We expected that this method would yield a dataset that would include genes with a known role in Salmonella infection as well as identification of expression changes in genes whose role in host response was unexpected. We identified alterations in 38 genes whose functions are as yet unknown. Undoubtedly, the function of these transcripts will be better understood as our understanding of the bovine genome continues to improve and may enhance our understanding of host response to Salmonella infection. The microarray data presented here were validated by confirmation of a portion of the altered genes using real time PCR as well as consistency of our results with previously published data. The limitations of this approach are that only changes in expression of genes spotted on the array are measured and that the intestinal tissue used is comprised of multiple cell types, such that it is not possible to differentiate gene expression from one cell type, such as PMNs, from another type, such as epithelial cells; however, we have employed laser-capture microdissection in other studies to evaluate specific gene expression in specific intestinal cell types. Additionally, the data from this study are limited to gene transcription and do not describe alterations in protein expression or state of activation or phosphorylation that may affect activity in the pathways discussed. Studies to address these issues are currently underway in our labs. Ideally, unbiased evaluation of gene expression would include expression data for all genes but at the time that this study was performed, deep-sequencing techniques were cost-prohibitive. Upcoming studies are in progress to utilize these methods and to further develop our understanding of bovine host response to Salmonella.Using microarray analysis, we were able to generate a temporal gene expression profile for 12,257 genes concurrently. Analysis of a relatively large number of genes reduces investigator bias and allows the generation of a gene expression model. Selection of the genes printed on the microarray was independent of our expectations about the effects of Salmonella and the sipA, sopABDE2 mutant Limitations of our study included the use of four animals instead of a larger number of animals. Previous studies in our laboratory indicated that four animals would be sufficient to detect significant differences between wild type in silico model for host response to both wild type S. Typhimurium and a ΔsipA sopABDE2 mutant. SipA, SopA, SopB, SopD, SopE2 were previously demonstrated to have a combined role in the generation of fluid, induction of cytokines and C-X-C chemokines, and PMN recruitment in bovine ligated ileal loops Salmonella effectors, SopB and SopE2, and the products of three bovine genes, RAC1, MAPK1, and AKT2. Although this subset of interactions does not explain the entire cascade of interactions observed in the data presented herein, they do serve to validate the model and the DBN approach Our original hypothesis and intent was to generate a profile of host gene expression associated with inflammation and diarrhea using two Salmonella strains, wild type and a mutant that generates reduced fluid secretion and inflammatory responses during intestinal infection. Although we have generated gene expression profiles for these two strains, we have not correlated activation or repression of these pathways with the production of fluid in the bovine intestine as a measure of diarrhea. In vitro studies employing gene silencing are underway to determine the roles of the 49 pathways identified as differing between the WT and MUT conditions.The primary result of this work was the establishment of a temporal gene expression profile and development of an CXCL9, CXCL10, and CXCL11. This is consistent with previous work in both rhesus macaques and mice which demonstrated that IL17, IL12, and CXCL10 are increased in response to wild type S. Typhimurium and that this response is ablated in animals with diminished numbers of CD4+ T cells in the intestinal mucosa CXCL9, CXCL10, and CXCL11 was observed in WT and MUT loops  selecting potential intervening gene targets; 2) guiding future experiments for validating host response models; and 3) identifying dynamic gene expression patterns for early diagnostics.In conclusion, this report catalogued the temporal pattern of intestinal gene expression in response to wild type S. Typhimurium in a natural Table S1z-score for All Genes. The z-scores for the individual comparison of gene expression between wild type inoculated loops and control loops, mutant inoculated loops and control loops, and between wild type inoculated and mutant inoculated loops for each gene represented on the microarray are provided. WT = Comparison of gene expression in wild type inoculated loop versus uninfected control loops. MUT = Comparison of gene expression in ΔsipA, sopABDE2 mutant inoculated loops versus uninfected control loops. WvM = Comparison between wild type inoculated loops and ΔsipA, sopABDE2 mutant inoculated loops.(XLSX)Click here for additional data file.Table S2Fold-Change for All Genes. The fold-change in gene expression between wild type inoculated loops and control loops, mutant inoculated loops and control loops, and between wild type inoculated and mutant inoculated loops for each gene represented on the microarray are provided. WT = Comparison of gene expression in wild type inoculated loop versus uninfected control loops. MUT = Comparison of gene expression in ΔsipA, sopABDE2 mutant inoculated loops versus uninfected control loops. WvM = Comparison between wild type inoculated loops with ΔsipA, sopABDE2 mutant inoculated loops.(XLSX)Click here for additional data file.Table S3Detailed gene list for  Summary of significant genes determined by t test using cutoff p value ≤0.05 and |fold-change| ≥1.5 in response to infection with wild type Salmonella Typhimurium or an isogenic ΔsipA, sopABDE2 mutant. UI = Uninfected control loops inoculated with Luria-Bertani broth. WT = Wild type inoculated loop versus uninfected control loops. MUT = ΔsipA, sopABDE2 mutant inoculated loops versus uninfected control loops.(XLSX)Click here for additional data file.Table S4Detailed gene list for  Summary of significant genes determined by Bayesian inference z-score using cutoff |z-score| ≥1.96 and |fold-change| ≥1.5 in response to infection with wild type Salmonella Typhimurium or an isogenic ΔsipA, sopABDE2 mutant. UI = Uninfected control loops inoculated with Luria-Bertani broth. WT = Wild type inoculated loop versus uninfected control loops. MUT = ΔsipA, sopABDE2 mutant inoculated loops versus uninfected control loops.(XLSX)Click here for additional data file.Table S5Detailed gene list for  Summary of significantly altered genes that overlap between the two statistical methods ( and ). Genes with significant z-score changes and significant p value and 1.5-fold-change in response to infection with wild type Salmonella Typhimurium or an isogenic ΔsipA, sopABDE2 mutant. UI = Uninfected control loops inoculated with Luria-Bertani broth. WT = Wild type inoculated loop versus uninfected control loops. For genes to be on this list, they had to be found significant based on both statistical analysis methods .(XLSX)Click here for additional data file.Table S6z-score of pathways that differed significantly between loops inoculated with wild type Salmonella Typhimurium or an isogenic ΔsipA, sopABDE2 mutant over time. Pathways are based on Kyoto Encyclopedia of Genes and Genomes descriptions and pathway numbers. Symbol_Count refers to the total number of gene symbols present in a pathway. Observed_Count refers to the number of genes that were observed to be significantly altered at a minimum of one time point. Probe_Count refers to the number of spots on the array that were significantly altered at a minimum of one time point (time in minutes).(XLSX)Click here for additional data file.Table S7Pathways associated with key mechanistic genes. Key mechanistic genes (determined by Bayesian z-score) from selected pathways identified by dynamic Bayesian Modeling. Pathways are based on Kyoto Encyclopedia of Genes and Genomes descriptions and pathway numbers.(XLSX)Click here for additional data file.File S1Description of the methods used in the integrated platform termed the BioSignature Discovery System (BioSignatureDS™) .(DOC)Click here for additional data file.