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Modulation of the Akt Pathway Reveals a Novel Link with PERK/eIF2α, which Is Relevant during Hypoxia

The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.

Introduction
Akt (also known as protein kinase B or PKB) is a serine/ threonine kinase member of the AGC family of protein kinases, which plays a central role in growth, proliferation, protein translation and cell survival [1][2][3]. Akt is recruited to the plasma membrane by phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) generated by activated PI3K. Once at the membrane, Akt is phosphorylated on Thr308 [4] by PDK1 and on Ser473 by mTORC2 [5]. Akt can also be phosphorylated at other sites, which are important for its kinase activity [6,7]. Activated Akt phosphorylates multiple targets in the cytoplasm, nucleus, mitochondria and at the surface of the endoplasmic reticulum membrane (ER) [8,9]. Deregulation of the Akt pathway is associated with a variety of human cancers, and mouse models with activated Akt support its role in cancer development [10][11][12]. Several inhibitors of the Akt pathway have been developed as therapeutic treatments, some of which are currently being tested in clinical trials [13][14][15][16]. One of these inhibitors is the benzimidazole derivative Akt-IV (also known as ChemBridge 5233705 or Akt inhibitor IV) [17], which has potent anticancer and antiviral activity [18][19][20]. Although the direct target of Akt-IV is not known, it has been proposed to bind the ATP pocket of a kinase upstream of Akt but downstream of PI3K, possibly that of PDK1 [17]. Akt-IV has been shown to inhibit the phosphorylation and activity of Akt. However, at low concentrations, Akt-IV promotes the hyperphosphorylation of Akt [20]. The mechanisms behind these seemingly contradictory effects of Akt-IV on the Akt protein and its antiviral and antiproliferative activities are poorly understood.
Like the Akt pathway, the unfolded protein response (UPR) is involved in the regulation of metabolism, protein translation, cell death and survival [21,22], and it is thought to be important in the development of different malignant neoplasms such as multiple myeloma, prostate and breast cancer [23,24]. The accumulation of unfolded proteins in the lumen of the ER triggers a multipronged signal transduction response aimed at reestablishing cellular homeostasis. This includes a rapid reduction in the protein load in the ER, which is accomplished by lowering protein synthesis and translocation into the ER, and an increase in the capacity of the ER to fold proteins by upregulating the expression of foldases and chaperones. If homeostasis cannot be reestablished, the UPR can induce cell death, probably to protect the organism from rogue cells that express misfolded proteins. Three ER stress transducers have been identified: inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6) and protein kinase RNA (PKR)-like ER kinase (PERK). These integral membrane proteins sense the protein folding status in the ER lumen and communicate this information to cytosolic target proteins that translocate to the nucleus to modulate gene expression [22,25,26]. The UPR was historically viewed as a stress response system but a growing body of work suggests that it also functions in the maintenance of basal cellular homeostasis [21]. In this view, the UPR could be activated and its output modulated by signals other than misfolded proteins. In accordance with this notion, P58-IPK and a novel cytosolic isoform of BIP have been described to interact and regulate PERK from the cytosolic side [27][28][29].
Both the UPR and the Akt signaling pathways regulate protein translation, albeit in opposing ways. Akt promotes translation by two paths. First, it phosphorylates and activates mTORC1, which in turn inactivates 4EBP by phosphorylating it in at least four sites [2]. Unphosphorylated 4EBP blocks translation by binding to the cap binding protein eIF4E, the rate limiting step in cap-dependent translation. Second, Akt inactivates the glycogen synthetase kinase 3b (GSK3b), the major kinase that phosphorylates and inactivates the eukaryotic initiation factor 2 (eIF2) and its activator, the guanine exchange factor eIF2B [3]. In contrast, the UPR, via PERK, blocks translation initiation by directly phosphorylating one of the subunits of eIF2 trimer, eIF2a. eIF2 containing phosphorylated eIF2a inhibits eIF2B, preventing further activation of eIF2 [26]. Although the UPR and the Akt pathway have long been known to regulate similar cellular systems and influence cell fate, a functional link between the two pathways has only recently emerged. The UPR has been reported to activate [30][31][32][33][34][35] or inhibit [36][37][38] the Akt pathway depending on the nature and severity of the ER insult. It was recently proposed that Akt phosphorylates and inhibits PERK [39], although the role of the Akt pathway in regulating the UPR remains poorly understood.
In this work we used a series of chemical modifiers of the Akt pathway and tested whether Akt signaling can regulate the UPR. We found that Akt-IV strongly activates all the three branches of the UPR, with the PERK branch showing a dependence on the presence and activity of Akt. Our results suggest that Akt is a cytosolic regulator of the UPR and that the signaling between these two pathways may control the balance of pro-survival and pro-apoptotic signals thereby regulating cell fate decisions.

Pharmacological modulation of Akt with Akt-IV activates the UPR
To test the hypothesis that the Akt pathway regulates the UPR, we used a series of small molecules that differentially target the PI3K/Akt pathway (Fig. 1A, B). We used the classic PI3K inhibitor LY294002 [40], Akt-VIII, a direct and selective inhibitor of Akt [41], and Akt-IV. We evaluated the effects of these drugs on the activation of the three branches of the UPR in HEK293T cells in culture.
Akt-IV triggered a strong and sustained (2 to 24 h) activation of the IRE1 branch, as detected by monitoring the splicing of Xbp-1 mRNA by RT-PCR ( Fig. 2A). Neither LY294002 nor Akt-VIII showed any effects on Xbp-1 mRNA splicing. We evaluated the activation of the ATF6 branch by measuring the cleavage of a FLAG-tagged ATF6 by western blot (WB) (Fig. 2B) and the relocalization from the ER to the nucleus of a YFP-ATF6 reporter (Fig. S1A). Akt-IV induced both the cleavage of ATF6-FLAG (Fig. 2B) and the re-localization of the fluorescent reporter ( Fig. 2C and Fig. S1B). In contrast, neither LY294002 nor Akt-VIII induced the cleavage of ATF6-FLAG (Fig. 2B). Finally, we found that Akt-IV rapidly activated PERK, as assessed by a mobility shift in an SDS-PAGE gel, and the phosphorylation of the PERK target eIF2a (Fig. 2D and 2E). As with the IRE1 and ATF6 branches, LY294002 and Akt-VIII had little or no effect on the PERK branch (Fig. 2D). Even at longer incubation times these compounds failed to activate the PERK branch at levels comparable to those of Akt-IV (Fig. S2). As expected, all three compounds inhibited the phosphorylation of Akt at Ser473 and of its downstream target GSK3b at Ser9 (Fig. 2D), making the effects of Akt-IV on the UPR all the more striking.
We noted that just before PERK branch activation, Akt-IV caused an early transient activation of Akt, as assessed by the increase in phosphorylation of Ser473 of Akt and on Ser9 of GSK3b (Fig. 2D). This activation peaked between 1 and 5 minutes post stimulation with Akt-IV and was specific to this compound since neither LY294002 nor Akt-VIII provoked such behavior (Fig. 2D). If this transient activation of Akt is important for the induction of eIF2a phosphorylation needs further confirmation.
eIF2a phosphorylation induced in response to Akt-IV depends on PERK and Akt but not on PI3K Since all three compounds work as Akt inhibitors (Fig. 2D), but only Akt-IV activates the UPR, we tested the possibility that Akt-IV regulates the UPR in an Akt-independent manner. To do that, we evaluated the effects of Akt-IV on mouse embryonic fibroblasts (MEFs) lacking Akt1 and Akt2, two of the three isoforms of Akt (Akt DKO, Fig. S3). These cells have no basal activation of Xbp-1 mRNA splicing nor have they increased basal PERK activity as judged by eIF2a phosphorylation. Treatment of Akt DKO cells with Akt-IV induced the splicing of Xbp-1 as well as the mobility shift of PERK to a similar extent as in wild type MEFs ( Fig. 3A and 3B) indicating that these effects are independent of Akt1 and Akt2. However, the absence of these two isoforms impaired the capacity of Akt-IV to induce the phosphorylation of eIF2a (Fig. 3B) showing that in the case of the effect on eIF2a, Akt1 or Akt2 kinases were required. The remaining Akt and pAkt signal observed by western blot and immunofluorescence in these cells ( Fig. 3B and Fig. S3) is likely due to the fact that Akt DKO cells still express Akt3. To test whether the kinase activity of Akt is required as well, we transfected wild type MEFs with a vector containing a dominant negative kinase dead allele of Akt (HA-Akt-KM) [42] and determined the effect of Akt-IV on eIF2a phosphorylation. As in Akt DKO cells, the phosphorylation of eIF2a induced by Akt-IV was greatly reduced in cells overexpressing HA-Akt-KM compared to wild type cells (3 fold decrease in peIF2a/eIF2a levels compared to control, Fig. 3C) whereas the mobility shift of PERK was unaffected. Moreover, over-expression of wild type HA-Akt potentiated the effect of Akt-IV on eIF2a phosphorylation (3 fold increase in peIF2a/eIF2a levels compared to control, Fig. 3C), without affecting phosphorylation of eIF2a in untreated cells.
Thus far, we have shown that Akt is necessary for eIF2a phosphorylation, but not for the mobility shift of PERK. This raises the possibility that Akt might cause this effect in a PERK independent manner. In fact, eIF2a is known to be phosphorylated independently of the UPR by GCN2, HRI and PKR [43]. To test whether PERK was the kinase responsible for phosphorylating eIF2a in the presence of Akt-IV, we treated PERK knock out MEFs (PERK2/2) with Akt-IV (Fig. 3D). We found that the ability of Akt-IV to induce eIF2a phosphorylation was greatly impaired in PERK2/2 MEFs as compared to wild type cells.
The residual phosphorylation of eIF2a visible at later time points (1 h) is likely due to one or more of the other eIF2a kinases [44].
Our data are consistent with a model in which Akt-IV promotes activation of the PERK/eIF2a branch by triggering Akt kinase activity. To test whether the effects of Akt-IV on PERK and eIF2a require the canonical activation of Akt by PI3K, we pre-treated wild type MEFs with the PI3K inhibitor LY294002 before stimulating with Akt-IV. As expected, this pre-treatment inhibited the phosphorylation of Akt on Ser473 (Fig. 3E). However, it did not affect the ability of Akt-IV to induce the phosphorylation of eIF2a (Fig. 3E).
Taken together, these results demonstrate that the effect of Akt-IV on eIF2a phosphorylation is PI3K-independent but Akt-and PERK-dependent.

PERK is an Akt substrate
One possibility is that the eIF2a phosphorylation elicited by Akt-IV is due to the activation and phosphorylation of PERK by Akt or one of its substrates. Alternatively, Akt and PERK could act in parallel, such that the phosphorylation of eIF2a in the presence of Akt-IV requires both. If the former model were true, PERK might be a direct target of Akt, what could be detected in an in vitro kinase assay. In support of this notion, sequence analysis revealed that while neither IRE1 nor ATF6 contain putative cytosolic Akt phosphorylation sites, human PERK contains eight sequences that match the Akt consensus sequence (RxRxxS/T) [45]. Seven of these sequences, conserved in rats and mice, are located in its cytosolic domain (Fig. 4A) and thus are accessible to Akt. Interestingly, three of these sites (human S554, S1093 and S1095) have been found to be phosphorylated in vivo according to PhosphoSitePlusH [45,46]. An in vitro kinase assay with the wild type HA tagged Akt1 kinase domain immunoprecipitated from transfected HEK293T cells and a recombinant catalytically inactive fragment of PERK (aa 537 to 1114) revealed that, indeed, Akt can phosphorylate PERK (Fig. 4B). Immunoprecipitated kinase dead HA-tagged Akt1 kinase domain failed to phosphorylate PERK, confirming the dependence on Akt activity (Fig. 4B).
Supporting an in vivo role of Akt in phosphorylating PERK, fluorescence microscopy experiments with Akt1-CFP and the ER membrane marker ATF6-YFP confirmed previous reports that, in addition to localizing to the cytosol, nucleus and plasma membrane, Akt localizes to the ER membrane (Fig. S4). Furthermore, we detected co-localization of PERK and phosphorylated Akt substrates in cells transfected with myc-PERK and immunostained with an anti-myc antibody and an antibody that Akt phosphorylation and activation result from its recruitment to PIP 3 at plasma membrane, after which it exerts cytoplasmic and nuclear functions. Accumulation of PIP 3 classically follows ligand (L) binding to tyrosin kinase cell-surface receptors (RTK), adapter proteins (AP) recruitment to RTK and finally, PI3K activation to phosphorylate PIP 2 to PIP 3 . While Akt can be activated by the UPR it is not known if Akt can also regulate the UPR. doi:10.1371/journal.pone.0069668.g001 Figure 2. Pharmacological modulation of Akt with Akt-IV activates all three UPR branches: PERK responds first. (A) When activated, IRE1 processes Xbp1 mRNA by a non-conventional cytoplasmic splicing reaction, changing Xbp1 open reading frame. HEK293T cells were treated with Akt-IV (10 mM), Akt-VIII (5 mM) or LY294002 (20 mM) for the indicated times. Xbp1 mRNA splicing was detected by RT-PCR. Xbp1s: spliced form (activated IRE1); Xbp1u: unspliced form (inactive IRE1). (B) When activated, ATF6 translocates to the Golgi apparatus where it is cleaved to release a fragment that enters the nucleus where it functions as a transcription factor. HEK293 cells were transfected with ATF6-Flag plasmid and 24 h later they were treated with Akt-IV (10 mM), Akt-VIII (5 mM), LY294002 (20 mM), or thapsigargin (Tg; 100 nM) for the indicated times. Western blots (WB) using antibodies against FLAG and actin are shown for every case (B, upper panel). ATF6: uncleaved protein; ATF6f: cleaved form. (C) HEK293T cells were transfected with a plasmid that expresses the YFP-NLS-mATF6short reporter (top, see Fig. S1A for details). Forty-eight hours post-transfection cells were treated for the indicated times with Akt-IV and then fixed, DNA was stained with DAPI and cells were imaged (lower panel); Yellow, YFP-ATF6; Blue, DNA; scale bar, 5 mm. For all cases cells treated with DMSO were used as a control (Control). (D) When activated, PERK is autophosphorylated at multiple residues and activated to phosphorylate eIF2a. HEK293T cells were treated with Akt-IV (10 mM), Akt-VIII (5 mM) or LY294002 (20 mM) for the indicated times. Protein extracts were analyzed by WB using the indicated antibodies. Data in the plot corresponds to ratio of phosphorylated total abundance of each of the indicated proteins (normalized to the initial value) in cells treated with the indicated drugs for different times. Error bars correspond to the standard error of three independent experiments. (E) HEK293T cells were treated for 5 h with Akt-IV. peIF2a abundance was detected by immunofluorescence. Green, peIF2a; Blue, DNA; scale bar, 5 mm. Data are representative of at least three independent experiments. doi:10.1371/journal.pone.0069668.g002  recognizes proteins containing a phosphorylated Akt target motif (RxRxxS*/T*) [47] (Fig. 4C). In untreated cells, we detected a structured reticulated cytoplasmic staining with the phospho-Akt substrate antibody. The rather strong signal is likely due to high Akt activity normally present in HEK293T cells [48]. At the same time, the PERK-myc signal was detected in only a few cells due to the low transfection efficiency of this construct. The time-course of the signal revealed by the phospho-Akt substrate antibody showed a peak and decline behavior: after an initial increase seen 15 minutes post treatment, the signal diminished to levels below those seen in untreated cells. This behavior is similar to what we observed by western blot using anti phospho-Ser473-Akt (Fig. 2D), supporting the idea that this antibody was a good reagent to follow in vivo Akt activity. In contrast to the majority of cells, those cells that overexpressed PERK-myc did not exhibit this peak and decline behavior. In these cells, the phospho-Akt substrate signal concentrated in the same spots where the myc-signal was detected.
These results indicate that PERK might be a direct target of Akt in vivo.

Akt-IV induces apoptosis in a UPR-dependent manner
Both the Akt pathway and the UPR control cell fate by inducing pro-survival and/or pro-apoptotic signals. Akt-IV has also been described to induce cell death in different cell types [49][50][51][52]. To determine the effects of Akt-IV on apoptotic cell death in our system, we treated HEK293T cells for 7 hours with Akt-IV and determined by WB the extent of caspase 3 and PARP cleavage. Akt-IV caused strong activation (cleavage) of caspase 3 and PARP, which were blocked by the caspase inhibitor ZVAD (Fig. 5A). Consistent with these results, we found that Akt-IV treatment induces the formation of cell blebs (Fig. 5B). These transient globular protrusions of the plasma membrane are seen at the onset of the certain stress-associated processes, such as cell injury, cell invasion, hypoxia, high concentrations of free radicals and apoptosis in some cell lines [53]. Interestingly, Akt-YFP transiently localizes to these blebs in HeLa cells within 15 minutes of treatment with Akt-IV (Fig. 5C). Consistently, phosphorylated Akt substrates and eIF2a also exhibit a transient enrichment in cell blebs after treatment with Akt-IV treatment ( Fig. 5D and 5E), but not with other inhibitors such as Akt-VIII (Fig. S5).
To determine the role of the UPR in the cell death induced by Akt-IV, we evaluated the viability of wild type, IRE1 (IRE12/2) and PERK (PERK2/2) knock out MEFs using a propidium iodide incorporation assay measured by flow cytometry. Cell death induced by a 12 h treatment with Akt-IV was greatly reduced in the absence of IRE1 or PERK (Fig. 5F), indicating that one of the mechanisms by which Akt-IV induces cell death is UPR dependent.

Akt mediates PERK/eIF2a activation induced by hypoxia
The internal environment of solid tumors can be hypoxic and can lead to the activation of mechanisms within the tumor to cope with this stress. In general, adaptation to hypoxia is associated to poor response to therapy [54]. During hypoxia, Akt is activated by phosphorylation of Akt in the non-canonical Thr450 by JNK kinase [55,56]. In the same conditions, eIF2a is phosphorylated in a PERK-dependent manner [57]. Furthermore, hypoxia is associated with cell blebbing and cell death [58]. Thus, given our results, we hypothesized that Akt might be required for eIF2a phosphorylation during hypoxia.
To test this hypothesis, we incubated wild type and Akt DKO MEF cells in low oxygen conditions (0.1% O 2 ) for 1, 2 or 4 hours and evaluated the levels of eIF2a phosphorylation. In wild type MEFs, hypoxia induced an increase in phosphorylated eIF2a, but not a PERK mobility shift. It is possible that PERK autophosphorylation under these conditions is either transient or low. However, in Akt DKO MEFs, hypoxic conditions did not affect neither PERK nor eIF2a phosphorylation (Fig. 6A). This result supports our idea that Akt mediates PERK/eIF2a activation during hypoxia.

Discussion
Here we report the finding of a link that connects Akt with the PERK branch of the UPR. Our results also shed light into the molecular mechanism of action of Akt-IV, a drug with anticancer and antiviral properties with therapeutic potential. We provide evidence supporting the idea that a non-traditional activation of Akt leads to PERK-dependent phosphorylation of eIF2. And more important, we also show that this connection is relevant during a physiological condition such as hypoxia.
Recently, Mounir et al. provided initial evidence that Akt regulates PERK signaling [39]. Contrary to our results, the authors reported that Akt phosphorylates and inhibits PERK function. They observed higher levels of eIF2a phosphorylation in Akt DKO MEF cells compared to wild type cells, consistent with Akt being a PERK inhibitor. We have not observed these differences (Fig. 3B), although our source of knock out cells was different [59,60]. In contrast, we found that the overexpression of a dominant negative form of Akt in wild type MEFs largely blocked eIF2a phosphorylation in response to Akt-IV, consistent with a stimulatory, rather than an inhibitory, action of Akt kinase over PERK (Fig. 3C). Additionally, the authors described that treatment with other Akt inhibitors like Akt-VIII and LY294002 also induced phosphorylation of eIF2a after several hours of treatment. We only detected an effect using the Akt-IV compound (occurring in minutes), with marginal effects using the other Akt inhibitors. It is worth mentioning that they used a very high concentration of Akt-VIII (ten times higher than us and more than twenty times higher than in the paper that originally described Akt-VIII [41]). One potential explanation for our discrepancies is that Akt might phosphorylate different residues of PERK depending on the cell context; some of these could be stimulatory while others inhibitory. This idea would be consistent with the presence of several consensus sites for Akt phosphorylation in the cytosolic domain of PERK (Fig. 4A), and some of which have been shown to be phosphorylated in vivo [46,61]. It has been shown that in some cell contexts a prolonged treatment with PI3K inhibitors reactivates Akt to some extent [62]. Thus, the delayed effect of Akt-VIII and LY294002 on eIF2a reported by Mounir et al. may be due to a nontraditional reactivation of Akt instead of inhibition. Further work will be needed in order to address this issue. Nonetheless, it seems now confirmed that Akt targets and phosphorylates PERK modulating its activity and establishing a connection between these two pathways.
protein was incubated with 10 mCi of [c-32 P] ATP for 30 min and with 1 mg of GST or PERK GST protein as substrate. Phosphate incorporation was analyzed by SDS-PAGE and autoradiography. Akt levels were determined by WB while GST-PERK levels were revealed by Coommassie blue staining. (C) HEK293T cells were transfected with pCDNA-Myc-PERK and treated for 15 min with Akt-IV (10 mM). Cells were fixed and immunostained for pAkt substrate and Myc tag. Green, pAkt substrate; Red, Myc-PERK; scale bar, 5 mm. Data are representative of at least three independent experiments. doi:10.1371/journal.pone.0069668.g004   (1). Subsequently, UPR is activated (2). Akt presence and activity are necessary for eIF2a phosphorylation due to the existence of a connection between Akt and PERK/eIF2a signaling pathways. IRE1 (4) and ATF6 (5) are activated are later times. At the end, activation of IRE1 and PERK and dephosphorylation of Akt and GSK3b are associated with cell death (6). doi:10.1371/journal.pone.0069668.g006 We found that in response to Akt-IV, cells develop cell blebs, to which Akt relocalizes in a transient manner, at a time coincident with the onset of eIF2a phosphorylation (Fig. 2C and 5B-C). Determining if the formation of these blebs is important for this action of Akt will require future work. Nevertheless, this finding supports the current view that Akt-IV probably targets PDK1 [17], since it has been shown that PDK1 is involved in cell blebbing [63]. In the same line, though it needs further confirmation, it seems possible that the activation of the UPR by Akt-IV is via PDK1. In support of this hypothesis, other pharmacological modulators of PDK1 also activate the UPR pathway [64][65][66]. Since these compounds, like Akt-IV, activate Akt1 independent branches (such as IRE1), it seems likely that PDK1 targets the UPR via Akt dependent and independent pathways.
Several non-canonical mechanisms of Akt activation have been recently reported, such as Akt ubiquitination and Akt phosphorylation in residues different from Thr308 and Ser473. In fact, many kinases, including JNK, Ack1, ERK, CaM kinase and PKA are able to phosphorylate Akt, and some of them do so independently from PI3K activity [7,52,[67][68][69][70][71]. Remarkably, in the case of non-traditional activation of Akt induced by Akt-IV, because of the induced eIF2a phosphorylation, it would promote translation inhibition, contrary to the classic function of Akt in stimulating protein synthesis. Rapid and transient activation of the classic Akt pathway (phosphorylation in S473 and of Akt major target GSK3b) precedes persistent activation of PERK/eIF2a pathway, which lasts even after inhibition of Akt (Fig. 2D). This can be explained both by a persistent non-canonical activation of Akt (independent of S473 phosphorylation) and/or by a transient non-canonical activation of Akt, which phosphorylates alternative targets like PERK, which in turn displays a low dephosphorylation rate.
We speculate that both cell death and translation inhibition induced by Akt-IV could be associated with either the late Akt dephosphorylation at S473, as has been described by the group of Luo et al [72] or with a non-canonical activation Akt, directing its kinase activity to alternative targets, such as PERK. We show that the absence of PERK greatly reduces cell death induced by Akt-IV, further supporting this alternative (Fig. 5F). Independently of whether IRE12/2 and PERK2/2 cells have a reduced apoptosis potential or not, what it is clear is that Akt-IV needs the presence of these proteins to induce cell death. Here, we also show that Akt-IV displays effects that are independent of Akt, such as IRE1 dependent Xbp1 mRNA splicing and PERK electrophoretic mobility shift. We speculate that Akt-IV -perhaps mediated by PDK1 as suggested above-might in fact cause protein unfolding at the ER lumen in an Akt independent manner, affecting all the branches in different manners. In this regard, the shift induced by Akt-IV on PERK mobility in an SDS-PAGE persisted on Akt DK0 cells as well as in cells transfected with HA-Akt-KM ( Fig. 3B and 3C), indicating that the phosphorylation of PERK induced by Akt-IV responsible for the shift is independent of the presence of Akt1/2. In addition, this result also showed that the classic mobility shift might not always indicate full PERK activation, since in this case, even though the PERK band shifted, phosphorylation of eIF2a was greatly reduced. Thus, we speculate that Akt signaling activates PERK in a manner that might complement the activation associated with the classical mobility shift.
The design of drugs that target different components of the Akt signaling pathway is of great value for the therapeutic treatment of cancer [13][14][15][16][17]50,73,74], and Akt-IV has been shown to exhibit potent anticancer and antiviral activity [13][14][15][16][17]50,73,74]. For this reason, new analogues of Akt-IV have been designed with enhanced antiviral/antiproliferative activity and low cytotoxicity in normal cells [50]. Dunn et al. found that Akt-IV elicited Akt phosphorylation and blocked viral replication. They proposed that Akt-IV antiviral activity was unrelated to its action on Akt [17,20]. They based this conclusion solely in their observation that other drugs, like LY294002, neither have this antiviral action nor prevented it, despite blocking Akt phosphorylation, similar to what we observed for Akt-IV and the UPR activation ( Fig. 2A-D). However, using MEF Akt DKO cells and a dominant negative form of Akt (Fig. 3A and B), we found that activation of the PERK branch actually required Akt kinase activity. In this regard, it is worth mentioning that the UPR and PERK in particular, play an important role in the expression of viral proteins [75][76][77]. Thus, in the light of our results it would be interesting to test if the antiviral activity of Akt-IV does indeed depend on Akt especially on this newly discovered Akt/PERK pathway.
In summary, our data suggests that Akt-IV triggers cell blebbing and a rapid and non-traditional activation of Akt, which stimulates the PERK branch of the UPR. Akt-IV also activates the IRE1 and ATF6 branches, maybe through the promotion of unfolded proteins at the ER. Akt-IV also promotes a late inhibition of Akt traditional activity (evaluated by loss of pAkt S473 and pGSK3b S9 phosphorylation) which in combination with the UPR eventually result in cell death via apoptosis (Fig. 6B). It has been previously shown that PERK-mediated phosphorylation of eIF2a during hypoxia is important for long-term survival [57]. Here we found that Akt is necessary for eIF2a phosphorylation in these conditions (Fig. 6A), providing an important physiological context for the Akt-PERK interaction. The link between Akt and UPR pathways suggests a constant feedback between them, since ER stress and UPR have already been shown to alter Akt activity [24,35,78,79]. This feedback provides a mechanistic basis for previous suggestions that UPR plays a key role in the maintenance of basal cellular homeostasis to a level far beyond to its role in ER protein folding [21,24]. Cross-talk between these key signaling cascades is not surprising since both pathways play crucial functions in apoptosis, cell survival, protein translation and tumor growth [10][11][12]23,24]. Consistently, the connection between ER stress and the Akt downstream target mTOR was recently highlighted [80]. We suggest that the Akt/UPR connection functions as a master control mechanism of cell decision-making, illustrating the remarkable flexibility of signaling pathways.

Chemicals, reagents and treatments
Akt-IV (10 mM), Akt-VIII (5 mM) and LY294002 (20 mM) were obtained from Calbiochem. Thapsigargin (Tg) was obtained from Sigma. Caspase Inhibitor Z-VAD-FMK (ZVAD) (100 mM) is from Promega. The cells were plated (2610 5 cells per well for 6 well plates, 4610 4 cells per well for 24 well plates and 1610 4 cells per well for 96 well glass-bottom imaging plates) and grown for 24 h before transfection and/or treatment for the specified times.

Construction of fluorescent reporters
YFP-NLS-mATF6short reporter was constructed by PCR amplification of mouse ATF6 transmembrane and ER luminal domains (short ATF6). Forward primer contained an SV40 NLS sequence upstream of ATF6 specific sequence. This PCR product was digested with HindIII and cloned into pEYFP-C1 (Clontech). The primers used were as follows:

Cell Imaging and Immunofluorescence
For fluorescent reporter assays and immunofluorescence, cells were plated into 96 well glass-bottom imaging plates and transfected -when indicated-24 h later with lipofectamine according the manufacter instructions. After treatment, cells were fixed as described [86] with 4% paraformaldehyde in PBS for 5 min at room temperature, washed twice with PBS, permeabilized for 5 min with 0.2% Triton X-100 in PBS and blocked for 30 min with 3% BSA in PBS. After incubation for 1 h with primary and secondary antibodies (Alexa 594 or Alexa 488 labeled anti-mouse antibodies) in blocking solution for 1 h each, cells were extensively washed with PBS and nuclei were stained with DAPI. Images were captured on an Olympus IX-81 fluorescence microscope equipped with a 606 oil immersion objective and a Coolsnap HQ 2 CCD camera (Photometrics). Acquisition was carried out with MetaMorphH Microscopy Automation & Image Analysis Software.

Expression of recombinant proteins, immunoprecipitation and PERK in vitro Phosphorylation Assays
Expression and purification of GST and GST fusion proteins as well as Akt in vitro kinase assays were performed as described [86]. Briefly, GST and GST-PERK (K618R) were over-expressed in the E. coli Rosetta pLysS BL21 strain (Novagen) by incubation at 37uC until the optical density was 0.5 and induction with isopropyl-b-thiogalactopyranoside during 3 h. Following bacterial lysis, soluble proteins were affinity purified under nondenaturing conditions, on a glutathione-sepharose 4B-CL resin (Amersham-Pharmacia) according to the manufacturer instructions. For Akt kinase assays, HEK293T in 100-mm plates were transfected with pCMV6-HA-Akt, pCMV6-HA-Akt KM or were mock transfected. After 48 h cells were washed with cold PBS and lysed at 4uC in Akt lysis buffer (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 1% Triton X100, 10% glycerol, 20 mM NaF, 2 mM sodium vanadate, 1 mM DTT and 25 mM b-glycerophosphate plus a protease inhibitor cocktail (Roche)). WT and KM mutant HA-Akt were immunopurified from cleared lysates with an anti-HA antibody (MMS-101R, Covance) and Gamma-Bind Sepharose beads (Santa Cruz Biotechnology). Lysates from mock transfected cells were subject to the same procedure and used as a negative control. After extensive wash, the immunoprecipitate was incubated with 10 mCi of [c-32 P] ATP for 30 minutes in 30 ml kinase buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 20 mM ATP) at 25uC using 1 mg of GST or GST fusion protein as substrate. The reactions were terminated by addition of 56sample buffer and boiling. Phosphate incorporation was analyzed by sample electrophoresis on a 12% SDS-PAGE and autoradiography.

Cell viability assay
After the corresponding treatment the cells were incubated with 1 mg/ml of propidium iodide (PI). PI incorporation was measured after 15 min by flow citometry using FACScanto II cytometer and FACS DIVA program.

Hypoxia induction
MEF WT and Akt DKO cells were plated in p6 culture dishes at a density of 1610 5 cells/ml. After approximately 18 h the culture dishes were placed in the hypoxic culture chamber (BioSpherix). The oxygen concentration in the chamber was maintained with an oxygen sensor (Proox Model 110) and a carbon dioxide sensor (ProCO 2 model 120).


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Non-genomic effects of progesterone on Rho kinase II in rat gastric smooth muscle cells

Various studies have shown that pregnancy is associated with gastrointestinal complaints that might result from disturbance of the normal contractile pattern of smooth muscle. Progesterone is an important steroid hormone, which plays a crucial role in female pregnancy. Progesterone affects muscle cells by genomic mechanisms, through nuclear receptors, and non-genomic mechanisms, through unidentified pathways. Non-genomic actions were defined as those occurring within 10 min of progesterone exposure. The aim of the present study was to investigate the non-genomic effect of progesterone on Rho kinase II activity in gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague Dawley rats were used. Dispersed gastric smooth muscle cells were treated with progesterone or acetylcholine (ACh) separately. Cells designated for progesterone treatment were incubated with 1 μM progesterone for 10 min. Rho kinase II expression and both basal and ACh-induced Rho kinase II activity were measured via specifically designed enzyme-linked immunosorbent assay (ELISA) and activity assay kits respectively in both control and progesterone-treated groups. Progesterone inhibited the ACh-induced, but not the basal, Rho kinase II activity in dispersed gastric smooth muscle cells without affecting its expression level. This study suggested that progesterone can rapidly affect the contractile activity of isolated gastric smooth muscle cells in rats via inhibition of the Rho kinase II pathway.

Introduction
Pregnancy is usually associated with various gastrointestinal (GI) complaints such as nausea, vomiting, reduced colonic activity resulting in varying degrees of constipation, and various gastric emptying disorders (1)(2)(3). There is an accumulating body of evidence to suggest that the myoelectric and motor activities of the GI smooth muscle are disturbed during pregnancy. In support of this suggestion, it has been found that pregnancy is associated with decreased gallbladder contractivity (4,5), lowered esophageal sphincter pressure (6)(7)(8), reduced gastric emptying (9,10), and reduced small intestinal (11) and colonic transit times (12). However, the exact molecular mechanisms for such pregnancy-associated GI disorders are still poorly understood.
Progesterone and estrogen are significantly elevated in the serum of pregnant females. These hormones play central roles in the maintenance of pregnancy and the initiation of parturition by modulating myometrial contractility and excitability. In addition, recent studies have shown that sex hormones target other body organs besides the myometrium, such as the GI tract, bladder, and blood vessels (13)(14)(15).
Functionally, progesterone affects mammalian cells by both genomic and non-genomic mechanisms. The genomic actions of progesterone are mediated via two sub-nuclear receptors, A and B, which act as transcription factors (16). Genomic actions of progesterone on the GI smooth muscle might explain some disturbances that complicate pregnancy and female functional disorders. For example, it has been shown that progesterone signaling regulates G protein expression level in female slow transit chronic constipation, which correlates with an over-expression of progesterone receptors (17).
The mechanisms responsible for the non-genomic effects of progesterone are not fully understood. Nongenomic effects of progesterone have been defined as those occurring within 10 min of exposure (18,19). It has been proposed that progesterone interacts with plasmalemmal receptors and might lead to rapid activation of tyrosine kinases and phospholipases (20), mitogen-activated protein kinase (MAPK) (21), or inhibition of membrane transport systems (22). Furthermore, the types of changes induced by the non-genomic actions may be tissue specific, as diverse effects have been demonstrated in muscle, neural, endocrine, and reproductive cells (22)(23)(24).
Physiologically, smooth muscle is an important component of the GI tract and maintenance of its normal contractile behavior is essential for proper GI functions. Rho kinase II, a serine/threonine kinase, is an important downstream effector of the small G protein RhoA that has been found to be important in developing smooth muscle tone by maintaining the level of myosin light chain (MLC 20 ) phosphorylation, -the essential step in smooth muscle contraction (25). The importance of the RhoA/Rho kinase pathway has been demonstrated in the pathogenesis of cardiovascular disorders and as a target in the development of new drugs such as fasudil, a Rho kinase inhibitor (25,26). Exploring changes in Rho/Rho kinase pathway expression and activity in GI smooth muscle during pregnancy and the effect of this on muscle contractility could be an important step for a better understanding of the GI complaints that accompany pregnancy.
Although numerous studies have examined the effect of progesterone on GI smooth muscle, its effect on gastric Rho kinase II has not been explored. Therefore, the present study was designed to investigate the nongenomic action of progesterone on the Rho kinase pathway in smooth muscle cells of the stomach. Because progesterone may affect various types of gastric cells, studying the role of progesterone on a single cell type in a multicellular preparation could be difficult. For this reason, this study was performed on single smooth muscle cells freshly isolated from the stomach of rat. Insights into the molecular basis of abnormal smooth muscle function will prove invaluable in the treatment of GI motility disorders associated with pregnancy.

Materials and Methods
Male Sprague Dawley (SD) rats were provided by the animal house of Jordan University of Science and Technology (JUST). Male rats were used in this study to avoid possible confounding influence from differing circulating progesterone levels in cycling female rats. All procedures were approved by the Animal Care and Use Committee (ACUC) at JUST. Male SD rats (6 weeks of age, 200-250 g) were sacrificed by an overdose of ether.
Assays were performed using a Rho kinase II assay kit (Cell Biolabs, INC., CA, USA), a Rho kinase II ELISA kit (Cusabio Biotech, Newark, DE, USA) and a Dc protein assay kit (Bio-Rad, Hercules, CA, USA).
The Anti-calponin antibody (ab46794) was purchased from Abcam, Cambridge, MA, USA. The 500-μm Nitex mesh was purchased from Amazon. All remaining materials were purchased from Sigma, St Louis, MO, USA.
Progesterone was dissolved and diluted in 99% ethanol while acetylcholine (ACh) was dissolved in distilled water.

Preparation of dispersed gastric smooth muscle cells
Smooth muscle cells were isolated from the circular muscle layer of the rat stomach by sequential enzymatic digestion, filtration, and centrifugation as described previously (27,28). Briefly, strips of circular muscle from the stomach were dissected and incubated at 31°C for 30 min in HEPES medium containing 120 mM Eagle's essential amino acid mixture, 0.1% collagenase, and 0.01% soybean trypsin inhibitor. The tissue was continuously gassed with 100% oxygen during the entire isolation procedure. After the partly digested strips were washed twice with 50 ml of enzyme-free medium, the muscle cells were allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500-μm Nitex mesh and centrifuged twice at 350 g for 10 min to eliminate broken cells and organelles. The cells were counted in a hemocytometer and it is estimated that 95% of the cells excluded trypan blue. All the experiments were done within 2-3 h of cell dispersion.

Identification of smooth muscle cells
The smooth muscle identity of rat gastric muscle cells was verified by immunohistochemical staining of paraffin-embedded rat smooth muscle using ab46794 at 1/100 dilution with anti-calponin antibody.

Progesterone treatment
Aliquots (0.4 ml) of dispersed gastric smooth muscle cells were prepared. Aliquots were randomly distributed into either control or treatment groups. Aliquots which were designated for progesterone treatment were incubated with progesterone (1 µM) for 10 min in the presence or absence of ACh.

Measurement of Rho kinase II activity
Rho kinase II activity was analyzed by an enzyme immunoassay, using Cell Biolabs' 96-well Rho kinase II activity assay kit. Experiments were done according to the manufacture's protocol, using 10 µl of protein lysate. The total starting protein concentration for every sample was 1 mg/ml.

Measurement of Rho kinase II expression
Muscle cells were solubilized in Triton X-100-based lysis buffer plus protease and phosphatase inhibitors (100 μg/ml phenylmethanesulfonylfluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml leupeptin, 30 mM sodium fluoride and 3 mM sodium vanadate). After centrifugation of the lysates at 20,000 g for 10 min at 4°C, the protein concentrations of the supernatant were determined with a Dc protein assay kit from Bio-Rad. Samples of equal amounts of proteins were quantitated by ELISA according to the manufacturers' instructions.

Analysis of data
Each experiment was performed on gastric smooth muscle cells that were harvested from six rats. Statistical analysis of all experiments was performed using Prism 5.0 software, GraphPad Software, San Diego, CA. For Rho kinase activity experiments, a one-way analysis of variance (ANOVA) was performed. Where the ANOVA was statistically significant, it was followed by Fisher's post-hoc analysis to determine the significance of differences between experimental groups. For the Rho kinase expression experiments, an unpaired student t-test was used to reveal significant differences between the compared groups. A P<0.05 was required for statistical significance in all the experiments. All data are shown as mean ± standard error of the mean (S.E.M).

Smooth Muscle Identity
The smooth muscle identity of rat gastric muscle cells was verified by immunohistostaining with anticalponin antibody. The results showed that greater than 95% of cells stained positive for calponin (Fig. 1).

Rho kinase II expression
To determine whether progesterone had any effect on Rho kinase II expression profile we examined the expression level of Rho kinase II protein in control and progesterone-treated cells by ELISA. Despite the lower agonist-stimulated Rho kinase II activity in progesterone-treated cells compared to control, the expression of Rho kinase II protein was not different in both groups of cells (P>0.05, n=6) (Fig. 3). Consistent with the indifferent expression level of Rho kinase II protein, basal Rho kinase II activity was similar in both groups (P>0.05, n=6) (Fig. 2).

Discussion
It is well-known now that pregnancy is commonly associated with GI complaints such as nausea, vomiting, and improper gastric emptying of solids and liquids. Recent research has reported disturbances in the myoelectric, mechanical, and motor properties and activities of the GI smooth muscle during pregnancy (4)(5)(6)(7)(8)(9)(10)(11)(12).
Still, the precise mechanism for such gut effects is poorly understood. In this study we found that progesterone  ACh, by two-way ANOVA followed by Bonferroni multiple comparison test). treatment for 10 min rapidly decreased the ACh-induced activity level of Rho kinase II in rat gastric smooth muscle cells without affecting Rho kinase II expression. Based on these findings, we propose that this rapid progesterone inhibitory effect may contribute to the change in gastric motility during pregnancy.
This rapid hormonal effect on Rho kinase II represents mostly non-genomic action of progesterone, as the short incubation time of the hormone would not allow changes in protein expression levels to occur (18,19). Our expression data negate an effect for short progesterone treatment on Rho kinase II protein levels. In support of these findings, basal Rho kinase II activity was not affected by progesterone incubation. Indeed, several previous reports have indicated that progesterone induces rapid, within 10 min, non-genomic effects in a variety of tissue types. For example, Bielefeldt et al. (22) found, using a human intestinal smooth muscle cell line, that progesterone reduced calcium currents consistent with blocking the L-type calcium channel.
Both of these effects occurred very rapidly (within 1 min) and were not blocked by progesterone antagonists, which would impede genomic actions of progesterone and other progestins (19,22). In addition, Xio et al. (30) reported that progesterone transiently inhibited calcium release from storage sites of colonic muscle cells and blocked the contraction to cholecystokinin (CCK-8) and neurokinin A (NKA). Moreover, a group of researchers showed in a well-designed experiment that cell-impermeant albumin-conjugated progesterone decreased thromboxane A2 receptor agonist-stimulated vascular smooth muscle calcium responses (31). This conjugated progesterone is believed not to rapidly cross the cell membrane and thus acts extracellulary. These findings might explain the rapid vasodilator action of progesterone in the primate coronary artery and isolated vascular smooth muscle. In spermatozoa, progesterone was also found to increase intracellular calcium levels by acting on a distinct non-genomic cell surface receptor. It is thought that this rapid effect of progesterone initiates the acrosomal reaction (19). These data strongly suggest the existence of an independent surface membrane progesterone receptor distinct from the classical nuclear progesterone receptor that is part of the transcriptionactivating superfamily. Furthermore, a progesterone binding membrane protein was isolated and cloned from porcine coronary artery muscle cells (32). Such a protein might be also expressed in the smooth muscle of other organs such as the stomach and could provide a possible explanation for the observed rapid non-genomic effects of progesterone on gastric Rho kinase II. Whether progesterone mediates its non-genomic action via affecting other membrane receptors such as G protein receptors is unknown so far.
Rho kinase II, the predominant Rho kinase expressed in smooth muscle, has been found to be important in developing smooth muscle tone by maintaining the level of MLC 20 phosphorylation, the essential step in smooth muscle contraction (25). There is an accumulating body of evidence from previous reports to support a genomic inhibitory effect of progesterone on the contractility of smooth muscle in various organs such as the stomach (33), colon (12,33), and gallbladder (34). This hormone effect might contribute to gastric dysmotility during pregnancy. Most importantly, progesterone was shown to induce rapid relaxation of KCl-induced contraction of rat aortic rings that was not mediated by gene transcription mechanisms. This relaxant effect of progesterone on aortic rings was partially dependent on endothelial NO production (35).
A non-genomic effect of progesterone on gastric smooth muscle contractility has not been investigated before. This study has shown for the first time that Rho kinase II is a target for rapid progesterone action in isolated smooth muscle cells of the stomach. Indeed, studying progesterone-mediated changes in the Rho kinase pathway activity and thus muscle contractility in the GI tract could be an important step for a better understanding of the GI complaints that accompany pregnancy. In conclusion, our results indicate that progesterone inhibited Rho kinase II activity in gastric smooth muscle cells occurs via non-genomic actions. Knowing the importance of the enzyme Rho kinase II in maintaining smooth muscle contraction and in support of the previous reports of progesterone action in the GI tract, we suggest that progesterone could affect the contractile activity of gastric smooth muscle cells in rats by inhibiting the Rho kinase II pathway. Future contraction studies using selective Rho kinase II inhibitors such as Y27632 and different concentrations of progesterone might further reinforce these findings.


Basic research: Issues with animal experimentations

In vivo studies using the animals are helpful in developing the treatment strategies as they are important link between the successful in vitro testing and safe human use. Various research projects in the field of fixation of fractures, development of newer biomaterials, chemotherapeutic drugs, use of stem cells in nonunion of fractures and cartilage defects etc., have hugely depended on animal experimentation. The employment of animals in experiments is both scientific and ethical issue. There must be reasonable reasons to show that it will significantly advance the present knowledge and lead to improvement in care. The regulatory bodies exist for humane use and care of animals used for experiments e.g., International Council for Laboratory Animal Science, Council for International Organizations of Medical Sciences, International Union of Biological Sciences, International Committee on Laboratory Animals. In India, Indian National Science Academy, Indian Council of Medical Research, National Centre for Laboratory Animal Sciences promote high standards of laboratory animal quality, care and health. The Committee for the Purpose of Control and Supervision on Experiments on Animals guidelines are well defined and is a must read document for any one interested to carry out research with animal facilities.

Review Article
intRoduction I n vivo studies using the animals provide invaluable information about various disease processes and help in development of treatment strategies. Virtually every medical achievement in the twentieth century relied on the use of animals in some way. 1 The Institute for Laboratory Animal Research of the U.S. National Academy of Sciences argue that even sophisticated computers are unable to model interactions between molecules, cells, tissues, organs, organisms and the environment, making animal research necessary in many areas. 2 Animal studies have been used in the past and shall continued to be used in the future for a number of applications in the field of orthopedics. Human testing cannot be done for any novel treatment unless adequate safety has been ensured. Any new implant material should match various safety standards in terms of biocompatibility, mechanical stability, lack of local and systemic toxicity. In vitro studies are quintessential initial step in testing new implant materials but results from in vitro studies are hard to apply to the in vivo situation. Many aspects of biocompatibility and safety like tissue reaction to implant, systemic toxicity, long term safety, late carcinogenicity, effect of controlled physiologic loading etc., cannot be tested in an in vitro situation. 3 Hence, extensive in vitro, as well as in vivo studies [animal studies] are essential, prior to clinical application in humans. Animal studies bridge the knowledge gap between successful in vitro testing and safe human use. Innumerable advances in the field of orthopedics and allied sciences have been critically dependent on animal experiments. Advances in internal fixation of fractures, newer implant materials, chemotherapeutic drugs for various neoplastic conditions, use of stem cells in nonunion of fractures and cartilage defects etc., have hugely depended on animal experimentation. 4

ScoPe
By law, any new drug has to pass through a series of toxicological analysis in animals before being introduced to clinical research and usage. The results from animal studies can be extrapolated to human since the internal milieu of humans are similar to animals. 5 After a successful in vitro tests of a new treatment strategy [stem cells, drugs etc.], it has to show its effect in an animal model before it is introduced into clinical research. Healing of bone gaps by stem cells, characterization of new implant materials all have to undergo animal trials prior to human application. Healing potential of tissue engineered stem cells, chondrogenic construct has to be tested in cartilage defect model in rabbits or dogs to demonstrate its efficacy prior to human trials. 6 Biodegradable materials are analyzed in vitro and in vivo to determine various characteristics of the material like rate of degradation, enzymatic reactions, immunological reactions, effect of physiological loading etc., Larger animals [sheep, goat] are used for testing of joint replacement components and surface and to study the innovative methods of fixation of bone fragments.

Selection of an animal model
An ideal animal model is one in which the anatomy and physiology match the specific study design. The pathogenesis and disease progression should parallel that of humans and so should the histopathology response. 4 The in vivo milieu should closely mimic the human clinical situation. Invertebrate animals are preferred over vertebrate animals for experiments; however, commonly used animals for experiments are rats, mice, rabbits, goats, sheep, pigs, dogs, cats and primates. 7 Rabbits are commonly used animal in tissue engineering research and in orthopedic practice. They are vertebrate animals with an appropriate size for surgical operation, hence are suitable for studies on healing of gap nonunion in bones, repair of damaged articular cartilage, reconstruction of ligaments and tendons, and spinal fusion. [8][9][10][11] Rat is also in use for tissue engineering research due to its low cost and easier maintenance. 4 It is used for studies on fracture healing, 12 biocompatibility studies. 13 and repair of bone defects. 14 Mouse, a small rodent has been extensively been used as its genome can be easily manipulated and because of its ease of handling. Regular mice or immuno compromised nude mice have been used in studies on bone 15 and cartilage healing. 16 Generally, larger animals impose more housing and handling difficulties than small animals such as rats and rabbits, and are more expensive. The factors involved in selection of the animal model includes the study design, cost, ability of the facility to handle the animals, resistance of the animal to diseases [specially in long term studies] and transportation.

ethical iSSueS
Before an animal experiment is designed there must be reasonable expectation that it will significantly advance the present knowledge and understanding about the subject and lead to improvement in care. 5 Public opinion polls have consistently shown that a majority of people approve the use of animals in biomedical research that does not cause pain to the animal and leads to new treatments and cures. 2 Animals should be used in research only when there are no suitable alternatives. The designing of research projects should be such that minimal required number of animals should be used and in a manner that conform to established standards of animal care. Animal care and ethical committee should be formed in all institutes and its due approval should be made essential prior to starting the animal studies. This unit subsequently got shifted to National Institute of Nutrition, Hyderabad, wherein in 1986 it received additional support from Department of Biotechnology, and developed into a National Centre for Laboratory Animal Sciences (NCLAS). India enacted its first animal law as early as 1960, called the 'Prevention of Cruelty to Animals Act'. It was subsequently amended in 1982, which provided for the prevention of cruelty to animals in general. Chapter 4 of act deals with control of experimentation on animals. The act also empowers committee formed under it to take care of the legal and ethical aspects of experimental animals being used in research and enact preventive actions when there is violation of the law. 5 The ministry of social justice and empowerment, New Delhi, issued a notification 18 on fifteenth February, 2001, where some guidelines were defined. In this guidelines, the word "experiment" means any program/project involving use of an animal for the acquisition of knowledge of a biological, psychological, ethological, physical or chemical nature. The establishment on registration for the purpose of performing experiments on animals shall comply with the conditions as may be specified at the time of registration by the committee and every registered establishment shall maintain a register of particulars about the animals used for conducting experiments. A detailed specifications for housing, feeding and maintenance of various species to be used in animal experimentation as notified by the committee, shall be adhered to by the registered establishment. It also specifies that the experiments shall be performed in every case by or under the supervision of a person duly qualified, that is, degree holders in medicine or veterinary science, post graduate and above in life sciences/pharmaceutical sciences or any other natural sciences, degree or diploma holders in pharmacy, diploma or certificate in laboratory animal techniques sciences from a recognized institution as identified by committee. It also mentions that experiments shall not be performed for the sole purpose of attaining or retaining manual skill. The Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines 19 are well defined and is a must read document for any one likes to carry out research with animal facilities. It provides the guidelines regarding the animal procurement, animal care, requirement of physical facilities and the environmental issues. The guidelines also deal with the issues of their food, water, sanitation, and transportation. The guidelines regarding anesthesia, standard operating procedures, euthanasia are helpful for any orthopedic surgeon or research institutions if they wish to carry the research using the animals.

Guidelines for care of animals
In order to reduce the impact of research on animals, the principles of three Rs as advocated by Russel and Burch (1959) 20 are still considered as good laboratory practice. The three Rs are: Reduction, Refinement and Replacement. Reduction means reducing the number of animals used in experiments. Refining the experiment to reduce their suffering and whenever possible, replacing experiments on animals with alternative techniques.

The Indian scenario
In India animals have been considered sacred. The animal experimentations are in primitive stage in India due to poor infrastructure in most institutes, lack of expertise in handling animals, dearth of adequate funding and lack of interest in basic research among orthopedic surgeons. Most experiments involve small laboratory-bred animals and are approved by the Institutional Animal Ethics Committee (IAEC). These committees are formed as per the guidelines of the Indian National Science Academy, and include a nominee of the CPCSEA. The current rules (2000) empowers IAECs to allow experiments only on small laboratory bred animals-guinea pigs, rabbits, rats, mice, hamsters, and invertebrates. 21 For all other animals, permission must be sought from a subcommittee of the CPCSEA. 19 The Medical Council of India (2010) has allowed medical colleges to use software instead of animals for teaching and training undergraduate students. The condition can be improved by more focus on animal experiments at medical colleges, better awareness about need and methodology of animal experiments, more funding, stringent laws regarding unnecessary harm to animals and their strict implementation.

Challenges in the field
Though animal experiments have contributed immensely towards our understanding of various disease processes and in developing better treatment methods, the opposition to use of animals for research purposes has always existed. This movement is gaining a strong foothold in our country too. Organizations like PETA [People for Ethical Treatment of Animals], IFAW [International Fund for Animal Welfare] etc., have protested against animal experiments. Appropriate permissions from animal welfare boards, maintenance of highest standards of care of animals and being abreast with the current law on animals and their use is essential to avoid trouble from the animal rights activists.
The set up for conducting animal studies is costly and faculty must be trained in handling animals well. In the West, 1 or 2 year diploma courses are available to train biology, medical and veterinary graduates. In India, short term courses ranging from six to twelve weeks are run by national bodies for those involved in animal husbandry and animal studies. National Centre for Laboratory Animal Sciences (NCLAS) at Hyderabad under the ICMR and Central Drug Research Institute (CDRI), Lucknow under the Council for Scientific and Industrial Research (CSIR) host such training programs on a regular basis. 5 The animal experiments are relevant to an average orthopedic surgeon. An average orthopedic surgeon may not come into direct contact with animal studies but he should know the importance of animal studies in his day to day practice. New drugs for osteoporosis, osteoarthrosis, antibiotics, cancer chemotherapy etc., have been derived from animal research. New implant materials for fracture fixation, bearing surfaces for joint replacement, biodegradable interference screws for ligament reconstruction, stem cell therapy for nonunion of bones are only a few examples to show the bearing animal studies have on the day to day practice of an average orthopedic surgeon. Thus, all of us as the orthopedic community should be aware of the issues associated with animal studies, recent developments, legal and ethical aspects associated with the same. All of us should pay due respect and be deeply indebted to the animal kingdom, our fellow species for making our daily practice of orthopedics so much more better and this world a much better place to live in.


text:
Detection of Specific IgA Antibodies against a Novel Deamidated 8-Mer Gliadin Peptide in Blood Plasma Samples from Celiac Patients

We studied whether celiac disease (CD) patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer) was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD), 17 CD patients on a gluten-free diet (GFD), 10 healthy controls (C) and 23 patients with other gastrointestinal pathology (GP)) and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients). Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD.

Introduction
Celiac disease (CD) is a gluten-sensitive enteropathy that develops in genetically susceptible individuals following exposure to dietary wheat gluten and similar proteins from barley, rye and some varieties of oats [1][2][3] (Highlights S1).
Prolamins constitute eighty percent of total gluten proteins. They are soluble in ethanol and rich in glutamine (Q) and proline (P) residues. Their names varies based on the source cereal (gliadin from wheat, secalin from rye, hordein from barley and avenin from oats) and they are classified in α-, γand ω-prolamins according to their electrophoretic mobility. The remaining 20% of the total gluten proteins are insoluble in ethanol and are divided in high molecular weight (HMW) and low molecular weight (LMW) glutenins.
CD is characterized by villous atrophy, crypt hyperplasia and infiltration of inflammatory cells, both in the epithelium and in the mucosal lamina propria of the small intestine. The disease might affect approximately 1% of the Caucasian population. At present, the only treatment for CD is a life-long strict glutenfree diet (GFD), which in most cases leads to a complete remission of the disease.
The inflammatory reaction appears to be driven by activation of Th1-like-CD4+ T cells that recognize gluten peptides modified by the enzyme tissue transglutaminase (tTG) in the context of human histocompatibility leucocyte antigen (HLA) region namely the HLA-DQ2/DQ8 molecules [4,5]. Deamidation is important for binding of gliadin-derived peptides to HLA DQ2/DQ8 molecules and subsequently for the stimulation of T cells [4]. Several gliadin-derived peptides have been identified as ligands for the disease-associated HLA-DQ molecules [6]. Whereas the T cell response in CD is relatively well understood, less is known about the B cell response [7]. Mucosal B cells are triggered to produce antibodies against food antigens, anti-gliadin (AGA), anti-deamidated gliadin peptides (DGP); and against self molecules as tTG. At the mucosal compartments humoral responses are mainly mediated by IgA antibodies so they are more specific than IgG antibodies as serological markers in gastrointestinal diseases like CD.
The diagnosis of CD is based on 3 pillars: i) mucosal alterations as determined by histological evaluation of duodenal biopsy, ii) genetic susceptibility (HLA-DQ2/DQ8) and iii) a positive serology (antibodies against tTG and antiendomisium) [8]. Despite small bowel biopsy is still the gold standard for CD diagnosis, endoscopy is uncomfortable and expensive. Therefore, research has been focused on developing less-invasive markers for its correct diagnosis.
Many approaches have led to the identification of several gluten peptides that can stimulate T cells from CD patients. Such peptides were found in α-, γ-and ω-gliadins as well as in glutenins. While α-and ω-gliadin-derived peptides are immunodominant in adults, responses to the LMW glutenins and γ-gliadins are frequently observed in children [9,10]. The study however of gliadin-derived peptides specifically generated in the duodenum of CD patients improves our current knowledge about gluten peptides since they may be used for development of specific serological markers with a clinical application and/or to target them in vivo to prevent their immunogenic properties in the celiac duodenum.
We have previously described for the first time to our knowledge a specific pattern of gliadin-degrading metalloproteases in the duodenal mucosa of CD patients, not found in the duodenum from non-CD patients [11]. Therefore CD patients (both aCD and GFD-patients) who have gliadin intolerance, carry CD-specific duodenal gliadin-degrading proteases suggesting a potential implication for such enzymes in CD pathogenesis.
The origin of the gliadin-degrading activity in CD is still unknown. One of the most accepted hypothesis is the possible contribution of specific CD mucosal-associated bacteria [11] which could be explained by the differences in the microbiota from patients with active celiac disease and on a GFD reported recently [12].
In order to study the possible implication of these proteases, independently of their origin, in the development of CD, here we studied whether a 8-mer gliadin peptide released by the action of duodenal CD-specific gliadin-degrading proteases could be specific epitopes of IgA antibodies found in the plasma of CD patients. We also studied if deamidation of the 8mer gliadin peptide increased its reactivity against the IgA Abs and if the latter were altered in CD patients following a GFD.
In this manuscript we report for the first time to our knowledge an 8-mer gliadin-derived peptide specifically generated in the CD duodenum, that likely deamidated by tTG, is an epitope of specific IgAs found in plasma samples from CD patients, providing a potential utility as diagnostic biomarkers and/or markers to monitor GFD compliance.

1: Study patients
Four groups of patients have been included in this study: active CD-patients (aCD), CD-patients on a gluten-free diet (GFD), healthy controls (C) and patients with other gastrointestinal pathology (GP). The first one (aCD) included celiac patients with a recent diagnosis of the disease, therefore on a normal gluten-containing diet. They had clinical symptoms, positive serology (IgA anti-TGt and/or IgA antiendomisium (EmA) antibodies), genetic susceptibility (HLA-DQ2/DQ8) and all but one had compatible lesion in the duodenal biopsy (Marsh I-III). Remainder cases fulfilled the ESPGHAN criteria (2012) to avoid intestinal biopsy. Celiac patients on a gluten-free diet (GFD), presented a reduction of symptoms, a normalization of the serological markers and a recovery of the histological lesion. Patients with other gastrointestinal pathology (PGI) were patients with a clinical profile compatible with CD but where diagnosis was later discarded, suffering another intestinal disease. These patients had negative serology and mild mucosal alterations non compatibles with CD. Finally, a group of healthy controls was included. Among each study group, patients were separated according to the age in adults (mean age 35) and children (mean age 7).
Tissue and plasma samples included in this retrospective study (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012) were obtained from the Gastroenterology and Pediatric Services from the Hospital Clínico Universitario and the Hospital Universitario Rio Hortega, Valladolid, Spain. Written consent was obtained from adult patients and parents or legal tutors from children. The study protocol was approved by the Ethics Committee of both, the Hospital Clínico Universitario and the Faculty of Medicine, University of Valladolid.
Biopsy samples. tissue samples (Table 1) were obtained according to the revised ESPGHAN criteria [13] from aCDpatients (n=2), GFD-patients (n=2) and GP-patients (n=2). All groups included one child and one adult. Duodenal explants were immediately submerged in 0.5 ml of RNALater® solution (Ambion Inc, Texas, USA) and stored at -20°C until protein extraction.
Plasma samples. Plasma samples were obtained from blood samples treated with EDTA and were obtained according to the same inclusion criteria than tissue samples. Plasma samples ( Table 2) were collected from 81 children [31 aCDpatients, 17 GFD-patients, 10 C-individuals and 23 GPpatients] and from 101 adults [16 aCD-patients, 12 GFDpatients, 27 C-individuals and 46 GP-patients], In all cases plasma samples were immediately stored at -20°C until tested.

2: HLA Genotyping
Genomic DNA was purified from whole blood (UltraClean® Blood DNA Isolation Kit, Mo Bio, California, USA) and HLA-DQ2 genotyping performed by PCR amplification using sequence specific primers (MWG Biotech, Ebersberg, Germany) and the Kapa Taq kit (Kapa Biosystems, Boston, USA) [14]. Analysis of HLA-DQ8 haplotype was only performed in those DQ2 negative patients, using the DQB1 Only SSP2LQB1 kit (Micro SSP TM DNA Typing Trays, One Lambda InC, California, USA).

4: Zymogram analysis
Proteins from biopsy samples were isolated using the Trizol Reagent (Applied Biosystems, California, USA) according to the manufacturer's protocol and quantified by the Bradford reaction. A total of 8 µg of protein samples were subsequently separated in 15% acrilamide/bisacrilamide (37.5:1) gel electrophoresis gliadin zymograms containing 0.1% of protein (gliadin, Sigma-Aldrich, Missouri, USA) using a mini-Protean II system (BioRad Laboratories Inc, California, USA). Gels were then washed twice (t=15' each) with 2.5% Triton-X-100 to remove SDS and allow protein renaturalization, and incubated overnight at 37°C with the reaction buffer (50 mM Tris, 5 mM CaCl 2 , pH 7.5). Protease activity was revealed by staining with 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad, California, USA) in a mixture of acetic acid:methanol:water (1:3:6), and destaining in the same solution mixture without the dye. A molecular weight marker was loaded in every gel (Kaleidoscope pre-stained standard, BioRad, California, USA). Additionally, stacking gel was kept during all the process since the lack of staining, compared to the running gel, was used as an internal control. All samples were separated at 200 Volts for 50 min in the running gel.

5: Fingerprinting and ion-trap Mass Spectometry
Duodenal CD specific proteases (26 and 82 kDa) from a previously described gliadin-degrading pattern [11] were removed from the substrate gel after electrophoresis and analyzed by a combined fingerprinting using a MALDI-TOF Reflex™ IV Bruker (Bruker-Franzen Analytic GmbH, Bremen, Germany) and ion-trap MS on a 3000 plus (Bruker Daltonics, Bremen, Germany) coupled to a Famos-Switchos-Ultimate (LCPackings, Amsterdam, The Netherlands) chromatographic system.

6: Protein and peptide sequence analysis
Protein identification was carried out by searching in the database of the National Center for Biotechnology Information (NCBI) using BLASTP algorithm v.2.2.20. Merops peptidase database (v 9.8) was used to analyze the cleaving points of the identified peptides.
The plates were washed with phosphate buffered saline (PBS)-Tween 20 and blocked with a solution (PBS-5% non-fat dry milk) for 1 h at room temperature (RT). Next, plasma samples (1/50) were added to the wells. After 1 h of incubation at RT, the plates were washed and horseradish peroxidaseconjugated rabbit anti-human IgA (catalog. No. P0216, DakoCytomation) was added (1/500) to duplicate wells and incubated for 1 h at RT. Then the plates were washed, and substrate solution (TMB, Sigma) was added. After 30 min of incubation at RT in the dark, the reaction was stopped with 0.3 M sulphuric acid, and the absorbance at 450 nm was measured (microplate reader UVM340, Asys Hitech GmbH, Eugendorf, Austria).

9: Statistical analysis
Each plasma sample was analyzed by triplicate and the mean value calculated. Positive and negative controls (CDpatients and C-individuals, respectively) and buffer blanks were included in each assay. An absorbance cutoff value was defined for each age group. It was calculated using the GPpatients (mean ± 2 x standard deviation) with the aim of discriminate, with the higher specificity possible, between that group and CD-patients. Only samples with an absorbance value ≥ cutoff value were considered positives. Unpaired, twotailed Student's t-test was applied and p<0.05 was considered as statistically significant. Specificity [(true negatives / (true negatives + false positives)) x 100]s and sensitivity [true positives / (true positives + false negatives)] x 100] were calculated for each age group.

1: Duodenal gliadin-degrading proteases specific from celiac patients release an 8-mer gliadin peptide
Gliadin zymogram analysis confirmed that the gliadindegrading protease pattern was specifically found in the duodenum of all 4 CD patients irrespectively of their status (aCD/GFD) or age (adults/children) while remained absent in protein samples obtained from non-CD pathogenic control ( Figure S1) as previously reported [11]. In order to identify gliadin peptides specifically released by such enzymes, fingerprinting was applied to the most intense gliadin-degrading band obtained from the zymogram matrix (26 kDa) from a GFD-patient. The fingerprinting profiles did not allow any match with any known protein. Therefore, a secondary MALDI-TOF ion-trap MS was applied and 3 peptides (8-, 15-and 18-mer) were identified (Table 4 and Figure S2).
To determinate the nature of the cleaving points generated in the 3 peptides resulting from the above analysis, we conducted a search on the peptidase database MEROPS [15]. As shown in Table 4, the C-end of the 15-mer peptide and both ends of the 18-mer peptide were potential cleaving points for trypsin so they may have been generated by the trypsin digestion applied during MALDI-TOF analysis. However, the N-end of the 15-mer peptide and both ends of the 8-mer peptide lack any described trypsin cleaving point so they did not seem to have been released during technical processing of the samples but on the contrary were likely to have been released by the action of the CD-specific duodenal gliadin-degrading proteases. A second independent analysis was also performed on the 82 kDa gliadin-degrading protease obtained from an aCD-patient revealing again the 8-mer gliadin peptide (Table 4). Since such peptide was found in two independent experiments and its cleaving points were likely to be derived from the CD-specific duodenal gliadin degrading proteases we decided to study it in depth.

2: Identification of the 8-mer peptide in proteins from toxic cereals for celiac patients
The sequence alignment made with the BLASTP program of the NCBI server [16] revealed several homologous sequences to the 8-mer gliadin peptide which remained restricted to prolamin and glutenin proteins from cereals which are toxic to CD patients like wheat, rye and barley species. None of the 8mer homologous sequences were found in non toxic cereals to CD patients, i.e. rice or maize. A full length copy of the 8-mer peptide was identified in 12 different species from the true grasses genera: Hordeum, Triticum, Lophopyrum, Aegilops and Secale. The 8-mer was more abundant in prolamins from wheat (ω-gliadin and ω-secalin in Triticum) with 196 peptides matched. Results are detailed in Table 5 and 6.

3: The 8-mer peptide overlaps with 3 known gluten T cell epitopes
We carried out a search for the known CD relevant gluten T cell epitopes restricted by HLA-DQ molecules [6] in proteins containing the 8-mer peptide. The 8-mer peptide appeared overlapped or next to three T-cell epitopes (Table 7), and the most repeated was QQPFPQQPQ [17] which overlapped in 34.5% of the 8-mer peptides matched in ω-gliadin, 13.1% in ωsecalin, 14.3% in hordein and 33.4% in glutenin sequences. Two more T-cell epitopes were close to the 8-mer peptide despite not overlapping, QQPQQPFPQ and PFPQPQQPF [17,18], which were more repeated in ω-secalin.

5: Detection of IgA anti-DGP 8-mer antibodies in plasma samples from celiac children
The ELISA test designed in this article allowed us to identify IgA Abs which recognize DGP 8-mer as a specific antigen in the plasma from CD patients. Having optimized the technique, we first studied the presence of specific IgA anti-DGP 8-mer Abs in plasma from celiac children (Table 2, Figure 1 (A)). The absorbance values (mean ± SEM) at O.D 450 nm obtained for Table 7. Identification and analysis of the alignment between the 8-mer peptide and three gluten T-cell epitopes identified previously. Comparison of the mean absorbance levels between aCDpatients and the remainder groups showed significant differences (p<0.001). IgA anti-DGP 8-mer Abs not only differentiated between CD patients and non-CD patients, but also between active celiac patients and those celiac patients who have initiated a GFD. The modified ELISA method used for detect IgA anti-DGP 8-mer antibodies showed 94% of specificity and 93.5% of sensitivity in children.

6: Detection of IgA anti-DGP 8-mer antibodies in plasma samples from celiac adults
CD is a common disorder in children and adults, and shows age-related differences at initial diagnosis. Given that IgA anti-TGt levels correlate both with the degree of villous atrophy and with the patient's age [22], we studied if the IgA anti-DGP 8mer ELISA test could be also applied to adult patients ( Table  2).
Results obtained in the adult population are shown in Figure  1  When mean absorbance levels obtained by aCD-patients and the remainder groups were compared, significant differences were obtained (p<0.001). The ELISA test showed a specificity of 98.8% and a sensitivity of 81.3% in adult population.

Discussion
We have previously described a CD-specific duodenal gliadin-degrading pattern with seven proteases with molecular weight's ranging from 20 kDa to 92 kDa [11]. In this work we describe for the first time to our knowledge a new gliadinderived 8-mer peptide (FPLQPQQP) released after gliadin digestion by duodenal CD-specific proteases. In addition, it remained restricted to prolamin and glutenin proteins from toxic cereals to CD patients (wheat, barley and rye). Among the more than 250 homologous sequences to the 8-mer peptide matched most of them was expressed in ω-prolamins (gliadin and secalin). To study if the 8-mer peptide could be found in already described toxic peptides for CD patients, we carried out a search for the known CD relevant gluten T-cell epitopes restricted by HLA-DQ molecules [6]. Since several HLA-DQ restricted gluten T-cell epitopes overlapped or were close to the 8-mer peptide, we wondered if IgA antibodies against the 8mer peptide could be found in the plasma from CD patients.
The first ELISA assays in which native and deamidated 8mer peptide were compared, showed an increase in the IgA anti 8-mer levels in CD patients when the peptide was deamidated in position 6. The increase in the reactivity of gliadin peptides in CD has been observed in several studies of native and deamidated peptides [23,24], and the effect of deamidation has been confirmed in the induction of a Th2 response in a mice model [25]. For that reason, we designed different peptides containing at least one copy of the DGP 8mer and combined with terminal modifications (Biomedal S.L) in order to select those more effective in the cover of the plate and in the binding to the antibodies. Finally, the combination of two peptides was selected as the optimum recombinant antigen to develop the assay which allowed as to detect IgA anti-DGP 8-mer in CD-patients.
Among pediatric population, levels of IgA anti-DGP 8-mer Abs were negative only in 2 aCD-patients (aCD-30 and aCD-31 in Table 8). A possible explanation for one of these cases is that the patient number 31 has a celiac sister so it is possible that he was already following (at least partially) either a GFD or a low-gluten containing diet as most of close relatives of CD patients do. Regarding GFD-patients, IgA anti-DGP 8-mer Abs were positive only in 2 of 17 children. One of them (GFD-32 in Table 9) turned out to be a case of not response to the glutenfree diet in addition to suffering prolonged diarrhea and also having negative IgA anti-tTG levels. The other child on a GFD with positive IgA anti-DGP 8-mer levels (GFD-33 in Table 9), had IgA anti-tTG levels one unit above the cutoff value, and 6 months later, IgA anti-tTG plasma levels became negative.
Ten children had positive levels of IgA anti-tTG despite being on a GFD and having clinical recovery. However, all of them were negative for IgA anti-DGP 8-mer Abs suggesting the possible application of IgA anti-DGP 8-mer antibodies as a marker of GFD compliance in children. Nevertheless, further studies are required to confirm this point, including an assessment of the patients and a direct and controlled comparison of the tTG enzyme and DGP 8-mer peptide IgA antibodies, with direct controls of GFD compliance by fecal detection of gluten peptides [26].
Although IgA anti-tTG antibodies have been proposed as markers of GFD compliance, their effectiveness is still not clear since they have been reported both well [27] or poorly [28] correlated with dietary transgressions. The normalization of IgA anti-tTG levels occurs, approximately, after 6 months on a strict GFD. Nevertheless, IgA anti-DGP 8-mer Abs were not detected in a child after a month on a GFD, while he was positive for IgA ant-tTG. In agreement with our data, Comino et al [26] estimated the time of total gluten toxic-peptide excretion to be between 2 and 6 days and the biological half-live for IgA antibodies in human plasma has been calculated in 5.9 and 4.5 days for IgA1 and IgA2 subclasses respectively [29].
Specificity is the critical parameter to discriminate CD from other gastrointestinal pathologies, and sensitivity is crucial for finding new patients. Among the 23 GP-patients studied, only one patient, who was son of a CD woman, was positive for IgA anti-DGP 8-mer test. According to a recent report published in 2012 by the ESPGHAN [13] and other studies of antibodies against DGP [30,31], the specificity is ranged between 86.3% and 93.1%, while sensitivity is in the range of 80.7% to 95.1%. Since our results in pediatric population were similar to those observed in other DGP studies, we next wondered if our ELISA test could also be applied to the adult population where CD is more frequently diagnosed in older population (20% of the celiac adults are older than 65 years). Therefore, classical gastrointestinal symptoms characteristics of CD are usually accompanied by other manifestations and pathologies usually linked with the age including liver cirrhosis, inflammatory bowel disease and diabetes mellitus, which have been related with CD in several studies [32][33][34][35][36]. Since CD diagnosis in adults is getting more complicated and false positive reactions for IgA anti-tTG have been reported we also studied 46 plasma samples from adult non-celiac patients but with other gastrointestinal pathology. All of them were negative for IgA anti-DGP 8-mer while some of them had positive levels of IgA anti-tTG revealing therefore a specificity of 100% in such cohort.
The highest absorbance values in pediatric population (Figure 1 (A)) were almost 4 fold over those observed in the adult one (Figure 1 (B)). However IgA anti-DGP 8-mer antibodies could provide an additional degree of specificity in both populations, compared to other serological markers, due to the following reasons: tissue transglutaminase enzyme is expressed in a constitutive manner in everyone [37] because of its functions in apoptosis and cellular differentiation and anti-tTG antibodies may persist independently of the GFD compliance or could be generated in other pathologies. In Table 9. Clinical data from celiac patients on a gluten-free diet (n=17) from the pediatric population (n=48).  addition, DGP usually are derived from whole gluten digested with commercial enzymes like pepsin, trypsin, chymotrypsin, elastase and carboxypeptidase and subsequently modified with TG2 [20]. On the other hand, the 8-mer peptide is generated by specific duodenal proteases of celiac patients.

Conclusion
In this study we have identified, for the first time to our knowledge plasma CD-specific IgA Abs that recognize as specific antigen a novel deamidated gliadin peptide (FPLQPEQP), derived from an 8-mer peptide generated during duodenal degradation of gliadin by CD-specific duodenal proteases. The native peptide (FPLQPQQP) remained restricted to prolamin and glutenin proteins from toxic cereals to CD patients and was particularly expressed in ω-prolamins. The 8-mer peptide overlapped with a previously identified gluten T cell epitope and its ability to develop a humoral immune response in vivo in CD patients was confirmed by the detection of IgA anti-DGP 8-mer Abs in plasma samples from those patients.
The DGP 8-mer could be used as a novel specific CD antigen for the diagnosis of CD and monitoring of the compliance of GFD. We have designed an ELISA test which detects IgA anti-DGP 8-mer Abs in plasma samples from CDpatients with a high specificity (94% in children and 98.8% in adults) and sensitivity (93.5% in children and 81.3% in adults). Future studies with larger number of samples and additional relevant data (i.e. use of novel tools for GFD compliance) will assess the full potential of the novel diagnostic tool for celiac diagnosis and management.

Supporting Information
Highlights S1. Significance of this study. Significance of this study in the context of what is already known about this subject and the new findings described in this manuscript. (DOC) Figure S1. the gliadin-degrading protease pattern. Gliadin zymogram analysis (A) of whole protein duodenal biopsy explants from active celiac patients (aCD, lines 1 and 2), celiac patients on a gluten-free diet (GFD, lines 3 and 4) and patients with other gastrointestinal pathology but non-celiac disease (GP, lines 5 and 6). The gliadin-degrading protease pattern, characterized by 7 CD-specific proteases (from 92 to 20 kDa), was found in aCD and GFD patients while was absent in GPpatients. The fingerprinting and ion-trap mass spectrometry analyses of 26 kDa protease from a GFD-patient (line 3) and 82 kDa protease from an aCD-patient (line 2) allowed the identification of three peptides