Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved.Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors.Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.The online version of this article (doi:10.1186/1471-2164-15-1049) contains supplementary material, which is available to authorized users. Plant miRNAs are indispensable for the control of wide variety of biological functions, including development, hormone responses, feedback mechanisms and biotic and abiotic stresses , 2. MostmiRNAs are generated in a stepwise manner from long non-coding, genome-encoded PolII transcripts. Primary miRNA transcripts of varied length must form a secondary structure that can be recognized by Dicer-like protein 1 (DCL1) and its partners. DCL1 complex cleaves the secondary structured RNA to a pre-miRNA structure having a stem with almost complete complementarity and a loop. Another processing step in the cytoplasm produces miRNA:miRNA* duplex of predominantly 21 nts with 2 nt overhangs. While miRNA* is usually degraded, miRNAs associate with Argonaute (AGO) proteins to form RNA-induced silencing complexes (RISC). Most of the plant miRNAs appear to be targeting mRNAs for degradation, typically ‘slicing’ target mRNA between position 10 and 11. There are also many reports of plant miRNA:mRNA complexes leading to translational inhibition [Arabidopsis and other related plants [Many factors in miRNA pathway have been characterized, but there is limited information on the self-regulation of miRNA pathway itself. Three miRNAs have been reported to regulate DCL and AGO by cleaving transcripts of corresponding targets as part of a robust feedback mechanism. miR162 has been implicated in targeting DCL1 , miR168 d plants –19. The Arabidopsis. Thus, it appears that Arabidopsis has a refined feedback regulatory loop that balances AGO1 and miR168 accumulation. In addition, miR168 expression is regulated by invading viruses. Upon infection with wide range of viruses, miR168 levels go up to dramatic levels quite quickly, leading to the repression of AGO1 translation [Arabidopsis, although an in-depth analysis is not forthcoming.A relatively well-known feedback mechanism involves AGO1 homeostasis that is controlled by coordinated action of miR168 , 20 and nslation , has notN. tabacum) has isoforms with insertion of transposon-like sequences that are likely to reduce their processing, providing a clue to the loss of miRNAs. Furthermore, when we analyzed miR168 sequences across 68 plant species representing almost all major plant families, a clade specific abundance of miR168 was observed. The maximum abundance and diversity of miR168 was among monocots. Monocots have more numbers of AGO1 members than dicots, but few of them are not readily targeted by miR168 creating functional difference. The regulation of AGO2 and AGO3 mRNAs by miR403 is specific to very few plants. miR403 is absent among monocots and many eudicot lineages. Absence of miR403 among monocots indicates an inverse relationship with that of miR168, since miR168 is highly expressed among all monocots studied. In those plants where miR403 is present, the sequences are less diverse and plants have fewer isoforms of this miRNA indicating recent evolution of miR403. Together, this analysis indicates that miR168/miR403 relationships with their targets as observed in Arabidopsis are likely to be specific only to few plant lineages and plants have evolved every shade of such regulation providing case for altered transcription factor regulations and disease resistance.The induction of miRNAs by viruses to meddle with AGO expression indicates that pathogens use these miRNAs particularly to suppress host silencing. This implies that variation in expression levels or targeting abilities of miRNAs among plant lineages has the potential to be the basis of susceptibility or resistance against pathogens. In order to understand these relationships, we analyzed sequence diversity, copy numbers and expression levels of miR168 and miR403 among plant species for which a small RNA dataset is available. Surprisingly, our results suggest that mature sequences of miR168 can be classified into three clades. Presence of three clades is also evident after aligning precursor sequences of these miRNAs. Variation in miR168 sequences seems to correlate well with their AGO1 mRNA targeting abilities. Strikingly, tobacco (Solanaceae members. Solanaceae members exhibited similar sequence in 14th position (G) like other dicots, but had similarity at 21st position (C) identical to monocots. The miR168* sequences, on the other hand, had uniform sequence diversity among monocots. Distinct miR168* sequences for Solanaceae members were not observed. Among Solanaceae members, Nicotiana tabacum showed an unusual sequence diversity with two forms  showing mature miRNA sequences similar to other dicots while three additional isoforms  having a Solanaceae specific sequence signature. Two sequences from the dicot clade, miR168 from Brassica napus and Medicago truncatula, had variations that could not be compared to three clades and are likely results of rapid independent evolution.In order to understand the sequence diversity of miR168, we used sequences from miRBase (version 20) as well as from genome-wide transcriptome data reported from plants that have been studied. A total of 58 sequences were obtained representing 31 families of plants. Among these, 16 were newly designated sequences. All 58 miR168 and miR168* sequences were used for sequence alignment , Cardamina flexuosa (Brassicaceae), Citrus clementine (Rutaceae), Theobroma cacao  and Brachypodium distachyon (Poaceae), functional significance of which is unknown.If the miRNAs and their targets co-evolved with their target genes in different plant lineages as proposed , 23, 24,Populus trichocarpa, Malus domestica, Theobroma cacao showed very high tapir scores of 10.5, 12 and 7, respectively, and correspondingly low MFe ratios indicating that their AGO1 mRNAs may not be targets of their miR168.It is unclear whether miRNAs from one plant species target their mRNAs to the same levels as in other species as such studies have not been carried out. In order to understand the significance of variations in miR168 mature sequences we predicted the miR168 targeting abilities among AGO1 mRNAs of all plant species that have clearly identified miR168 sequences. We used both pssRNAtarget  as well Zea mays such as AGO1c and AGO1d are not targeted by zma miR168, but AGO1a and b are good targets  is clearly not a target of bdi miR168 (more than 3 mismatches in seed region). These differences among monocots might contribute to the diversity in their miRNA pathways since AGO1 is a major player in miRNA stabilization and action. We also predicted targets for few miR168 examples from each of the three clades with AGO1 mRNAs from the corresponding plants  have unusually long (up to 290 nt) loops.The sequence and distance between miRNAs and miRNA* must indicate evolutionary history of miRNAs . HoweverSolanaceae, those with shorter loops had structures similar to other dicots, but as expected the structures of miR168 isoforms with long loops were complex. The nta miR168d and e precursors have long loop sequences similar to each other but extremely different from any other miR168 precursor  from few dicots  on the other hand, seem to have the common dicot specific form as the most abundant. Musa not only has a dicot specific form as most abundant form, but also has accumulation of miR168 levels matching those of dicots in that it has low accumulation. The monocot specific form surprisingly is found also among Cycads (Cycas rumphii), Gingkophyta (Gingko biloba) and Pinophyta (Picea abies). These species represent forms that are ancient to monocot/dicot divergence and it is easy to speculate that these two forms are ancient. Magnoliids (Aristolochia and Persea) show abundance of either mature miR168 form depending on the species. Depending on the plant lineage some miR168 forms could have evolved and expressed better than the other forms. Vitis vinifera , a eudicot, has higher expression of monocot specific form unlike other eudicots for those a sequence information is available, is an example wherein both forms co-exist.We used small RNA datasets derived from 42 plant species representing 25 plant families to compare expression and diversity of miR168 sequences. Next generation small RNA sequence datasets from the corresponding species were taken from GEO and other published sources . The abundance of miR168 and those reads that match to miR168 with 1 or 2 mismatches were taken into consideration  as genoArabidopsis and members of Solanaceae such as Solanum spp. and Nicotiana spp. Most abundant form of miR168 among Solanaceae was the form that is distinct from other dicots. These forms have also been reported recently in a global study using small RNA datasets from many vascular plants [Mimulus guttatus (Lamiaceae), Lactuca sativa  have the highest accumulation of miR168.Surprisingly, abundance of miR168 was comparatively low among r plants . Among dArabidopsis that floral tissues accumulate more 24 nt siRNAs than miRNAs, miR168 levels are generally low among floral samples of all plant species analyzed. Unexpectedly, rice (Oryza sativa) and sorghum (Sorghum bicolor) are some of the few species where miR168 was consistently expressed highly among both leaf and floral tissues  when compared to all other species. This lack of diversity in sequences arises from its recent evolution as this miRNA is present only in few lineages of eudicots. Some plant species have many copies of miR403, for example Vitis has 5 copies, all with the same mature sequence but with diverse miR* sequences. The most common and abundant form of the miR403 is 5’-UUAGAUUCACGCACAAACUCG-3’. Few interesting deviations from this sequence were observed in Solanaceae members (S. pennellii and S. tubersum) and some Malvids (Carica papaya) sharing 5’-CUAGAUUCACGCACAAACUCG-3’ as the major miR403 isoform. This isoform is extremely interesting in that it has an unusual 5’ terminal nucleotide as C. One more abundant form specific to Gossypium arboreum is 5’-UUAGAUUCACGCACAAACUCA-3’, thus, Malvids seem to have the most diversity in miR403 sequences.A similar search for miR403 in miRBase as well as transcript databases recovered 35 sequences Figure . miR403 Cucurbita, Phaseolus and Medicago spp.) have no expression of miR403 , a member of Malphigiales that belong to Fabidae have high expression of miR403. Vitales have very high expression both in vegetative and reproductive tissues .Surprisingly, while two sets of Rosiids called Malvids and Vitales have abundant miR403, some Fabids  and Vitales , while it is ineffective with score of excess of 10 among Fabids .Arabidopsis where AGO2 gets induced during viral infections [Rice stripe virus and Rice dwarf virus infections in rice [On the other hand, absence of miR403 among monocots correlates well with the expression levels of AGO2. In rice, AGO2 expression is very high among all tissues unlike in Arabidopsis where AGO2 expression is largely confined to siliques and at much lower levels in leaves . It remafections , there i in rice . This suN. tabacum. The common dicot forms of miR168 in tobacco (isoforms d and e) have insertions of MITE-like transposons. A direct result of this insertion corresponds to reduced accumulation of mature miRNA forms in all tissue types. The Solanaceae-specific forms on the other hand , does not have MITE-like insertions and are expressed at high levels. A duplication event of miR168 in tobacco might have ended up with isoforms having two different miR168 sequences. One set (d and e) while retaining mature miR168 of dicot ancestor, attracted transposons in the loop region. Tobacco seems to be a hotbed for miR168 duplication since it already has the most diverse miR168 precursors. In addition, there are many tobacco genomic regions matching miR168 sequences present in the genome, sometimes their miR* sequences either missing or present a long distance downstream or upstream. These may represent the lost miR168 isoforms. It is possible that miR168 example that is observed in tobacco is seen in other miRNAs where some forms are lost during duplication and subsequent loss.A case for loss of miRNAs can be argued based on the miR168 diversity in Transposons invite siRNAs and methylation. Invasive Transposons such as MITES can be detected and neutralized using RNA-directed DNA methylation . This maArabidopsis is specific only among few plant lineages. Plants have evolved every shade of this regulation providing case for varying miRNA levels, thus influencing transcription factor and other activities regulated by miRNAs. The nature of this interaction may also influence disease resistance due to the way viruses use these miRNAs to manage the arms race with their host plants.Our study indicates that intricacies of AGO targeting by miRNAs as observed in http://srna-tools.cmp.uea.ac.uk/. Sequences of miR168 and miR403 were obtained from miRBase release 20 [The UEA small RNA analysis toolkit  was usedlease 20 , 44 and lease 20 . Previoulease 20 ) as welllease 20  and TAPIlease 20 . To findlease 20 , Solanaclease 20 , Refseq lease 20  as well lease 20 .Analysis of abundance of miR168 and miR403 members were performed through miRProf analysis of published large-scale data sets derived from various plant species available through the Gene Expression Omnibus (GEO) platform , 50. Thehttps://code.google.com/p/jmodeltest2/). For the J-model test the precursor alignment was given as the input in “.aln” format. It calculates for the variations in the nucleotide sequences and gives the log-likelihood scores for all the models for phylogenetic tree construction.The phylogenetic tree was constructed using the MEGA 6.0 software with Neighbor Joining Method with 1000 bootstrap replications. The model used was Jukes Cantor that had the highest log-likelihood score according to the J-model Test Additional file 2: Figure S1: Phylogenetic analysis of precursors of miR168. (PPTX 62 KB)Additional file 3: Figure S2: Targeting abilities of miR168 among selected plants indicating mosaic targeting among multiple AGO1 members in monocots. (PPTX 675 KB)Additional file 4: Figure S3: Length of loops (distance between miRNA and miRNA*) among 58 miR168 precursors from diverse plants. (PPTX 94 KB)Additional file 5: Figure S4: Secondary structures of 58 precursors of miR168 indicating clade-specific changes in the loop region. (PPTX 620 KB)Solanaceae indicating site of MITE insertion. (PPTX 79 KB)Additional file 6: Figure S5: Multiple sequence alignment of miR168 precursors from Additional file 7: Figure S6: Abundance and sequence diversity of miR168 members across plant families in reproductive tissues. (PPTX 88 KB)Additional file 8: Figure S7: miR168 is not significantly induced in rice upon infection with viruses. (PPTX 63 KB)Additional file 9: Figure S8: Abundance and sequence diversity of miR403 members across plant families in reproductive tissues. (PPTX 6 MB)Additional file 10: Figure S9: Ago2 targeting abilities of miR403 across plants. (PPTX 239 KB)Additional file 11: Figure S10: Ago3 targeting by miR403. (PPTX 51 KB)


Breastfeeding is recommended by the World Health Organization regardless of mother’s hepatitis B virus (HBV) status since breast milk is not considered as an efficient pathway for HBV perinatal transmission.3We present here a case of an HIV-HBV co-infected mother who exposed her child to a significant HBV infectious inoculum via breast milk, likely due to a hepatitis flare or an acute HBV infection.3 log10 HBV DNA IU/mL) whereas, ALT level was normal (12 IU/ml). HBsAg levels were also quantified in breast milk (>101 IU/ml) using a quantitative assay . A dramatic decrease in the levels of HBV DNA and HBsAg was observed in both serum and breast milk at week 12 post-delivery  that steadily decreased at months 1, 3, and 6 postpartum . HBV DNA was detected in breast milk from three HBV viremic women (33%) and four out of 24 breast milk samples (17%). When detectable, HBV DNA levels in breast milk were low in all cases ranging from 1.18 to 2.20 log10 IU/ml, suggesting that level of HBV DNA in breast milk might always be negligible. The rate of HBV infection was also assessed among 23 HIV-HBV exposed children along the two years study period. Diagnosis of transient HBV infection was made in four children whereas other three children acquired HBV infection during the postnatal period (first HBsAg positive test at 12 months in one case and 24 months in the other two cases). Pirillo et al observed that those 7 children were exposed to the highest HBV infectious inoculum, not only at delivery, but also during all the postnatal period, since their mothers presented the highest levels of HBV DNA in serum. However, no correlation was found between HBsAg positivity and HBV DNA levels in breast milk.Recently, HBV DNA was tested in serum and breast milk using a standardized quantitative PCR method in HIV-HBV coinfected mothers who were under lamivudine-containing antiretroviral regimen from the 25th week of pregnancyIn our case, exposure to a larger amount of HBV infectious particles via breast milk was detected, but the child, who was correctly vaccinated, was not infected with HBV. This result seems to confirm that breastfeeding is not an effective route of HBV transmission during the perinatal period, as previously reported in clinical studies.11Hepatitis flares or acute HBV infections in chronic HBV infected patients do not usually required antiviral treatment since most of the episodes resolve spontaneously.Exposure to a significant HBV infectious inoculum through breastfeeding should not always be considered negligible in children from HIV-HBV coinfected mothers who do not receive any active antiviral treatment against HBV. However, cumulative exposure during lactation does not seem to contraindicate breastfeeding among correctly vaccinated children despite the immaturity of their intestinal mucosa.


Myotis lucifugus to seek evidence for infiltration of these retroviruses in other mammalian species (>100) currently represented in the genome sequence database. We provide multiple lines of evidence for the cross-ordinal transmission of a gammaretrovirus endogenized independently in the lineages of vespertilionid bats, felid cats and pangolin ~13–25 million years ago. Following its initial introduction, the ERV amplified extensively in parallel in both bat and cat lineages, generating hundreds of species-specific insertions throughout evolution. However, despite being derived from the same viral species, phylogenetic and selection analyses suggest that the ERV experienced different amplification dynamics in the two mammalian lineages. In the cat lineage, the ERV appears to have expanded primarily by retrotransposition of a single proviral progenitor that lost infectious capacity shortly after endogenization. In the bat lineage, the ERV followed a more complex path of germline invasion characterized by both retrotransposition and multiple infection events. The results also suggest that some of the bat ERVs have maintained infectious capacity for extended period of time and may be still infectious today. This study provides one of the most rigorously documented cases of cross-ordinal transmission of a mammalian retrovirus. It also illustrates how the same retrovirus species has transitioned multiple times from an infectious pathogen to a genomic parasite (i.e. retrotransposon), yet experiencing different invasion dynamics in different mammalian hosts.Endogenous retroviruses (ERVs) arise from retroviruses chromosomally integrated in the host germline. ERVs are common in vertebrate genomes and provide a valuable fossil record of past retroviral infections to investigate the biology and evolution of retroviruses over a deep time scale, including cross-species transmission events. Here we took advantage of a catalog of ERVs we recently produced for the bat  The cross-species transmission of viruses poses a continuous threat to public health. Bats are increasingly recognized as a major reservoir for zoonotic RNA viruses, including rabies, Ebola, and possibly MERS, but little is known about their capacity to harbor and transmit retroviruses. Here we investigated past incidents of cross-species transmission involving bat retroviruses, by screening for the presence of endogenous retroviruses (ERVs) previously identified in the genome of the little brown bat in more than 100 diverse mammal species. This screen revealed an intriguing case of a gammaretrovirus that independently infiltrated the germ line of species belonging to three mammalian orders: vesper bat, felid cat and pangolin. We found that the ERV initiated its genomic invasion of the three lineages around the same timeframe ~13–25 million years ago, but experienced a different fate in each lineage. In the pangolin lineage, the ERV’s genomic propagation stalled shortly after endogenization, while it amplified continuously throughout felid and vesper bat evolution to generate hundreds of species-specific insertions in each lineage. Furthermore, in the cat lineage genomic amplification appears to have occurred predominantly via retrotransposition; while in bats the ERV has expanded via a mixture of retrotransposition and reinfection activity that may still be ongoing. Most viral diseases of humans are zoonotic: they stem from CST of viruses from domestic or wild animals . The expiral CST ,4. The piral CST . Other riral CST –10.Retroviruses are unique amongst animal viruses in that chromosomal integration of so-called proviruses is an obligatory step in their replication cycle . As a coMany ancient CST events have been inferred by comparing ERV sequences across species –28. MostMyotis lucifugus [Bats (order Chiroptera) are increasingly regarded as exceptionally potent reservoirs of zoonotic viruses –40. Indeucifugus   (642 nt) from members of each of the 86 MLERV subfamilies previously identified [M. lucifugus, the most significant hits  were obtained with a query representing the MLERV1 family [Felis catus) [Panthera tigris) [Manis pentadactyla). In addition, and less surprisingly, many highly significant hits to MLERV1 were also obtained in the genomes of vespertilionid bat species closely related to M. lucifugus .To detect possible CST events involving (RVT_1) 6 nt from (RVT_1) 6 nt from s catus) , Amur ti tigris)  and Chinbrandtii ; David’s davidii ; big broManis pentadactyla ERV1 and deposited its consensus sequence in Repbase. For simplicity, we refer to all the elements detected in vespertilionid bats as MLERV1 and all the elements in different felids as FcERV_γ6.Further examination revealed that the hits in the feline genomes corresponded to an endogenous gammaretrovirus family initially described in the domestic cat. Two proviruses of this family were initially documented in cat as FERVmlu1 and FERVmlu2 . In 2011M. lucifugus). It should be noted that the vast majority of proviruses we inferred to be full-length (based on the occurrence of a pair of LTRs within 10 kb) contain sequencing/assembly gaps. Thus we cannot ascertain whether they contain all the coding domains of a complete provirus.To determine the ERV copy number in each species, we used the LTR sequences to mask their corresponding genome assembly using the Repeatmasker program and parsed the positional output to infer the number of putative full-length proviruses (i.e. containing two LTRs) and solitary (solo) LTR see . The resTo illustrate the exceptional level of sequence similarity among MLERV1, FcERV_γ6 and MPERV1, we generated nucleotide pairwise alignments of FcERV_γ6 and MLERV1 and of FcERV_γ6 and MPERV1 using the most closely related full-length proviruses from each family and performed a sliding window analysis of nucleotide identity across the two pairwise alignments . As a coThe overall level of nucleotide similarity between MLERV1, FcERV_γ6 and MPERV1 is strongly incongruent with a scenario of vertical inheritance of an ancestral ERV present in the common ancestor of chiropterans, felids and pangolins, which dates back to ~85 million year ago (MYA) ,55. FurtMyotis considered here.To gain further insights into the evolutionary history of these ERVs, we next sought to estimate when they first infiltrated their host genomes. Given that the likelihood of the same endogenous retrovirus to integrate at the same exact genomic location independently in different lineages is negligible, the presence of an element at orthologous position in different species can be interpreted as having inserted prior to their divergence time ,57. ConvWe first examined the sharing of FcERV_γ6 elements between the cat and tiger, which diverged ~10.8 MYA . Out of M. lucifugus can be divided into 3 subfamilies. Here we performed a systematic analysis of the presence/absence of MLERV1 elements (including solo LTRs) from the 3 subfamilies in the genome assemblies of three other vespertilionid bats currently available: Brandt’s myotis (Myotis brandtii), David’s bat (Myotis davidii) and the big brown bat (Eptesicus fuscus), which have been estimated to diverge from M. lucifugus ~10 MYA, ~13 MYA and ~25 MYA, respectively [Myotis genomes  of MLERV1 elements in the 3 Myotis genomes, could not be identified at all in the E. fuscus genome. Indeed, all 29 elements detected in E. fuscus cluster with either one of the other two subfamilies  MY . However ~10 MYA ,64, but  ~10 MYA ,72. IndeTo further characterize the evolution history of MLERV1, FcERV_γ6 and MPERV1, we examined the phylogenetic relationship of elements within these families using a maximum-likelihood tree built from an alignment of their 3’ LTR sequences. We used only ‘complete’ proviruses , since we observed that including tiger provirus and solo LTRs did not yield any new major clade in the phylogeny . Also thThe unrooted tree resulting from the phylogenetic analysis  clearly To further explore the history of the FcERV_γ6 and MLERV1 families, we next turn to an analysis of selection regimes that have acted on their coding sequences during their amplification. Such analysis can help discern whether ERVs have spread primarily through reinfection or retrotransposition events because the latter mechanism, which is strictly intracellular, is predicted to be associated with the loss of envelope function. Indeed, the envelope protein binds to host cell membrane receptor to promote virion entry in the host cell and therefore is required for most retroviral infection . Thus, pTo evaluate how natural selection may have constrained the different coding regions of MLERV1 and FcERV_γ6, we computed the dN/dS ratio (ω) applying the branch model implemented in PAML, where dN denotes the non-synonymous substitution rate and dS denotes the synonymous substitution rate, along the branches of the phylogeny of FcERV_γ6 and MLERV1 elements for each of their predicted coding domains . ω valuep < 0.05), with the notable exception of Gag matrix and envelope domains . First, elements found in these species display a level of nucleotide sequence similarity (~85%) along their entire length that is comparable to that observed between closely related retroviruses that have undergone very recent CST (such as SIVcpz and HIV-1). Such a level of sequence similarity between ERVs inhabiting species diverged by ~85 MYA  is incomIt is conceivable that this retrovirus could have transferred directly between these ancestral species because their geographic distribution likely overlapped in Eurasia during the estimated period of initial ERV infiltration ~13–25 MYA) ,62,83. G5 MYA ,62KIT gene currently segregating in domestic cats is responsible for the “Dominant White” and white spotting pigmentation phenotypes [KIT locus lacks envelope domain and clusters with other recently active FcERV_γ6 copies in our phylogenetic analysis  introduced in their common ancestor or if they result from independent acquisition of the same retrovirus. On the one hand, the observation that both E. fuscus and Myotis harbor elements from two diverged subfamilies may be interpreted as evidence that these subfamilies descend from a single progenitor ERV acquired in the common ancestor of these species. On the other hand, the fact that none of the MLERV1 insertions are shared (orthologous) between E. fuscus and any of the 3 Myotis genomes   from an infectious pathogen to a genomic parasite (i.e. a retrotransposon). The biological factors and sequence of events underlying such transition remain poorly understood. In a seminal study, Ribet et al. showed that the loss of envelope gene combined to the gain of an endoplasmic reticulum targeting signal were apparently sufficient for an infectious progenitor of the mouse IAP elements to turn into a highly active retrotransposon . Magiorkdavidii) . Thus, oOne important difference is that the shift between infection and retrotransposition in the MERV1 family was apparently accompanied by little changes in the sequence of MLERV1 elements. Indeed, members of the MLERV1_2 and MLERV1_3 subfamilies diverge by ~15–20% in their RT domain nucleotide sequences with Kimura correction. By comparison, infecting and retrotransposing IAP subfamilies diverge more substantially (~65% in RT domain). Thus our findings suggest that the transition between the two modes of ERV amplification can occur relatively fast during ERV evolution.KIT insertion segregating in domestic cats). Meanwhile, in the bat lineage, the ERV followed a more complex evolutionary path characterized by multiple episodes of reinfection, and at least one burst of amplification by retrotransposition. These observations beg the question whether the loss of infectious capacity of an ERV and its conversion to a retrotransposon is a purely stochastic process, largely owing to the stochastic mutation of gag matrix and loss of envelope functions, or possibly the characteristics of different proviral ancestors, or if it can be influenced by some biological characteristics of the host species? For instance, it has been recently reported that the level of endogenous retroviral activity may be partly governed by host body size [An intriguing finding of this study is that the same or very similar retrovirus was endogenized in three different mammalian hosts, but followed quite different evolutionary trajectories in the three species lineages. In the pangolin lineage, the ERV family failed to amplify (only 2 detectable copies) and was essentially ‘dead-on-arrival’. In the cat lineage, the ERV progenitor apparently lost its infectious capacity shortly after endogenization and subsequently amplified to high copy numbers by retrotransposition through an extended period of time ranging from at least 10 MYA (256 insertions orthologous in cat and tiger) to modern times .To comprehensively retrieve all proviruses and solo LTRs related to the FcERV_γ6/MLERV1/MPERV1 families in each of their respective genomes, we run RepeatMasker  with defIn the pangolin genome, our initial MEGAblast search yielded only 2 significant hits to the MLERV1 RVT_1 domain, but many more related ERVs could be retrieved using the RT domain from these two initial hits in reiterative blast searches against the pangolin genome assembly. To examine the relationship of these RT elements to each other and to the MLERV1 and FcERV_γ6 families, we conducted a phylogenetic analysis using the Maximum Likelihood package PhyML3.1 with the GTR+Γ model . The resAll identified ERVs are available as bed format .To generate the sliding window analysis shown in https://github.com/xzhuo/orthologusLTR.git).Orthologous ERV loci were detected similarly as we described previously . Briefly−9 yr-1 [−9 yr-1 [−9 yr-1 [Sequence divergence between 5’ and 3’ LTRs from the same provirus was computed as previously described . To infe−9 yr-1  and 1.8 [−9 yr-1  for the [−9 yr-1  to inferThe maximum-likelihood phylogenies presented for LTR sequences were built using PhyML3.1 and the support for each node is determined with an approximate likelihood ratio test . The mulERV coding regions were predicted using HMMER3 in all 6 reading frames  to delinCoding region integrity was assessed by calculating the frequency of stop codon or frameshift indels per codon. We calculated the total length of each domain in every subfamily, and counted the occurrence of stop codon and frameshift indels. The mean frequency and 95% confidence interval is calculated using the Poisson distribution .S1 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 FigY axis represents frequency of stop codon or frameshift indel per codon in each domain, four different subfamilies are illustrated with different colors. Error bars represent 95% confidence interval.(PDF)Click here for additional data file.S5 FigAll members of MPERV1 are enclosed within an arch, and the aLRT support of MPERV1 is labeled at the branch node.(PDF)Click here for additional data file.S6 FigE. fuscus cluster with either MLERV1_1 or MLERV1_2.Three subfamilies of MLERV1 are illustrated. SoloLTRs in (PDF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity.In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca Therefore, it is critical to develop experimental methods to study the primary phase of bTBI.Blast-induced traumatic brain injury (bTBI) continues to be a worldwide health problem. bTBI can be complex, resulting from one or more physical phases of the blast phenomenon. Even those experiencing low-level blast explosions, such as those produced by explosives used to breach fortifications, can develop neurocognitive symptoms without evidence of neurotrauma3452+ ion-selective fluorescent indicators, changes in intracellular free calcium following simulated blast were detected. We previously showed that a) cultured human brain cells are indifferent to transient shockwave pressures known to cause mild bTBI, b) when sufficient shear forces are simultaneously induced with the shockwave pressure, central nervous system (CNS) cells respond with increased intracellular Ca2+ that propagates from cell to cell; and c) cell survival is unaffected 20 hours after shockwave exposure2+.To better study the primary phase of bTBI, we developed a pneumatic device that simulates an explosive blast by producing pressure transients similar to those observed in a free field explosion and is compatible with real-time fluorescence microscopy of cultured cells; this device can produce blast-like pressure transients with and without accompanying shear forces7Astrocytes respond rapidly to traumatic brain injury, having both beneficial and deleterious effects in a wide range of pathological conditions. Under normal conditions, astrocytes also have important roles in integrating information and feedback modulation exists between astrocytes and neurons911141516We show that simulated blast primarily affects calcium signaling in human astrocytes producing calcium waves that propagate via purinergic signaling. Dissociated human CNS cortex cells, gestational weeks 19–21, are more responsive than dissociated rat CNS cortex, embryonic day 18. Two genes, astrocyte GFAP and matrix metallopeptidase 9 (MMP-9), have increased expression in human cell cultures and may be involved in longer-term brain effects associated with mild bTBI.2+ are observed72+ occurred within the observation field  alone or TTX (1 μM), (2R)-amino-5-phosphonopentanoate, (APV) , and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX)  to block excitability, NMDA and non-NMDA glutamate receptors, respectively, had no significant effect on the calcium response (integrated Δ F(t)/F0 time course, n = 4 for each condition, p = 0.19, single factor ANOVA). Since astrocyte calcium signaling in response to mechanical strain or cell swelling is known to occur through purinergic receptors, we tested whether the observed propagation of increased cytoplasmic free Ca2+ in this preparation is a result of purinergic activity. Cells were laser wounded in the presence of apyrase (Sigma-Aldrich), an enzyme that rapidly degrades extracellular ATP. A significant dose-dependent reduction in the calcium response was observed with increasing concentrations of apyrase . Cellular ΔF Fluo-4 fluorescence before, immediately following, and 3 minutes after the addition of KCl in NB+B27 is shown in To examine the extent that calcium responses to simulated blast are propagated by neurons or astrocytes, calcium levels were monitored continuously using the fluorescence signal ΔF before, during and after blast . The pseWe investigated whether the calcium response to simulated blast is dependent upon a purinergic signaling pathway as observed using laser wounding. The effect of purinergic ATP (P2) receptor inhibitors was evaluated in the same well and field of view following a control blast. Calcium levels were monitored continuously during the experiment; an example of the peak fluorescence signal observed following a control blast is shown . To reprIn seven of eight trials in which a first blast response was detected in the astrocytes, subsequently identified using KCl, the addition of 100 μM PPADS significantly reduced the blast response . To de-cTo further quantify these changes in astrocytes, the distribution of decay times, exponential offsets, and calcium responsive cell fractions were evaluated. In the presence of PPADS, the astrocyte decay times were exponentially distributed with τ parameter 8.26 (0.49) seconds (fitted τ (95% confidence)). This represents an ~20% reduction in the τ parameter compared to matched controls. The distributions of decay times for matched controls and PPADS treated cells were significantly different for both astrocytes and neurons . The astrocyte exponential offset value was significantly lower following PPADS treatment (0.21 (0.00) vs. 0.68 (0.01), offset (95% confidence); PPADS vs. matched controls, respectively) indicating that the persistence of calcium was decreased in PPADS treated astrocytes after de-convolving propagation-dependent changes. The fraction of calcium responsive astrocytes was significantly reduced to 44% (23%) (mean (95% confidence)) corresponding to an ~54% reduction in the number of responsive astrocytes while the fraction of calcium responsive neurons remained unchanged  or the time-averaged activity of astrocytes and neurons following a second blast . Blocking the ATP P2 receptors with PPADS significantly reduced both the proportion of responsive astrocytes and the time-averaged response of astrocytes to blast.in vivo effects in humans compared to rat.Since human astrocytes differ from rat we compared the responses of rat and human astrocytes to simulated blast. Rat astrocytes produce focal, transient and minimally propagating calcium responses compare  to M1. CThe chronic neurocognitive effects that can develop after blast in the absence of brain injury evidence indicate that a brief blast exposure may lead to persistent changes in cellular function. Here we report, for the first time, gene expression changes in human dissociated CNS cultures, 24 hours following conditions simulating mild bTBI . Relativ1819Utilizing a microscope system that allows exposure of human CNS cells to blast-like pressure profiles that are comparable to those associated with mild bTBI7In our system, the response of human CNS cells to simulated blast is the initiation of a propagating calcium response from one or more sites within the culture well. Astrocytes are the main carriers of this calcium wave. Astrocytes respond with higher proportion and calcium load compared to neurons; the calcium elevation occurs on the time scale of minutes. Blocking P2 receptors with the antagonist PPADS significantly reduced both the proportion of responsive astrocytes and the time-averaged calcium response of astrocytes without affecting the proportion of responsive neurons. The reduction in the time-averaged response of neurons was smaller than that of astrocytes. The calcium wave often propagates from outside the field of view, and can reach the field of view from different directions and distances; consequently, the apparent response time can differ between different culture wells. It is not clear what mechanism initiates the cellular response; focal membrane damage, activation of mechanoreceptors, and disruption of local cell contacts may all produce the initiating disturbance that results in the wave(s) of calcium activityTo determine if the blast-induced calcium response is the result of a common signaling pathway initiated from focal response sites we used laser injury to create a spatially defined injury site where calcium wave propagation was initiated. The response to laser induced focal damage was the generation of calcium activity that propagated throughout the cellular culture. This propagated calcium activity was blocked by apyrase-dependent ATP hydrolysis and by the P2 receptor antagonists PPADS; both observations are consistent with P2 receptor activation contributing to the propagated calcium activity. Furthermore, the propagated calcium activity was not dependent on neuronal communication since blocking neuronal activity with TTX, APV and BNQX had no effect. In both simulated blast and laser wounding, the calcium propagated response and the dependence on astrocytes and P2 receptors blockers were similar, consistent with the hypothesis that the calcium activity propagates via astrocyte-dependent pathways23in vitro. The reasons for this difference may involve receptors, as there are known differences in receptors: the rat P2Y4 receptor responds to ATP as an agonist while in humans the same receptor responds to ATP as a competitive antagonistThe response to blast-like shock waves with concomitant shear forces differed between astrocytes from dissociated human CNS cortex, gestational weeks 19–21 and rat CNS cortex, embryonic day 18; human astrocytes have an increased calcium load and different propagation kinetics. These differences may result from species and/or developmental differences  effects observed in blast victims. In response to CNS injury, aberrant calcium elevations may be part of the pathological process. In rodent in vitro models, tertiary and secondary blast phases produce cellular responses involving astrocytes, including glial scar formation and formation of reactive astrocytes193031333436in vivo as a result of a TBI, a number of pathways and mechanisms have been proposed to explain the loss of brain function including glutamate excitotoxicity due to impaired glutamate uptake38392+ 45The mild bTBI response to the primary blast phase can be relatively short, on the order of minutes to hours, but the pathophysiological consequences can last for much longer times. In our 2+-dependent activation of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and Jun N-terminal kinase (JNK)In our system, which models the primary blast phase with no evidence for injury and cell death during the first 24 hours, we found up-regulation of GFAP without up-regulation of the calcium-dependent N-cadherin pathway. This lack of change in the calcium-dependent N-cadherin pathway over 24 hours is consistent with the time course reported following stab-wound injury in which up-regulation was observed only 4 days after injuryin vitro bTBI research primarily concentrated on injuries relevant to the secondary and tertiary  phases of blast injury193031in vitro conditions. Using a model system that replicates the physical properties of an explosive blast known to produce mild bTBI in humans, we have 1) determined a cell type (astrocytes) and pathway (purinergic) that propagates a calcium response in CNS cell cultures during the primary injury phase and 2) reported molecular changes (GFAP and MMP9 expression) that take place on a time scale much greater than the blast event. Since human astrocytes differ from rodent and other primate astrocytes structurallyPrevious Primary, Sprague Dawley rat  CNS cortex tissue was obtained from pregnant female rats euthanized by carbon dioxide asphyxiation; all procedures were carried out in accordance with the NIH guidelines for care and use of animals for experimental procedures approved by the NINDS Animal Use and Care Committee. Primary, human CNS tissue, gestational weeks 19–21, was obtained under surgical written informed consent in accordance to National Institutes of Health Institutional Review Board Exempt # 5116 under Johns Hopkins University approved protocols, based on its designation as pathological waste.Cultures from primary, Sprague Dawley rat CNS cortex tissue, embryonic day 18, (E18) were prepared as described previouslyPrimary, human cortex CNS tissue, gestational weeks 19–21 was dissociated using gentle titration in Hank’s Basic Salt Solution (HBSS), centrifuged, washed and re-suspended in neurobasal medium (NB) supplemented with B27 . Cells were plated at either a density of 50,000 cells/well in NB + B27 in optical coverslip bottomed 96 well plates  that were threaded using a 1/16 NPT tap or a density of 300,000 per 35 mm culture dish  for laser wounding experiments. The 96 well plates and 35 mm culture dishes were coated with PureCol . The PureCol coated plates were then coated with poly-D-lysine  as previously described2. At the end of the loading period, the cells were washed twice with NB+B27. An additional wash to the final well volume was done prior to imaging each well. Fluo-4 was excited using 480 nm light  and fluorescence emission collected  during an exposure time of 80–100 msec. The image acquisition rate was 1.0–0.3 Hz. An external isolation box  maintained temperature (37 °C) and blocked ambient light. A 96 well plate was mounted on the microscope stage using a custom clamp. To connect the pneumatic device to the 96 well plates, the microscope condenser was removed.The real time imaging system is a Ti wide-field inverted microscope with Perfect Focus  equipped with an EM camera  running NIS Elements  for data acquisition and experimental control. A 20x air objective  0.75) and 60x oil immersion objective (NA 1.49) were used for imaging; 2x2 area stitching was used with the 60x objective. The fluorescence indicator, Fluo-4  was used to monitor intracellular calcium. Before each experiment, cells were loaded in the incubator for 30 minutes with 6 μM Fluo-4 AM in NB+B27 and maintained at 37 °C with 95% air and 5% COoC) and blocked ambient light. Cells were maintained in a small environmental box (Oko Lab) supplied with a gas mixture of 95% air and 5% CO2. Cells were targeted and wounded using the fixed point-scan application in the LSM software for 5 seconds. The wounding power varied between 10–33% of the maximum laser power available at the sample. Wounding required that the laser be focused on the cell membrane because 5 μm deviations above or to the side were without observable effect. The wounding occurred at an energy threshold, required direct laser damage to the cell membrane, and was not the result of cavitation or indirect damage. Laser wounding creates a single injury site in which calcium activity propagates with a pattern of activity that was stereotypic and reproducible.When propagation of calcium activity from a defined initiation site was needed, laser wounding was used to injure cells and localize the initiating injury site to within the observation field in this same CNS culture system. A Zeiss LSM 410 laser scanning confocal system, converted to a 2-photon system , with a 40x oil immersion objective (NA 1.3) was used for laser wounding studies. An ultra-fast laser  tuned to 800 nm produced ~1.8 W prior to entering the microscope. An external isolation box  maintained temperature  sealing the wells of the plate, and preserving the gas environment inside each wellCells were identified using two procedures: immunocytochemistry and their calcium response to KCl depolarization. For immunocytochemistry, cells were fixed with 4% paraformaldehyde and then stained for lineage-specific antigens without removing the live-cell chamber from the microscope stage. Cells were blocked in 5% normal goat serum with 0.1% Triton X-100, then labeled with two neuron-specific markers, class III beta-tubulin, TUJ1,  and microtubule associated protein 2, MAP2-Neuronal marker , and a glia marker anti-Glial Fibrillary Acidic astrocyte marker, GFAP , and the nuclei marker, Hoechst . GFAP is known to be a cell specific astrocyte marker applicable for human fetal astrocytesFor the calcium response to depolarization an equal volume of NB+B27+200 mM KCl was added to the well resulting in an excess 100 mM KCl in NB+B27. The rationale for using KCl depolarization to identify neurons and astrocytes was the following. In a single experimental session, up to 10 individual wells (experiments) from a single 96 well plate were used. For immuno-cytological identification each well was fixed following that experiment; at the conclusion of the entire series of experiments immunostaining was performed and, for each well, the identical field of view needed to be identified and reimaged. This was a very elaborate and time-consuming process and there were cases in which it was difficult to recover the identical area of interest. On the other hand, identifying cell type with KCl was rapid and robust and only required adding KCl at the conclusion of the individual experiment prior to investigating the next well.Frames1−5). The variance/mean image captures the changes over time in a single image and quantifies the calcium activity observed; this derived image was used as an aid in identifying calcium-responsive cells. Cell identity was determined at the end of the blast experiment using immunostaining for neuronal and astrocytic markers, and/or calcium response to KCl depolarization. Single cell analysis was used to quantify and identify cell types correlated with calcium activity triggered by simulated blast. Initially, regions of interest derived from the correlation between marker specific cell type (immunofluorescence) and the responses to depolarization were used to create identifying masks that were superimposed over all cells in the blast image sequence. Subsequent analysis, using only KCl depolarization, made use of ImageJ macros that automated the tasks of alignment, thresholding at 0.85 of the intensity cumulative distribution, object ) identification, image mask creation, and time series analysis. Time series data were then analyzed in Matlab using programs that allowed rapid operator data curation into responding and non-responding data sets for analysis and display. Since two methods of analysis were used, manual analysis and automated detection using 2 sigma threshold levels, all automated ROI identifications were validated manually and adjusted, if needed. All kinetic data were validated against the non-normalized image sequence; aberrant data, usually due to moving debris, were dropped.Fluo-4 image sequences and immunofluorescence images, when acquired, were registered together using a rigid body transformation implemented in the ImageJ (NIH) plugin StackReg. To identify cells that responded with changes in Fluo-4 fluorescence to either a blast or KCl depolarization, the variance/mean intensity of a relevant time window from the image sequence before, during, and after the blast was created and, for KCl depolarization, a five frame average prior to KCl exposure was subtracted from each image (ΔF = F(t) − <F(t)>oC. Prior to RNA extraction, the wells were visually evaluated in the microscope in order to assess culture integrity; wells that showed cellular damage were excluded from analysis . RNA was collected using TRIzol reagents (Life Technologies) and then purified using RNeasy Mini Kit (Qiagen). The concentrations of RNA and 260/280 ratios were determined using a NanoDrop 2000 (Thermo Scientific). Equal amounts of RNA were used to make cDNA using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Oligonucleotide sequences specific for genes of interest were designed with the Vector NTI Advance 11.5 program (Invitrogen). qRT-PCR determinations were performed in triplicate on an Applied Biosystems 7500 RT-PCR system with AB Power SYBG 2x master mix. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as the reference for qRT-PCR analysis. Changes in the genes of interest were evaluated against variations in GAPDH detected in all samples. Calibration lines were determined for each probe using robust (bisquare) linear regression of threshold cycle vs. log amount. The weighted mean and weighted standard deviation of the reference probe GAPDH were determined from all measurements (4 independent samples each in triplicate). Rather than direct reference probe normalization , and the problems created by ignoring the reference probe distribution10(1)) with standard deviation, sigma, represents the expected variation of the reference probe; changes greater than 3 sigma in the paired test probes were considered significant increases compared to the reference probe. This approach is similar to previous methods that try to incorporate the statistical properties of the reference probe5354In order to evaluate changes in gene expression following exposure to simulated blast, two primary human CNS tissue samples were dissociated and plated in two 96 well plates. Cells were grown as described in the cell culture method section for 21 days; simulated blast was delivered as described previously. As control, all steps in the procedure were repeated excluding the simulated blast itself. Blast-treated and control experiments were alternated on a row-by-row basis and then the 96 well plate was returned to the incubator. Samples remained in the incubator for an additional 24 hours at 37 Significance between data sets was determined using ANOVA, t-test, or Kolmogorov-Smirnov and indicated in the text. The fraction of calcium responsive cells was evaluated after correcting for 0 and 1 fractions using Freeman-Tukey arcsin transformed, cell frequency weighted, pooled and back transformed averages; 0 and 1 fractions were assigned a value of 1/(4*n) and (1−1/(4*n)) where n is the total number of cells. Summary statistics were calculated over individual tissue averages using 1/variance weighting. 95% confidence intervals for the means were calculated using t*SEM where t is the two-tailed, 0.05 probability t-value and SEM is the standard error of the mean. Summary statistics, data analysis, and curve fitting were performed using the programs Excel  and Matlab ).How to cite this article: Ravin, R. et al. Blast shockwaves propagate Ca2+ activity via purinergic astrocyte networks in human central nervous system cells. Sci. Rep. 6, 25713; doi: 10.1038/srep25713 (2016).


These mice showed a pronounced resistance to obesity and to the development of insulin resistance in response to chronic high-fat feeding. Moreover, −/−Enpp1 mice exhibited increased levels of the bone-derived hormone osteocalcin, which increases β-cell proliferation and insulin secretion, thereby linking bone remodelling to metabolic homeostasis. These results support the involvement of NPP1 in the development of obesity and type 2 diabetes via its regulation of insulin sensitivity. The NPP1 pathway might hence represent a potential therapeutic target to treat insulin resistance in human metabolic disease. 1341Page Bone has emerged as an important endocrine regulator that can mediate the interplay between bone remodelling and energy metabolism. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) is a factor that regulates bone mineralisation and is elevated in individuals with insulin resistance, a condition associated with type 2 and obesity-related diabetes. Thus, this factor might play a role in the development of metabolic disease. Vicky MacRae and colleagues sought to investigate this by using


Glucocorticoids (GCS) remain one of the mainstay treatments in the management of ulcerative colitis (UC) but up to a third of patients will ultimately fail to respond and progress to a more severe and difficult to manage disease state. Previous clinical studies suggest that the Toll-Like Receptor 9 (TLR9) agonist DIMS0150 not only induces production of key anti-inflammatory cytokines as IL-10 but interestingly also enhances steroid sensitivity in steroid refractory UC patients. We investigated, in the context of a clinical study, whether a pre-selection of steroid response genes could identify steroid refractory UC subjects most likely to respond to DIMS0150 treatment.In a non-interventional pilot study, blood from steroid refractory UC patients and healthy volunteers was taken and thirty-four previously described steroid response genes were analysed by real time PCR analysis. To establish clinical utility of the identified biomarkers, a placebo controlled, randomized, double blinded study in active steroid dependent and steroid resistant UC patients on concomitant steroid therapies was used (EudraCT number: 2006-001846-15).We identified three potential biomarkers CD163, TSP-1 and IL-1RII whose response to steroids was significantly enhanced when DIMS0150 was applied. Thirty-four subjects were randomized to receive a single rectal administration of placebo or 30 mg of DIMS0150. Blood derived PBMCs were obtained prior to dosing and assayed for evidence of a steroid enhancing effect following steroid incubation in the presence of DIMS0150. Comparison to established steroid sensitivity marker IL-6 confirmed that clinical responders are steroid refractory UC patients. Upon study completion and un-blinding, the biomarker assay correctly predicted a clinical response in over 90% of the patients.Using specific steroid response biomarkers, GCS refractory UC patients most likely to benefit from DIMS0150 treatment could be identified and illustrates the usefulness of a personalized treatment approach. Ulcerative colitis (UC) is a disease characterised by chronic inflammation of the rectal and colonic mucosa. The medical management of UC is divided into treatment of active disease and maintenance of remission. The treatment of patients with UC aims to reduce inflammation and promote colon healing and mucosal recovery. In the majority of cases the disease may be controlled with conventional drugs including sulphasalazine (SASP), 5-aminosalicylic acid (5-ASA) and glucocorticosteroids (GCS) ,2. GCS aDNA based immunomodulatory sequence (DIMS0150) is a single stranded partially modified synthetic oligonucleotide of 19 bases in length. The drug functions as an immunomodulator by activating the Toll-Like Receptor 9 (TLR9) present in immune cells such as B-cells, macrophages and plasmacytoid dendritic cells (pDCs) that are found in abundance on mucosal surfaces such as the colonic mucosa. Through rectal administration of DIMS0150 in the form of an enema, the agent comes in direct contact with a large number of target cells thereby ensuring a robust immunomodulatory response. Activation of TLR9 by DIMS0150 results in the local production of potent anti-inflammatory cytokines such as IL-10 and type I interferons that have also interestingly been shown to increase steroid sensitivity in cells derived from steroid-resistant UC patients  and humaResults from two previous clinical studies in UC patients treated with a single rectal dose of DIMS0150 have indicated that steroid refractory UC patients benefit from the treatment. In the first clinical study, a single dose of DIMS0150 was given to steroid unresponsive IBD patients on concomitant steroid therapies . The stuin vitro assay. Additionally, to be able to re-affirm whether steroid refractory UC subjects on concomitant steroid therapies are the relevant target group or whether the combination of DIMS0150 and steroid therapy is needed, a phase IIa proof of concept (PoC) study was conducted in steroid dependent or steroid resistant UC patients on concomitant steroid therapies addressing DIMS0150 at a single dose level of 30 mg.A larger phase II study evaluated the ability of DIMS0150 at four dose levels  administered as a single rectal dose to induce clinical remission in 151 patients with mild or moderately active UC compared to placebo. No significant benefit was demonstrated at any dose level suggesting that the lack of efficacy was possibly due to the different patient target group (data not published). The target groups of these studies differed in two ways, firstly the second trial was conducted in less severe UC patients and secondly concomitant steroid therapy was an exclusion criteria. To investigate whether the effect of DIMS0150 in the first study could be linked to steroid sensitivity, we screened 34 known steroid response genes in an According to European Crohn’s and Colitis organisation (ECCO) guidelines, the definition of steroid resistance is a failure to respond to 0.75 mg/Kg body weight intravenous administered steroids given over 3 to 5 days . Likewisin vivo following DIMS0150 treatment in a case report from a named patient basis program and confirm that after treatment with DIMS0150, GCS can be used to successfully treat a new disease flare in a patient.The results from this PoC phase IIa study indicate that GCS refractory patients on concomitant GCS therapy respond more favourably to a single dose of DIMS0150 and the utility of the biomarkers CD163, TSP-1 and IL-6 in confirming the right target group and predicting a most likely response to DIMS0150 could be demonstrated. Using the biomarkers we could also demonstrate a steroid re-sensitizing effect A study including 9 steroid resistant active UC patients and 9 healthy volunteers  was performed to donate blood at one occasion in one study centre in Stockholm, Sweden.The study was placebo controlled, randomized, double-blinded and conducted in Sweden and Russia in 17 study centres. Steroid resistant or steroid dependent patients (Intention-To-Treat population ITT = 34) on a stable and tolerable steroid dose of at least 5 mg/day for 2 weeks with mild to moderate UC with DAI score [One chronic active treatment refractory UC patient  was treated three times with DIMS0150 with four weeks between dosing occasions [DIMS0150 is a fully synthetic oligodeoxynucleotide with the sequence 5′-G*G*A*ACA GTT CGT CCA T*G*G*C-3′ where (*) indicates phosphorothioate linkages, produced by Avecia  and prepared as study drug by Apoteksbolaget AB, Production & Laboratory, Umeå, Sweden. The randomisation code was produced by a computer-generated procedure, which used the method of randomly permuted blocks. Double-blinding against water was accomplished by giving all study products identical appearance, packaging and labelling.Whole blood (5 mL) from 26 patients of the PoC study  was collected in PAXtubes  just before drug administration and 4 hrs after. RNA isolation was performed according to the manufacturer’s guidelines using PAXgene Blood RNA Kit .From 32 patients of the PoC study, 30 mL blood was collected in sodium heparin tubes  and peripheral blood mononuclear cells (PBMC) isolated 24 hrs after collection by density gradient centrifugation using Ficoll-Paque Plus . PBMCs were washed three times in buffered saline solution, and resuspended in complete RPMI 1640 medium .6 cells/well), cells stimulated for 48 hrs with DIMS0150 (25 μM or 100 μM) in the presence or absence of Dexamethasone . Cell supernatant was collected and kept at -20°C for cytokine analysis. The cells were covered with 50 μl/well of RLT-lysis buffer (Qiagen) containing 1% of β-mercaptoethanol and kept at -20°C for mRNA isolation.PBMCs were seeded in 96 well plates  flex kit (Becton Dickinson) according to the manufacturer’s instructions on a FACSArray flow cytometer using FCAP Array software . IFN-α was analyzed using human IFN-α Multi-subtype ELISA kit  and IFN-β was detected with human IFN-β ELISA kit  according to the manufacturer’s instructions.RNA isolation was performed using Qiagen RNeasy RNA isolation kit and Qiacube (Qiagen) according to manufacturer’s guidelines.20NV-3′) was taken. The reactions were performed using Superscript II according to the manufacture’s guidelines .For first strand cDNA synthesis, 0.3-1.0 μg of total RNA/sample and 10pM of the Oligo-dT-primer  and γ-actin as internal control  was performed on an ABI 7500 Real-Time PCR system using Power SYBR®Green PCR Kit according to the manufacture’s guidelines (Applied Biosystems). Reactions were performed in triplicates using 1 μl 1:10 diluted cDNA per reaction and qPCR data were analyzed using relative quantification via 2Data analysis and graphing was performed in Microsoft Office EXCEL 2007 and GraphPad Prism 4.0c. The class membership analysis  was perfBlood samples from 9 steroid resistant UC patients and 9 healthy volunteers were obtained with ethical approval from EPN .The phase IIa study (EudraCT number: 2006-001846-15) was reviewed and approved by regional Independent Ethics Committees (IECs) and by the medical authorities in each country prior to inclusion of patients.Named patient basis treatment was performed under the responsibility of the treating physician and with approval of the local ethic committee .All patients had received written and verbal information concerning the study/treatment and signed an informed consent. From the named patient basis treated patient an additional written consent was obtained allowing the publication of individual clinical data.in vitro studies were performed on PBMCs isolated from healthy subjects and stimulated with DIMS0150. The results demonstrated a pronounced production of anti-inflammatory cytokines like IL-10 and type I interferons and the chemokine IP-10  and incubated with or without DIMS0150 (25 μM and 100 μM). To be selected as a potential biomarker for the sensitizing effect of DIMS0150, four criteria had to be fulfilled. Firstly, the individual variation in the expression of a gene in healthy volunteers should be minimal. Secondly, the effect of Dexamethasone stimulation should produce a reliable induction or repression. Thirdly, to discriminate between steroid resistance and normal sensitivity, a biomarker must demonstrate an impaired steroid response in the resistant group compared to healthy. Lastly, the biomarker should show a clear synergistic enhancement after co-stimulation with DIMS0150. Three genes  could be identified on the basis of their elevated expression levels when stimulated with DIMS0150 and Dexamethasone  were obtained and a panel of 34 potential steroid response genes ,19-32 weTo investigate the clinical usefulness of these steroid sensitivity biomarkers as surrogate markers for DIMS0150, a PoC phase IIa study conducted to confirm the target group of DIMS0150 was used. The objective of this study was to evaluate the clinical response following single dose treatment with DIMS0150 compared to placebo in steroid resistant or steroid dependent UC patients on concomitant steroid medication. Clinical responders were defined as patients showing a decrease in the Disease Activity Index (DAI)  score ofin vivo marker for the pharmacological activity of CpG containing oligonucleotides that act through the TLR9 pathway [in vitro  is known to be a sensitive  pathway -36. As Din vitro with Dexamethasone and/or DIMS0150 for 48 hrs and the expression levels of CD163, TSP-1, IL-1RII and IL-6 determined through qPCR analysis. In response to stimuli, levels of IL-6 are increased and the degree of suppression by GCS is indicative of the steroid sensitivity. Indeed, the ability of GCS to suppress the levels of induced IL-6 appears to be an accepted measure of steroid sensitivity as there appears to be a robust correlation with the clinical picture of steroid resistance [Blood derived PBMCs were isolated from 32 patients from the PoC phase IIa study at the time of screening, treated sistance ,13,14. Tin vitro observations that DIMS0150 is able to restore steroid sensitivity in steroid resistant patients suggesting that the patient group with reduced steroid response are steroid refractory.Prior to un-blinding, two types of responses in the IL-6 biomarker assay were observed Figure A. One grBoth biomarkers, IL-6 and CD163, were able to identify the same two groups, namely patients being resistant to steroids and those being similar to healthy controls and segregate between steroid refractory and sensitive patients. TSP-1 as well as IL-1RII could also segregate between two differing steroid sensitivity groups whereby the correlation between IL-6 and CD163 or TSP-1 was in both cases over 71% and IL-1RII showed only a correlation of about 45% (data not shown).in vitro steroid sensitivity had an actual clinical response to DIMS0150.Upon PoC phase IIa completion, we determined whether there was a correlation between patients showing a reduced steroid sensitivity in the biomarker assay before treatment and clinical response. In other words, is steroid resistance a pre-requisite for a response to DIMS0150 and could this be used as a predictor. Of equal interest was to determine whether those subjects that had previously demonstrated in the biomarker assay that DIMS0150 could restore their in vitro assay compared to the clinical outcome. From the 22 patients receiving DIMS0150, 10 patients had a clinical response at week 1 and/or week 4 and 12 patients had no clinical response either at week 1 or at week 4. For all the statistical analysis the first group of patients were classified as clinical responders and the second group as clinical non-responders. Table -8 and 10-6 M) and 25 μM DIMS0150.For this purpose, classification analyses were performed using the raw data from the relative quantification analysis of the performed in vitro steroid sensitivity was observed upon DIMS0150 incubation and clinical response. IL-1RII had less discriminative potential due to strong patient variations (data not shown). However, the highest prediction values were achieved when all three biomarkers CD163, TSP-1 and IL-6 were considered for steroid resistance and, taking into account restoration of steroid sensitivity as determined by CD163 and TSP-1. This gave an AUC of 0.98 with 10-6 M Dexamethasone and 25 μM DIMS0150 in contrast to an AUC of 0.83 for IL-6 alone at the same concentrations  were calculated from data derived from CD163, TSP-1 and IL-6 and the pc1/pc2 distribution plotted, reflecting the differences in the gene expression of these three genes in responding and non-responding patients  at wk1 and from 41% to 80% (8/10) at wk4. These data demonstrate a clear usefulness in being able to identify patients most likely to benefit from DIMS0150 treatment.in vivo during DIMS0150 treatment. After a further 14 weeks the patient experienced first disease deteriorations (increase of the CAI score from 0 to 4) and in light of the subject’s apparent improved steroid sensitivity, the treating physician was advised to increase the dose of GCS from 10 to 30 mg of Prednisolone/day. The patient regained complete remission within a week suggesting that following a period of re-sensitization to GCS, the patient was able to respond to the elevated dose given.In a case series of severe chronic active treatment refractory patients, treatment with DIMS0150 was provided under named patient basis , 4 weeksGastrointestinal disorders like UC can dramatically affect the quality of life , and inv. [The mechanisms behind steroid resistance are complex and numerous cytokines have been implicated as important factors. For example, Xystrakis and colleagues.  could sh. .Similar clinical observations have been demonstrated using type I interferons. For example, steroid resistant UC patients receiving daily intravenous injections of natural IFN-β experienced a rapid improvement of clinical symptoms . The abiin vitro, the same cytokines/chemokines implied to be important in regaining steroid sensitivity. The induction of IP-10 in vivo in the phase IIa study correlates well with the in vitro IP-10 analysis suggesting that treatment with DIMS0150 is likely to induce the same steroid enhancing cytokines as previously recorded.We could show that the TLR9 agonist DIMS0150 acts as an immunomodulatory compound by inducing IL-10, IL-6, type I interferons and IP-10 The biomarker CD163 belongs to a superfamily of cysteine-rich scavenger receptors (SRCR), several of which are involved in the innate immune response . CD163 iThrombospondin-1 belongs functionally to a group of diverse multidomain counteradhesive proteins influencing endothelial cell behaviour -48. It cTo assess the clinical utility of CD163, TSP-1 and IL-1RII as predictors of clinical response, a PoC phase IIa study in steroid refractory or steroid dependent UC patients on concomitant steroid treatment was performed and the results compared to those of the steroid sensitivity marker IL-6. Although this study was somewhat limited in size, it was nevertheless deemed sufficiently large enough to provide a robust assessment of the biomarkers. We hypothesized that DIMS0150 should enhance steroid sensitivity leading to improvements of symptoms and a reduced disease activity score and that prior analysis using the biomarker genes should enable a prediction of clinical response. The clinical outcome of the study showed that approximately half of the DIMS0150 treated patients 10 of 22) responded to the treatment and that this observation was very much in line with the biomarker data obtained at the time of screening. Both IL-6 and CD163 analysis strongly suggested the presence of two groups of patients being included in the study. One group demonstrated a clear picture of steroid resistance and the other showed a steroid response similar to healthy volunteers. Patients with a reduced response to steroids as determined by CD163, TSP-1 or IL-6 were statistically more likely to respond to the DIMS0150 treatment. The predictive potential of the biomarkers could be illustrated by classification analysis of the expression data compared to clinical response or non-response following DIMS0150 treatment. All three markers demonstrated a high potential as surrogate markers for a DIMS0150 response with CD163 and TSP-1 being slightly more sensitive than IL-6 because of their ability to demonstrate the steroid re-sensitizing effect of DIMS0150. As expected, the combination of all three markers gave the best result with an AUC of 0.98. This equates to a correct prediction of clinical response in 90% (9/10) of patients classified as being steroid refractory according to the biomarker assay. Conversely, 91% (10/11) of patients whose steroid sensitivity was comparable to healthy controls failed to respond to DIMS0150 treatment. A possible interpretation would be, patients classified with the biomarkers as steroid refractory are indeed steroid refractory patients. By contrast, patients that show no difference to healthy could be inferred as steroid dependent. The validity of these interpretations can only be properly tested through additional clinical studies where subjects are included using the clinical definitions of steroid resistance and dependence as given in the ECCO guidelines [ of 22 rein vitro, these in vivo data provide for the first time, evidence for a shift to improved steroid sensitivity in a steroid unresponsive patient following DIMS0150 treatment.Regarding the special case patient who received three doses of DIMS0150, the biomarker assay confirmed that the patient was steroid refractory at time of first dosing and classified as a potential responder to DIMS0150. Upon treatment with DIMS0150, a pronounced clinical response could be observed with the patient in complete remission at week 12. Further biomarker analysis at week 12 demonstrated that the patient most likely had regained steroid sensitivity that could be confirmed by treating an upcoming relapse successfully with GCS. While we have consistently recorded a steroid re-sensitizing effect for DIMS0150 Based on these promising data, a placebo-controlled, multiple dose, double-blind, randomized phase III clinical study (NCT01493960) is currently on-going to assess the efficacy and safety of DIMS0150 as an add-on to current practice in chronic active treatment refractory UC patients.in vivo shift in steroid sensitivity following DIMS0150 treatment and controlled steroid tapering combined with a long follow-up phase will gather information about reaching and length of steroid-free remission.This study will also provide a unique opportunity to gain further evidence for the observed The work presented here demonstrates that the target group of DIMS0150 are ulcerative colitis patients showing a reduced steroid sensitivity and a clear utility of using appropriate biomarkers for the selection of patients most likely to benefit from DIMS0150 treatment has been illustrated. Using such an approach represents a step towards a more personalized form of healthcare and may aid physicians in making the most optimum treatment choices for the patient.Nikolai V. Kouznetsov, Arezou Zargari, Alexander Gielen, Oliver von Stein and Petra von Stein received salaries from InDex Pharmaceuticals. Nikolai V. Kouznetsov, Arezou Zargari, Alexander Gielen and Petra von Stein have equity interest in Index Pharmaceuticals; Oliver D. von Stein and Robert Lofberg have stock ownership at Index Pharmaceuticals.PvS had substantial contribution in conception and design of work, data collection, data analysis and interpretation, drafting of manuscript; NK had substantial contribution in biomarker selection and screening, data collection, data analysis and drafting the manuscript; OvS participated substantially in conception and design of work, interpretation of data and critically revising the manuscript; RL was substantially involved in study design (blood collection studies and PoC study), acquisition of data and critical revision of manuscript; RB was substantially involved in study design (PoC study), acquisition of data and critical revision of manuscript; EM had substantial contribution in treatment of patient under name-patient basis (work design and acquisition of data) and critical revision of the manuscript; AZ and AWG were both substantially involved in data collection, data analysis and critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/14/79/prepub