Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis. Colorectal cancer (CRC) is one of the most common digestive cancers worldwide. Recently, combination therapy has improved the prognosis for CRC patients. However, the prognosis for advanced CRC with lymphatic metastasis remains poor because there are no effective therapies for this disease Autophagy is a highly conserved self-digestion process in eukaryotic cells that involves the degradation of old organelles and proteins to obtain energy. Increasing evidence suggests that the dysregulation of autophagic pathways is involved in various types of tumor clonal expansion and growth In this study, we used SW620 CRC cells to test the hypothesis that Ambra1 interacts with Beclin1 to promote autophagy and to inhibit apoptosis in CRC cell lines. We sought to determine whether autophagy occurs in SW620 CRC cells in response to apoptotic stimuli and whether Ambra1 regulates autophagy in SW620 cells by interacting with Beclin1. Our findings clearly suggest that Ambra1 functions at the intersection between autophagy and apoptosis. We found that Ambra1 interacts with Beclin1 to function as a pro-survival switch that inhibits apoptosis and induces autophagy, thereby preventing CRC cell death in response to apoptotic agents.2 humidified atmosphere. Unless otherwise indicated, cells were treated with 5 µg/ml etoposide  or 2 µM staurosporine , both of which are apoptosis-inducing agents. All of the above drugs were solubilized in DMSO. Calpain inhibitor (CL) and caspase inhibitor (z-VAD-fmk) were used as previous described The human SW620 colorectal cell line was purchased from ATCC  and cultured in Leibovitz’s L-15 medium  with 10% fetal bovine serum at 37°C in a 5% CO5′-AGAACTGCAAGATCTACAA-3′ and Ambra1 siRNA no. 2 5′-GGCCCTATGGTACTAACAA-3′. The cells were transfected with 100 pmol Ambra1 siRNA using Lipofectamine RNAi max  in 6-well plates. The negative control siRNA was transfected under the same conditions. The transfection was repeated for two days to achieve a high efficiency. Approximately 24 h after the second transfection, 20×104 cells/well were plated in 6-well plates and cultured with the indicated agents. Protein expression was measured by Western blot 48 h after transfection.The following siRNA oligonucleotides corresponding to human Ambra1 cDNA were purchased from Genepharma : Ambra1 siRNA no. 1 g for 10 min. The cells were subsequently lysed in cold lysis buffer  and sonicated for 5 s. The lysates were clarified by centrifugation at 12000×g for 30 min at 4°C. Equal amounts of cell lysate were resolved by 8 or 15% SDS-PAGE. The membranes were blocked for 1 h in 5% powdered milk in TBST . The membranes were subsequently immunoblotted using anti-Ambra1, anti-Beclin1, anti-LC3, anti-PARP, anti-Cathepsin D, anti-ATG5, anti-VPS34 and anti-β-actin antibodies . The PVDF membranes were incubated with a horseradish peroxide-conjugated anti-rabbit IgG antibody  for 1 h at room temperature. Immunoreactive bands were exposed to film . The band intensity was semi-quantified using BandScan software after scanning the blots .The cells were washed twice with PBS containing 1 mmol/l phenylmethylsulfonylfluoride (PMSF). Next, the cells were scraped off the dishes and pelleted at 500×Real-time PCR was performed as previously described The cells were fixed in 4% paraformaldehyde for 10 min and then washed with PBS for 5 min. The slides were immersed in 100 µg/ml digitonin for 15 min at room temperature and then washed. Subsequently, the cells were incubated with an anti-LC3 antibody  and washed 3 times. A FITC-conjugated anti-rabbit IgG antibody  was added to the cells, and the cells were washed after the incubation. Autophagosomes were examined using a fluorescence microscope .4 cells/well in 96-well plates. The negative control cells were treated with 1% DMSO. Fifteen microliters of MTT was added to each well after 48 h, and the cells were incubated for an additional 4 h. Subsequently, 100 µl of the solubilization solution/stop mix was added according to the manufacturer’s instructions , and the plates were incubated for 60 min. The absorbance was measured at 570 nm and 630 nm using an ELISA reader. The actual counts were calculated by subtracting the absorbance at 570 nm from that at 630 nm. Each assay was performed in triplicate, and the average absorbance was calculated.Cells were plated at a density of 1×106 cells/ml. Subsequently, 5 µl of annexin-V-PE and 5 µl of PI were added to 100 µl of the cell suspension, and the mixture was incubated for 15 min in the dark. After incubation, 400 µl of 1X binding buffer was added. The analyses were performed using a FACScan flow cytometer .The Annexin-V-PE Apoptosis Detection kit  was used to measure apoptosis. Cells were washed with PBS and resuspended in 1X binding buffer at a concentration of 1×10SW620 cells were cultured in serum-free medium for 2, 4 or 8 h. The cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 ml/L phosphate buffer (pH 7.4) followed by 1% osmium tetroxide. After dehydration, thin sections were stained with uranyl acetate and lead citrate and observed using a JEM 100 CX electron microscope .The supernatants from the CRC cell lysates were incubated with anti-sera bound to protein A-sepharose beads (Amersham Bioscience) for 2 h at 4°C and washed extensively. To elute the bound proteins, 100 mM glycine-HCl  was added, and the samples were washed with ice-cold acetone and resuspended in SDS sample buffer.The statistical analyses were performed with SPSS. Statistical significance was determined using Student’s t-test. A p-value equal to or less than 0.05 was considered significant.We induced autophagy by starvation. The SW620 cells were cultured in serum-free medium for various time periods. LC3 is a homolog of the yeast autophagy-related protein Apg8p To further confirm that the autophagosomes were induced by starvation, we examined serum-starved SW620 cells via fluorescence microscopy and transmission electron microscopy (TEM). An antibody against endogenous LC3 was used to detect autophagy by immunofluorescence. Endogenous LC3 was redistributed from a diffuse pattern to visible cytoplasmic puncta . Figure Numerous pro-autophagic proteins are cleaved during apoptosis In a previous study, calpains and caspases were shown to be responsible for Ambra1 cleavage in fibrosarcoma cell lines Ambra1 is an anti-apoptotic ATG protein that promotes autophagy To understand how Ambra1 triggers autophagy, we investigated the mechanism by which Ambra1 affected autophagocyte formation. The Beclin1-Class III PI3K complex initiates autophagocyte formation; however, it has not been determined whether Ambra1 interacts with this complex in CRC cell lines. We determined whether endogenous Ambra1 interacted with endogenous Beclin1 upon the induction of autophagy in CRC cells. The interaction between endogenous Ambra1 and endogenous Beclin1 was confirmed by co-immunoprecipitation in SW620 cells. Using anti-Ambra1 or anti-Beclin1 antibodies to pull down the protein complexes, we observed that the Ambra1-Beclin1 interaction was more pronounced in autophagy-stimulated cells than in non-stimulated cells . SimilarAmbra1 promotes autophagy in numerous cell types Autophagy plays a role in determining cellular fate by degrading old organelles, recycling cellular components and responding to stressful cellular conditions Previous studies have indicated that Ambra1 regulates autophagy in multiple cell types, including neuronal In this study, we demonstrated that the Ambra1-Beclin1 autophagic pathway that has been described in other cell types is conserved in SW620 CRC cells. Beclin1 is a phylogenetically conserved protein that is essential for autophagy. Beclin1 associates with PI3KCIII/Vps34 and other co-factors, such as Ambra1, to initiate autophagy An increasing number of factors have been identified that regulate both apoptosis and autophagy. For example, Bcl-2 is a well-known inhibitor of both autophagy and apoptosis Previous studies have reported that a functional deficiency in Ambra1 in mice results in developmental defects, embryonic death associated with impaired autophagy and increased apoptosis in vitro Our study confirmed that Ambra1 is an important factor in the pathways that regulate tumor cell survival in SW620 CRC cells. However, questions remain regarding the function of Ambra1 in the regulation of autophagy and apoptosis in these cells. We discovered one Ambra1-related autophagy pathway, but additional pathways may exist in CRC cells. Furthermore, the mechanism by which Ambra1 inhibits apoptosis has not been fully elucidated in these cell lines. Reports have suggested that Ambra1 and BCL-2 dynamically interact in the mitochondria to regulate apoptosis In conclusion, this study ascertained the role of Ambra1 as a regulator of autophagy and apoptosis in the SW620 CRC cell line. We demonstrated that Ambra1 is a key pro-survival factor in SW620 cells that promotes autophagy by binding to Beclin1 and inducing Beclin1-mediated autophagy. We determined that Ambra1 knockdown rendered SW620 CRC cells more susceptible to apoptosis, suggesting that Ambra1 may be an important negative regulator of apoptosis in these cells. Future studies are necessary to explore the detailed mechanisms that link the regulation of autophagy and apoptosis in these cancer cells. In addition, in vivo animal studies are essential for exploring the role of Ambra1 in the pro-survival pathways that contribute to CRC tumor growth, survival and chemoresistance.


Background: Nrf2 is required for normal dendritic cell immune functions.Results: Loss of Nrf2 alters DC function and results in hyperphosphorylation of CREB/ATF1 transcription factors that are responsive to p38 MAPK inhibition.Conclusion: The p38 MAPK-CREB/ATF1 axis contributes to Nrf2-mediated regulation of DC function.Significance: Defining the relevance of p38-CREB/ATF1 in Nrf2 signaling expands understanding of DC biology. −/− iDCs) is due to elevated ROS levels. Furthermore, it is unclear which intracellular signaling pathways are involved in Nrf2-mediated regulation of DC function. Using antioxidant vitamins to reset ROS levels in Nrf2−/− iDCs, we show that elevated ROS is not responsible for the altered phenotype and function of these DCs. Pharmacological inhibitors were used to explore the role of key MAPKs in mediating the altered phenotype and function in Nrf2−/− iDCs. We demonstrate that the increased co-stimulatory molecule expression (MHC II and CD86) and antigen-specific T cell activation capacity observed in Nrf2−/− iDCs was reversed by inhibition of p38 MAPK but not JNK. Importantly, we provide evidence for increased phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor 1 (ATF1), transcription factors that are downstream of p38 MAPK. The increased phosphorylation of CREB/ATF1 in Nrf2−/− iDCs was sensitive to p38 MAPK inhibition. We also show data to implicate heme oxygenase-1 as a potential molecular link between Nrf2 and CREB/ATF1. These results indicate that dysregulation of p38 MAPK-CREB/ATF1 signaling axis underlies the altered function and phenotype in Nrf2-deficient DCs. Our findings provide new insights into the mechanisms by which Nrf2 mediates regulation of DC function.Nrf2 is a redox-responsive transcription factor that has been implicated in the regulation of DC immune function. Loss of Nrf2 results in increased co-stimulatory molecule expression, enhanced T cell stimulatory capacity, and increased reactive oxygen species (ROS) levels in murine immature DCs (iDCs). It is unknown whether altered immune function of Nrf2-deficient DCs (Nrf2 Dendritic cells (DCs)Redox homeostasis is important for a variety of cellular functions such as proliferation, apoptosis, and intracellular signaling pathways, including MAPK signaling –12. In tWe and others , 26 haveAll reagents were from Sigma-Aldrich unless otherwise stated. FCS (Invitrogen), SB203580 and PD98059 , and tin protoporphyrin IX dichloride  were also purchased for the study.+/+ and Nrf2−/− mice were purchased from Riken BioResource Center  and maintained at the Biomedical Services Unit, University of Liverpool . Protocols described herein were undertaken in accordance with criteria outlined in license granted under the Animals (Scientific Procedures) Act 1986 (PPL 40/3379).Nrf2iverpool . Mice tr+/+ and Nrf2−/− mice according to published protocol (Bone marrow-derived iDCs were generated from Nrf2protocol . On day TC (Invitrogen) and αCD86FITC, or αMHCIIPE (BD Biosciences) antibodies for 30 min on ice, washed, acquired on a BD FACSCanto II flow cytometer (BD Biosciences), and analyzed using Cyflogic software .DCs were stained with fluorescent αCD11c+/+ and Nrf2−/− mice were stained using fluorescent ROS indicator, dihydroethidium, according to Ref. Basal or vitamin-treated iDCs from Nrf2+/+ and Nrf2−/− iDCs were pulsed with a dose range of antigenic peptide (NP68), washed, and co-cultured with F5 CD8 T cells for 72 h. [3H]Thymidine was added for the last 16 h. Cells were harvested onto glass fiber filter mats and read on a scintillation counter .F5 CD8 T cell proliferation was quantified as described previously . Briefly+/+ and Nrf2−/− iDCs were lysed, and 20 μg of lysate protein was resolved by SDS-PAGE, transferred to nitrocellulose membranes , blocked, and probed for the indicated proteins using the appropriate primary antibodies; phospho-p38, p38, phospho-CREB, CREB ; and α-tubulin (Santa Cruz Biotechnology) followed by horseradish peroxidase-conjugated secondary antibodies  and visualized using the ECL system (PerkinElmer Life Sciences).Nrf25 DCs per ml at 2 ml/well. After 48 h, cell-free supernatants from SB203580 untreated and treated groups with or without the Toll-like receptor agonist LPS were collected and stored at −20 °C until further analysis.Cell-free culture supernatants were used for measuring IL-10 cytokine concentrations by sandwich ELISA . On day 6, DCs were seeded onto 24-well plates in duplicates at a density of 8 × 10t test or one-way ANOVA. p values < 0.05 were considered to be statistically significant.Raw data obtained were analyzed using the unpaired +/+ and Nrf2−/− iDCs were treated with vitamins C and E for 48 h, and ROS levels measured by flow cytometry using the fluorescent ROS indicator dihydroethidium. A significant reduction in ROS levels was observed in vitamin-treated Nrf2−/− iDCs compared with untreated controls  as shown in Fig. 1A. A slight reduction in the ROS levels was also seen in vitamin-treated Nrf2+/+ iDCs, which was not statistically significant  . It is important to note that the ROS levels in vitamin-treated Nrf2−/− iDCs was equivalent to that in untreated Nrf2+/+ iDCs . To test whether elevated intracellular redox levels is responsible for increased DC co-stimulatory molecule expression, we measured MHC II and CD86 expression in Nrf2−/− iDCs following vitamins treatment. As shown in Fig. 1B, the expression of MHC II and CD86 under basal condition was significantly higher in the Nrf2−/− iDCs compared with Nrf2+/+ iDCs . However, there was no significant difference in the co-stimulatory molecules expression between untreated controls and vitamins treatment groups in both Nrf2+/+  and Nrf2−/− iDCs . This result indicates that restoring ROS levels in Nrf2−/− to Nrf2+/+ status did not reverse co-stimulatory molecule expression. We further investigated whether the lack of change in co-stimulatory molecule expression in Nrf2−/− iDCs to ROS reset is also reflected in its ability to induce antigen-specific T cell activation. To determine this, we utilized a TCR transgenic mouse model wherein the CD8 T cells express a T cell receptor (F5 TCR) that responds to an antigenic peptide, NP68, when presented by DCs . Lowering of ROS levels by vitamin treatment did not reduce the potential of NP68-bearing Nrf2−/− iDCs to stimulate F5 CD8 T cell proliferation . These results demonstrate that altered ROS status associated with loss of Nrf2 does not contribute to increased co-stimulatory molecules expression and T cell stimulatory potential of DCs.Loss of Nrf2 leads to increased co-stimulatory molecule expression, T cell stimulatory potential, and elevated ROS levels in iDCs , 26. We d by DCs . Using t−/− iDCs to that of the wild type levels  as indicated in Fig. 2A. The iDC-mediated antigen-specific CD8 T cell proliferation also remained unaltered following JNK inhibition in Nrf2−/− and Nrf2+/+ iDCs . However, inhibition of p38 MAPK activity caused a significant reduction in co-stimulatory molecule expression in Nrf2−/− iDCs (A) . Inhibition of p38 MAPK in Nrf2+/+ iDCs resulted in only a slight, statistically insignificant reduction in co-stimulatory molecules expression . Consistent with the changes in co-stimulatory molecule expression, inhibition of p38 MAPK resulted in significant reductions in DC-mediated antigen-specific CD8 T cell proliferation in Nrf2−/− iDCs with less pronounced effects on Nrf2+/+ iDCs (B). These observations suggest the contribution of p38 MAPK but not JNK in the Nrf2-dependent modulation of DC immune functions.Mitogen-activated protein kinase signaling comprising of ERK1/2, p38 MAPK, and JNK pathways coordinate DCs co-stimulatory molecules expression and DC-mediated T cell activation , 34, 35.−/− iDCs A (MHC II+/+ iDCs B. These −/− and Nrf2+/+ iDCs (A). Major downstream effectors of p38 MAPK are the transcription factors, CREB and ATF1. Serine phosphorylation of CREB and ATF1 by upstream MAPKs is required for their activation. We measured the phosphorylation state of CREB and ATF1 and observed that CREB and ATF1 were hyperphosphorylated under basal conditions in Nrf2−/− iDCs (B). A significant reduction in the phosphorylation of CREB and ATF1 was observed upon inhibiting p38 MAPK activity using a pharmacological inhibitor (SB203580) as seen in Fig. 4C. This suggests that CREB and ATF1 phosphorylation in DCs are dependent on p38 MAPK activity. There is evidence in other cell types that in addition to p38 MAPK, ERK1/2 can also mediate CREB/ATF1 phosphorylation. We tested the requirement of ERK1/2 for phosphorylation of CREB and ATF1 in the DCs under basal and LPS-stimulated conditions using an ERK1/2 inhibitor (PD98059). As seen in Fig. 4D, inhibition of ERK1/2 did not reduce the level of phospho-CREB/ATF1 under basal conditions. LPS stimulation markedly increased the phosphorylation of CREB/ATF1 in Nrf2+/+ and Nrf2−/− iDCs . Inhibition of ERK1/2 did not reduce the phosphorylation of CREB/ATF1 under LPS-stimulated conditions in both Nrf2+/+ and Nrf2−/− iDCs . Consistent with the previous result , p38 MAPK inhibition reduced CREB/ATF1 phosphorylation levels both basally and upon LPS stimulation. To test whether the basal hyperphosphorylation of CREB/ATF1 in Nrf2−/− iDCs could be the result of elevated ROS . These results indicate that Nrf2 is required for controlling the activity of the p38 MAPK-CREB/ATF1 signaling axis in DCs.As the activity of p38 MAPK is regulated by phosphorylation, we assessed the phosphorylation state of p38 MAPK. Western blotting analysis revealed marginal differences in p38 MAPK phosphorylation between Nrf2+/+ iDCs A. Major −/− iDCs B. A signated ROS , we trea−/− iDCs produce higher levels of IL-10 in comparison to Nrf2+/+ DCs under basal conditions (40.1 pg/ml versus 25.7 pg/ml p < 0.05). Furthermore, upon LPS stimulation, Nrf2−/− iDCs produced levels of IL-10, which was greater than that produced by LPS-stimulated Nrf2+/+ iDCs (96.3 pg/ml versus 73.3 pg/ml p < 0.05). Although basal production of IL-10 was not sensitive to p38 MAPK inhibition, a significant reduction in LPS-induced IL-10 production  was observed in both Nrf2+/+ and Nrf2−/−iDCs treated with SB203580 . In addition, inhibition of HO-1 activity was shown to increase the phosphorylation of CREB/ATF1 in Nrf2+/+ iDCs . This observation suggests that one of the mechanisms by which Nrf2 modulates co-stimulatory molecule expression and CREB/ATF phosphorylation is through its effect on HO-1 function.A key Nrf2-transcribed gene involved in cellular homeostasis is HO-1. To test whether HO-1 is involved in Nrf2-mediated regulation of co-stimulatory molecule expression, Nrf2−/− iDCs are therefore likely to result in cellular adaptive changes that cannot be simply reversed by rebalancing ROS levels but require more complex cellular reprogramming .Transactivation by CREB/ATF1 requires its association with cofactors such as CREB binding protein (CBP) . There a−/− iDCs , 54. A c−/− iDCs . The molIn summary, our data implicates p38 MAPK-CREB/ATF1 axis as a key signaling pathway that is regulated by Nrf2 in DCs. The molecules within this pathway, as well as HO-1, could represent targets for pharmacological intervention in disease states that arise from dysregulated Nrf2 function.


The effects of Pred were not mediated through cyclic nucleotide signaling, but rather seemed to evolve around selective regulation of P2Y12 ADP receptor signaling, intimating a novel mode of action. This study details the actions of Pred on platelets unveiling novel properties which could be relevant for this GC in controlling unwanted vascular and thrombotic diseases.We have previously reported presence of the glucocorticoid (GC) receptor (GR) alpha on blood platelets, and its ability to modulate platelet aggregation when activated by the synthetic GC prednisolone (Pred). In the present study we investigated the effects of Pred on broader aspects of platelet functions to unveil novel non-genomic actions on this cell type. Using whole blood assay we demonstrated that Pred was the only GC able to inhibit platelet aggregation and platelet–monocyte interactions. This latter effect was due to regulation of platelets, not monocytes. We next examined the effects of Pred on physiological actions of platelets, observing inhibition of platelet adhesion and spreading on collagen under static conditions. Moreover Pred inhibited thrombus formation under flow, suggesting potential important effects in haemostasis and thrombosis. Pred was unable to regulate platelet reactivity under conditions where the effects of platelet-derived ADP and TxA Of relevance, the inhibitory effect of Pred was reversed by pre-incubation with the GR antagonist mifepristone, suggesting the functional importance of the classical GR/GC complex also in the inhibition of platelet aggregation in platelet-rich plasma Our previous work has revealed the unexpected finding that GR activation can modulate platelet function Blood platelets play an established role in the thrombotic complications arising from atheroma-induced vascular rupture, and they can also contribute to the inflammatory responses leading to endothelial dysfunction 2Unless otherwise specified, all chemicals were from Sigma–Aldrich .2.1Human blood was collected from healthy volunteers, who had abstained from non-steroidal anti-inflammatory drugs medications at least for the preceding 7 days, using venepuncture into hirudin . All experiments were approved by the East London Research Ethics Committee (no. 05/Q0603/34).7 or 2.5 × 108 platelets/ml in modified Tyrodes buffer  unless otherwise stated.For preparation of washed platelets, blood was taken into acid citrate dextrose  as anticoagulant. Platelet-rich plasma (PRP) was obtained by centrifugation of whole blood at 200 g at 20 °C for 20 min. PRP was treated with citric acid (0.3 mM) and centrifuged at 1900 rpm for 12 min. The platelet pellet was then suspended in wash buffer  and spun once more. Platelets were finally re-suspended at a concentration of 5 × 102.2Whole blood impedance aggregometry was applied using a Multiplate™ analyzer . Thirty minutes after venepuncture, 300 μl of hirudinated blood was diluted 1:1 with saline  into a test cell at 37 °C and stirred for 3 min in presence of one or combination of GCs; prednisolone (Pred), dexamethasone (Dex), nitro-dexamethasone (Nitro-Dex), fludrocortisone (Fludro), triamcinolone (Triam) before the addition of either collagen , arachidonic acid  or adenosine-diphosphate . The electrical resistance between two pairs of silver coated wires was measured and plotted over a 6-min period. The Multiplate™ analyzer calculated the average area under the curve (AUC) for the two readings. Each test was conducted in duplicate for each donor sample.8 platelets/ml) were incubated for 5 min at 37 °C in a multichannel Chronolog Aggregometer  prior to stimulation to allow for temperature equilibration. The platelet suspension was then stimulated with ADP (5 μM) under stirring conditions (1000 rpm) and platelet aggregation was recorded for 4 min. The platelet aggregometer was recalibrated for every individual platelet sample used. The following incubation times were used for inhibitors added prior to stimulation of platelets; A3P5P (300 μM for 1 min), MRS2395 (10 μM for 1 min) and Pred (1 or 10 μM for 5 min) prior to the addition of ADP (10 μM).Alternatively, washed platelets  against human CD42b  and CD14  and either Pred or vehicle for 3 min at 25 °C. Platelets were stimulated by adding ADP (1–3 μM) and incubated for a further 20 min at 25 °C. Samples were fixed and red blood cells (RBC) lysed using BD FACS™ lysing solution  and kept at 4 °C up to analysis.4 1 mM, HEPES 10 mM) and added to tubes in equal volume. After addition of FITC-conjugated anti-human CD42b and PE-conjugated anti-human CD14, samples were stimulated with ADP (1–3 μM) and incubated for 20 min at 25 °C then fixed using paraformaldyde  and stored at 4 °C prior analysis. Flow cytometry was performed on a FacsCalibur analyzer . Platelets and monocytes were discriminated by forward and side light scatter, identified by their positive staining for FITC-CD42b or PE-CD14, respectively. Events double positive for FITC and PE identified platelet–monocyte aggregates and were recorded as a percentage of a total of 10,000 gated monocytes.In some cases, Pred was added to either the platelet or the leukocyte preparation separately. To this end, after removing PRP, the remaining of the blood was resuspended in RPMI 1640  (ratio 1:1) and mononuclear cells separated through a double density gradient as previously described 2.47/ml) the presence and absence of the appropriate inhibitors were adhered to collagen (10 μg/ml)-coated coverslips for 30 min at 37 °C. After incubation, coverslips were washed with PBS to remove non-adherent platelets. For experiments with Pred, the GC was added to platelets 5 min before addition to slides. The following incubation times were used for inhibitors added prior to adhesion of platelets: apyrase (2 U/ml for 1 min), indomethacin (10 μM for 5 min), ODQ (20 μM for 15 min) or myrPKI (500 nM for 15 min). Adherent platelets were fixed with 3.5% paraformaldehyde and permeablized with 0.2% Triton-X-100 in PBS. Platelets were stained for F-actin using TRITC-conjugated phalloidin and visualized using fluorescent microscopy . For each experiment, the numbers of platelets from eight random fields of view were added, and the results were calculated as mean number of adherent platelet per 0.01 mm2. The average surface area of the individual adherent platelets was calculated using Cell P software (Olympus).Platelets (5 × 102.58 platelets/ml) were pre-treated with either ODQ (20 μM) or myrPKI (500 nM) for 10 min and then incubated with either the nitric oxide donor, S-nitrosoglutathione  or prostaglandin E1 (1 μM) for 2 min. Platelets were lysed by the addition of Laemmli sample buffer (1×). Proteins were separated by sodium dodecylsulfate polyacryl-amide gel electrophoresis (SDS-PAGE) (7.5% polyacrylamide gels) and immunoblotted as previously described 239 or anti-VASP-phospho157 antibodies (1:1000).Washed platelets (2 × 102.68 platelets/ml) were incubated with dihexloxacarbocyanine  at 37 °C for 10 min and then incubated at 37 °C for 2 min with Pred (10 μM), before being reconstituted with autologous washed RBC. Flow studies were performed using glass capillary tubes , coated with immobilized collagen  for 12 h, blocked with BSA/PBS (5 mg/ml) for 1 h and washed under flow conditions with PBS for 4 min. Platelets were perfused through collagen-coated capillary tubes at 1000/s−1 for 4 min, followed by washing (4 min) at the same shear rate. Adherent platelets/thrombus formation was visualized using fluorescence microscopy . Data were calculated from the percentage of the area covered by adhering platelets in a defined area (surface coverage), since this methodology cannot fully discriminate between individual platelets and platelet aggregates Platelets . Increasing the concentration to supra-physiological levels such as 10 μM failed to augment the extent of inhibition afforded by Pred. In the same settings, aspirin (20 mg/ml) produced 70% inhibition of ADP platelet aggregation (not shown). In any case, the inhibitory effects of Pred were rapid, being established within 3 min of incubation and remained consistent for up to 60 min (longest time tested) post-steroid application (Incubation of whole blood with Pred (5 nM–10 μM) led to a concentration-dependent inhibition of ADP-induced platelet aggregation A and B. lication C. There lication C. In thelication .P < 0.05). Nitro-Dex also caused a significant inhibition of aggregation , although this is likely to be related to the NO-donor properties since the parent compound Dex alone had no effect. Fludrocortisone and triamcinolone were also ineffective platelet regulators  were observed with the 500 nM concentration. This GC retained its inhibitory effect also when a higher concentration of ADP (3 μM) was used, though the highest concentration tested of 5 μM was required to attain significant inhibition  , before reconstituting the co-culture system and stimulating with ADP (1 μM). When both cell types were treated with Pred, we observed a 30 ± 6% inhibition of aggregate formation ( < 0.05) C. When p < 0.05) C. Taken 3.32). When platelets were pre-incubated with Pred a concentration dependent inhibition of adhesion was observed. Threshold effects were observed with 1 μM Pred, with a number of adherent platelet reduced to 21.8 ± 4 platelets/0.01 mm2, while maximal effects occurred at 10 μM Pred, when adherent platelets fell to 6.8 ± 2.9  (2 (P < 0.05) in the presence of values Pred. The inhibitory effects of Pred (10 μM) on platelet adhesion to collagen were reversed by the GR antagonist, RU486  B. In add6 10 μM; C. Simila surface . Of inte3.422 , confirming that maximal adhesion requires substantial platelet activation driven by platelet-derived metabolites , while A3P5P and MRS2395 reduced aggregation to 46 ± 8 and 36 ± 7% respectively (P < 0.05). Interestingly when platelets were treated with prednisolone in combination with A3P5P, aggregation was reduced to 36 ± 5%, which was no different to when the inhibitors were used independently. In contrast, a combination of prednisolone with MRS2395 reduced aggregation to 22 ± 3%   in the presence of MRS2395 (10 μM) and A3P5P (300 μM) to inhibit the potential effects of ADP signaling through P2Yd alone) C. These 2 under both stirred and static conditions Previously, we and others have shown that cAMP and cGMP signaling pathways reduce the release and activity of platelet ADP and TxA3.5in vitro flow based assay. Under shear (1000 s−1), immobilized collagen (100 μg/ml) supported adhesion of numerous small thrombi that covered 14.7 ± 1.8% of the collagen-coated surface (P < 0.01).Finally, we examined the ability of Pred to regulate thrombus formation in response to collagen under shear conditions using an  surface . Pretrea4in vitro under conditions of flow.There is mounting evidence that GC can also exert biological effects through non-genomic modalities. Recently we demonstrated that the synthetic GC Pred modulated platelet aggregation, through a mechanisms that involved engagement of the classical GR Our first critical observation was that Pred regulated platelet activity in whole blood, suggesting that the agent could be active against platelets in the circulation, a finding with obvious clinical bearing. Using the whole blood protocol, Pred induced a significant inhibition of ADP-induced aggregation, but was ineffective when collagen and AA were used to stimulate aggregation. These data are in part consistent with observations conducted with PRP and the aggregation assay; in the whole blood protocol Pred afforded only a modest effect on collagen-induced aggregation, while it affected collagen-induced responses in PRP An increase of heterotypic leukocyte-platelet aggregates has been reported cardiovascular disease 2β1 which then allow stable adhesion and facilitate spreading 2 were abrogated. These data suggest strongly that Pred targets secondary signaling required for maximal platelet adhesion (hence the lack of complete inhibition) and is consistent with our observation that Pred preferentially targets ADP-induced aggregation and limit TxA2 synthesis At sites of vascular injury platelet accumulation begins with an interaction between the extracellular matrix collagen and the platelet receptor glycoprotein VI 12 inhibitor A3P5P, the ability of Pred to inhibit platelet was lost, while with combination of MRS2395, a P2Y1 antagonist, we observed an additive effect of Pred, strongly suggesting that this GC regulates AD-induced platelet activation through modulation of the P2Y12/Gi signaling pathway. We also found that in whole blood, Pred did not inhibit aggregation induced by either AA or collagen, which is in contrast to the data obtained using PRP. It is possible that the effects of Pred could be masked here due to the high concentrations of agonist required in whole blood assays, negating the reliance on secreted ADP. The influence of Pred on the complex relationship between secreted ADP and TxA2 is an area that requires further clarification.The argument is further strengthen by aggregation data using combinations of Pred with known ADP receptor antagonists MRS2395 and A3P5P. In the presence of the P2Y2 signaling and ADP bioavailability Previously we have observed that cyclic nucleotide pathways, particularly those activated through NO, also act through a similar mechanism and adhesion is regulated through reduced TxAsustained signaling, which takes places under static conditions. Thus, the inhibitory actions of Pred, may not be potent when competing with sustained activatory signaling, but under flow its inhibitory actions are magnified as it is only competing with transient signaling, enabling Pred to have a profound effect on thrombus formation.The adhesion and spreading of platelets on collagen are a prerequisite for subsequent thrombus formation, which is also known to require secondary signaling through ADP 12 providing guidance for our follow up studies. In any case, our current observations are highly relevant to cardiovascular and inflammatory clinical settings and set the basis for further investigation aiming to detail Pred mechanism of action. The last point could be particularly true in view of the fact that Pred is the most widely used GC in the clinical environment.In conclusion, Pred can inhibit platelet adhesion, spreading, aggregation, thrombus formation, and their interaction with monocytes, through a novel mechanism that targets secondary signaling. Initial evidence seems to point to the P2Y


B. tropicalis extract IgE antibodies (α-BtE IgE) were divided into two groups, according to the presence or absence of skin reactivity to B. tropicalis extract (BtE). The following parameters were investigated: total IgE levels; α-BtE IgE levels; an arbitrary α-BtE IgE/total IgE ratio; the proportion of carbohydrate-reactive α-BtE IgE; the proportion of α-BtE IgE that reacted with Ascaris lumbricoides extract (AlE); the production of IL-10 by BtE- and AlE-stimulated peripheral blood cells (PBMC).Two conditions are used as markers of atopy: the presence of circulating anti-allergen IgE antibodies and the presence of positive skin prick test (SPT) reactions to allergenic extracts. The correlation between these conditions is not absolute. This study aimed at investigating immunological parameters that may mediate this lack of correlation. Individuals whose sera contained anti--BtE IgE was significantly higher in the SPT-positive group (SPT+). A large overlap of α-BtE IgE levels was found in individuals of both groups, indicating that these levels alone cannot account for the differences in SPT outcome. Individuals of the two groups did not differ, statistically, in the proportion of α-BtE IgE that reacted with carbohydrate and in the production of IL-10 by BtE- and AlE-stimulated PBMC. Both groups had part of α-BtE IgE activity absorbed out by AlE, indicating the existence of cross-reactive IgE antibodies. However, the α-BtE IgE from the SPT-negative individuals (SPT-) was more absorbed with AlE than the α-BtE IgE from the SPT+ individuals. This finding may be ascribed to avidity differences of the α-BtE IgE that is present in the two groups of individuals, and could occur if at least part of the α-BtE IgE from the SPT- individuals were elicited by A. lumbricoides infection.Total IgE levels were similar in the two groups, but αBtE IgE avidities  may play a role in the down-modulation of type-I hypersensitivity reaction against aeroallergens described in helminth-infected individuals.The present results suggest that a low ratio of specific IgE to total IgE levels , and differences in α- The IL-10 concentrations in cultures with non-stimulated blood cells and with blood cells that were stimulated by BtE (40 μg/mL), AlE (10 μg/mL), bacillus Calmette-Guérin (BCG) lysate  or pokeweed mitogen , were measured using commercially available anti - IL-10 antibodies and a standard curve constructed with recombinant IL-10, by sandwich ELISA, following manufacturer's instructions . The detection range was 31.25 to 500.00 pg/mL.Venous blood samples were cultured at a dilution of 1:4 in RPMI  containing 10 mM glutamine  and 100 μg/mL of gentamicin . The cultures were performed within 6 hours of the blood collection, in a humidified atmosphere, with 5% COB. tropicalis and A. lumbricoides antigens was determined by a competitive ELISA. Briefly, different concentrations (0.3 to 300 μg/mL) of AlE, or the same volume of PBS, were incubated with the sera diluted at 1:2.5 overnight at 4°C. α-BtE IgE were semi-quantified in these treated sera in a final dilution of 1:5, as described above. As a control for the specificity of the reduction in α-BtE IgE levels, the concentration of total IgE was determined in the same sera. The proportions of α-BtE IgE and total IgE levels that were reduced by incubation with AlE were calculated comparing the absorbance obtained in control sera (sera not pre-incubated with AlE) and in sera pre-incubated with AlE. Results were expressed in percentage of reduction, calculated as follows: percentage of reduction in α-BtE IgE or total IgE level = [1 - (mean OD detected in the assay of duplicates of serum that had been pre-incubated with AlE/mean OD detected in the assay of untreated serum duplicates)] × 100.The presence of IgE antibodies that cross-reacted with -BtE IgE level and the average diameter of SPT reactivity was calculated. A p value equal to or less than 0.05 was considered statistically significant.All statistical analyses were performed using the SPSS Version 13.0 program . The Shapiro-Wilk test was used for verification of the data normality. The statistical significance of differences between the groups was assessed by the Mann-Whitney's U test. The Spearman's correlation between serum αBtE: B. tropicalis extract; α-BtE IgE: Anti-B. tropicalis extract IgE antibodies; AlE: Ascaris lumbricoides extract; IL-10: Interleukin 10SPT: Skin prick test; JCMP conducted the experiments under the guidance of NMAN and drafted the manuscript. SBJ helped in the design and in the conduction of the experiments. RVV performed all statistical analysis and reviewed the manuscript. MLB coordinated the field studies. LCPC helped in the design of the experiments and carefully reviewed the manuscript. NMAN conceived and designed the study and coordinated the research team. All authors read and approved the manuscript.


In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times.  Precision genome engineering requires highly specific nucleases that, even within the context of a complex genome, cleaves only a single site to prevent genotoxic off-target activity. So far, a variety of different architectures has been used to generate such highly specific nucleases -5. Althoin vivo by a genome-wide integration site analysis that mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or ILRG genes dATP  compared to the unaddressed site (-P-), radioactively labelled PCR fragments were generated using [α32P]dATP . 20 nM sE. coli strain XL10Gold (Stratagene) using 100 ng DNA for the transfection and 100 µl of the suspension per agar plate. The viability factor is defined by the ratio of the viable clones of E. coli cultures either expressing or not expressing the TALE-PvuII variants . The assay was repeated with the variants .ctcgccgaaaatgacccagag), 200 mM dNTPs, and 1 U of Phusion high fidelity DNA polymerase for 30 cycles. PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs). HEK293T cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). Cells were seeded in 6-well plates at a density of 500,000 cells/well. After 24 h, cells were transfected using polyethylenimin (PEI) as described before  with 1 µNuclease-associated toxicity was determined basically as previously described . BrieflyThe goal of our study was to generate a TALE-PvuII fusion protein that cleaves DNA only at a composite site consisting of the TALE target site and the PvuII recognition site (addressed site) but not at any other PvuII site (unaddressed site). For this purpose, trimmed versions of AvrBs3 , a well T46G or PvuIIT46G and variants thereof). The fusion proteins were expressed in E. coli, purified to homogeneity and tested for their DNA-cleavage activity towards both addressed and unaddressed PvuII sites. The ratio between these two activities is reflecting the specificity of the fusion protein. Two different types of substrates were used, plasmids and PCR products, the latter also radioactively labelled to quantify target cleavage specificity.Altogether, six different fusion proteins with PvuII and scPvuII, which all carry the T46G substitution that reduces star activity , were prAs the length of the linker between the binding and cleavage modules plays an important role for activity and specificity of fusion proteins ,54, we hT46G did not cleave unaddressed PvuII sites in detectable amounts even at a 10-fold excess of enzyme (80 nM) over substrate (8 nM). The scPvuIIT46G variant was somewhat less specific as compared to the PvuIIT46G variant  and a non-specific substrate (32 nM PCR fragment with an unaddressed PvuII site). As shown in For a quantitative determination of the specificity of AvrBs3-28-L-PvuIIT46G for an addressed over an unaddressed site, radioactively labelled PCR fragments were used. T46G for an addressed over an unaddressed site was >34,000-fold  was analyzed. We were interested to know whether AvrBs3-28-L-PvuIIT46G would also cleave a target sequence comprising an AvrBs4 recognition site. AvrBs4 has the same overall architecture as AvrBs3 but its DNA target sequence differs in 8 out of 19 positions from the AvrBs3 site. So far, only the specificity of AvrBs3-28-L-PvuIIT46G. The experiments reported above were all carried out with a “tripartite” substrate that contains a central PvuII site and two flanking AvrBs3 target sites (T3-6bp-P-6bp-T3). We wondered, whether a “bipartite” substrate that lacks one of the two flanking AvrBs3 target sites (T3-6bp-P) would also be cleaved by AvrBs3-28-L-PvuIIT46G fusion protein , but also for AvrBs3-28-PvuIIT46G (28-aa linker), which has a shorter linker between the AvrBs3 repeat array and PvuII module. AvrBs3-L-PvuIIT46G (16-aa linker) with the shortest linker, however, preferred the substrate with the 2-bp linker over the one with the 6-bp linker. The different linkers had no influence on the specificity of the fusion protein (data not shown).It cannot be precisely predicted by modeling of the TALE-PvuII protein , what thE. coli before testing their activity in human cells. In the viability assay, E. coli cells not expressing the PvuII DNA-methyltransferase (M.PvuII) are challenged with the PvuII fusion proteins. Clones growing in the absence of M.PvuII and in the presence of TALE-PvuII are picked. Some of them are subjected to further analysis: the sequence of the open reading frame coding for the TALE-PvuII protein is determined and the activity of the protein analyzed in vitro. The most active ones are then subjected to a cellular plasmid cleavage assay: in the present study G53R, A92T, G135W. As shown in T46G;G53R, AvrBs3-28-L-PvuIIT46G;A92T and AvrBs3-28-L-PvuIIT46G;G135W had similar activity in human cells. Nuclease-associated cytotoxicity was determined by co-transfecting an mCherry expression plasmid and comparing the decrease of mCherry positive cells to a control nuclease [We routinely check the activity of our PvuII fusion proteins in a viability assay in nuclease . Figure nuclease .Genome editing with engineered nucleases was chosen as Method of the Year 2011 by the editors of Nature Methods, as it is a powerful tool for studying biological processes . In prin2+ ions, cleaves the DNA downstream of the recognition site, 9 and 13 nucleotides away in the top and bottom strand, respectively. (ii.) FokI binds to its recognition site as a monomer and recruits another FokI monomer free in solution forming a dimer which cleaves the DNA in the presence of Mg2+ ions. (iii.) On a DNA substrate with two (or more) recognition sites, the active dimer can be formed from two FokI monomers each bound to a recognition site, which means that the DNA between the two recognition sites is looped out [2+ and is considerably faster on a substrate with two (or more) recognition sites than on a substrate with one recognition site [In vivo cleavage sites were identified that resembled the intended target site but could not be predicted in silico. It must be emphasized that off-target cleavage is likely to also be a problem with other engineered nucleases that use the catalytic domain of FokI as DNA-cleavage module, such as TALENs.The most widely used programmable nucleases are ZFNs and TALENs followed by the recently introduced CRISPR/Cas-based nucleases . ZFNs anoped out ,72; agaiion site . This woion site  and the ion site , and thuion site ,46,75. Oion site  and Pattion site . In vivoCAGCTG, is added to the TALE protein. The challenge of designing TALE-PvuII fusion proteins as highly specific programmable nucleases is to take precautions that a PvuII site that is not addressed by a TALE protein recognition site is not attacked by the PvuII module in the TALE-PvuII fusion protein. There are in principle two different strategies to achieve this, as shown for ZF-PvuII [After we had recently shown that in ZFNs the catalytic domain of FokI can be replaced by PvuII , we now ZF-PvuII  and I-ScZF-PvuII  fusion pPvuII is a homodimeric protein; this means that also the TALE-PvuII fusion proteins are homodimers. Because a single chain version (sc) of PvuII has been described , we alsoin vitro which can hardly be achieved in cellula. Still, this finding implies that precision genome engineering can be done by cleavage of a site consisting of one TALE target site adjacent to a PvuII site. If they are sufficiently specific, this would be an advantage over TALENs (and ZFNs) based on the catalytic domain of FokI, which require heterodimerization of two proteins with different target recognition domains. However, we have recently also engineered active obligate heterodimeric variants of TALEN-PvuII . This will allow us to target extended recognition sequences.Remarkably, the TALE-PvuII fusion protein also accepts half-sites, i.e. bipartite recognition sites, e.g. TALE-6bp-PvuII. We suggest that binding of a single AvrBs3 module to the TALE binding site of the substrate, leaving the other AvrBs3 module making non-specific contacts with the DNA, is sufficient to activate PvuII. This may be due to the rather high ratio of enzyme vs. substrate in vitro. To answer the question whether they are active in vivo, we have tested them in human cells using a cellular plasmid cleavage assay. This assay showed that all TALE-PvuII fusion proteins were active in HEK293T cells. When assaying cytotoxicity associated with expression of the TALE-PvuII fusion proteins, they exhibited almost as little toxicity as I-SceI and performed better than a toxic reference ZFN. PvuII-based TALENs (and ZFNs) require the presence of a PvuII site as part of a potential target site, which may be considered a disadvantage. However, since PvuII sites statistically occur on average within a few thousand base pairs, it should be possible to find a suitable target sites for TALE-PvuII fusion proteins.The TALE-PvuII fusion proteins that we have produced are highly active and specific in vivo for their specificity which means for off-target cleavage, as it was done for ZFNs in a genome-wide analysis [In the present paper, we demonstrated that TALENs can be produced with the restriction endonuclease PvuII as DNA-cleavage module instead of the most frequently used non-specific cleavage domain of FokI. The TALE-PvuII fusion protein does not exhibit any non-specific cleavage at unaddressed PvuII sites nor at any other site, as was demonstrated in experiments in which DNA carrying unaddressed PvuII sites was incubated with a high molar excess of the TALE-PvuII fusion protein for up to 24 hours. Under these conditions, addressed PvuII sites were cleaved within minutes. In principle, TALE-PvuII fusion proteins can be produced as homodimers or as monomers with scPvuII. The TALE-PvuII fusion proteins turned out to be active in HEK293T cells, with almost as little toxicity as I-SceI. Taken together, we suggest that TALE-PvuII fusion proteins should be considered as suitable alternatives to classical ZFNs and TALENs. Of course, highly specific nucleases, regardless of which DNA-cleavage module they contain, need to be tested analysis ,48. Finaanalysis , might bFigure S1Analysis of the final protein purification step by SDS-polyacrylamide gel electrophoresis.(PDF)Click here for additional data file.Table S1Overview of the substrates and primers used.(PDF)Click here for additional data file.


P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region  were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of  Numerous reports of vaccine failure are attributed to a mismatch between the genotype of the vaccine and the circulating target strains. This observation is congruent to the view that polyvalent vaccines protect broadly by inducing a multitude of type-specific antibodies. Polyvalent vaccines that can overcome antigenic diversity by refocusing antibody responses towards conserved functional epitopes are highly desirable. Development of an Apical Membrane Antigen-1 (AMA1) malaria vaccine has been impeded by extreme antigenic diversity in the field. We present here a solution to the AMA1 diversity problem. Antibodies against a mixture of only four naturally occurring AMA1 allelic proteins “Quadvax” inhibited invasion of red blood cells by a diverse panel of malaria parasites that represented the global diversity of AMA1 in the field. Competition experiments suggested that in addition to improving the diversity of strain-specific antibodies, the mechanism of broadened inhibition involved an increase in responses against conserved inhibitory epitopes. Monoclonal antibodies against the Quadvax inhibited invasion either by blocking the binding of AMA1 to its receptor RON2 or by blocking a crucial proteolytic processing event. Some mixtures of these antibodies were much more effective than expected and were shown to act synergistically. Together these two classes of functional invasion inhibitory epitopes can be targeted to engineer a more potent AMA1 vaccine. Plasmodium falciparum infection P. falciparum infection Despite the availability of effective drugs mortality caused by malaria remains a global health and economic concern In vitro growth inhibitory activity and AMA1-induced protection in animal models and in humans are highly strain-dependent Synthesized as an 83 kDa trans-membrane protein, native AMA1 undergoes maturation to a 66 kDa form, which then translocates to the merozoite surface To be successful, vaccines against pathogens that exhibit antigenic diversity require the inclusion of multiple components. The polio vaccine contains all three circulating serotypes, the influenza vaccine contains three seasonally prevalent serotypes whose antibodies are functionally non cross-reactive and human papilloma virus vaccine contains the four most pathogenic types. However, extreme diversity in AMA1 with over 200 prevailing haplotypes has precluded the inclusion of all AMA1 strains into a multivalent vaccine et al. showed that these populations do not clearly explain the patterns of cross-strain inhibition in growth or invasion inhibition assays (GIA) and all six populations may not be necessary for a multivalent vaccine to overcome antigenic diversity A sequence diversity based approach rationally classified AMA1 sequences using a clustering algorithm and suggested that no less than 6 populations would be required in a vaccine In view of the failure of AMA1 vaccines to induce broad protection against malaria, understanding of the structural basis of cross-strain invasion inhibition is required to make a case for developing second generation AMA1 vaccines. The aim of this study was to understand the structural basis of cross-strain invasion inhibition by mixed allele vaccine antibodies. To induce broadly inhibitory antibodies we mixed four diverse AMA1 allelic proteins to constitute the Quadvax or QV. The four allelic components of the QV  show limited cross-inhibition in a GIA 10 ELISA titers of the four monovalent vaccines, tested against their respective homologous target strains, were not different from those induced by QV . When the monovalent vaccine-induced titers were grouped together, the combined mean homologous strain titer was higher than the heterologous strain titer . To determine the antigenic breadth of the induced antibodies, individual rabbit sera were analyzed by ELISA against recombinant proteins corresponding to seven diverse AMA1 alleles . The QV in vitro , which confirmed that GIA was detecting antigenic escape in vitro . In contin vitro .et al. have shown broad inhibitory coverage against diverse strains Using a 4-Way pool of antibodies against the monovalent vaccines, given separately to rabbits using Freund's complete adjuvant, Drew 50) of heterologous strains as was required for 50% inhibition of 3D7 parasites  still showed some level of inhibition of FVO and M24 parasites, while the flow-through of anti-3D7 did not   .We also directly compared the relative inhibitory activities of the cross-reactive antibody fraction of anti-3D7 and anti-QV IgG affinity purified from pooled serum of 3 rabbits over a non-vaccine strain M24 AMA1 column . The netP. falciparum strains using the WRAIR flow-cytometric assay, inhibition across strains was significantly greater with the anti-QV IgG pool than with IgG induced by either of the two trivalent  and the bivalent vaccine (p<0.0001)  or three (3D7+FVO+HB3 and 3D7+FVO+W2mef) allelic proteins. Pooled IgG from each of the four vaccine groups were tested for inhibition of invasion against ten target parasite strains by the National Institutes of Health GIA reference laboratory using a parasite LDH based method following one invasion cycle <0.0001) . A high <0.0001) .The full-length AMA1 sequences, visualized on a dendrogram against 175 published AMA1 sequences from Asian, South American and African origin , showed P. falciparum sequences on a P. berghei AMA1 scaffold  which is known to have an identical fold to P. falciparum AMA1 T. gondii AMA1 (32% identity in domains I and II), known to have an identical fold in the core domain I+II region P. berghei and P. falciparum AMA1 possess identical folds even though their surfaces are antigenically non-cross-reactive , as defined by the disulphide bond structure To further characterize QV-induced antibodies, a panel of monoclonal antibodies (mAbs) were generated . Bindingscaffold . There i30 concentration) was about 10-fold lower for the three domain-1 mAbs, 1B10, 4E8 and 4E11  as compared to mAbs binding to other regions of AMA1  (30 = 0.08 mg/ml).QV-induced hybridoma supernatants were prescreened for cross-reactivity to the four vaccine homologous allelic proteins by ELISA and domain chimeras by dot blot (not shown). Representative mAbs against each domain, preferably those that cross-reacted with three or more allelic proteins, were expanded and tested in a GIA at 1 mg/ml against the 3D7 target strain. While some domain-1 mAbs were strain-specific and others cross-reactive, mAbs against domain-2 were exclusively strain-specific for 3D7 and mAbs against domain-3 were mostly cross-reactive . The two9 mg/ml) . HoweverP. falciparum domain-2 and 1e loops, but no reactivity to the Lin-D2 chimera containing the domain-2 loop was observed. This suggested that the epitope of the most potent domain-1 mAbs 1B10, 4E8 and 4E11 encompassed the 1e-loop (confirmed below). The moderately inhibitory domain-3 mAb 1E10 mapped to the polymorphic face  and the domain-2 loop (D2+1e chimera) hic face .Breadth of mAb recognition was tested by a dot blot against 7 AMA1 allelic proteins . The domSince mAbs 1B10, 4E8 or 4E11 bound to 3D7 AMA1 but not to M24 AMA1, we analyzed the polymorphic differences between 3D7 and M24 AMA1 and found a single difference at residue 230 within the 1e-loop. When residue 230 of the 3D7 AMA1 ectodomain displayed on phage was mutated to alanine all three mAbs showed reduced reactivity to the mutant phage compared to phage displaying wild-type 3D7 AMA1 . This co517  and at 2×IC30 concentration  (30 concentrations (1e-loop mAb mixtures in green or 1e-loop+domain-2 loop mAb mixtures in blue), the resulting inhibitions were not different from the 2×IC30 concentration of individual mAbs. However, when mAbs binding to spatially distant epitopes were mixed at their IC30 concentration (1e-loop+domain-3 mAbs in orange or domain-2 loop+domain-3 mAb in red), the average inhibitions were significantly higher than that of the 2×IC30 concentration of individual mAbs (p<0.05 corrected for multiple comparisons). The most potent inhibitory combination, mAb 1E10+4G2, was tested to confirm synergy using the “Bliss independence” equation recently used to discern synergistic antibody combinations by Williams et al.30 concentration of mAb 4G2 was mixed with a range of concentrations of mAb 1E10  . When pamAb 1E10  and syneUsing the strain-specific anti-3D7 as the reference, we conducted differential mapping of the polyclonal anti-QV inhibitory response. In a GIA against 3D7 strain, equivalent final concentration of 3D7 chimeric proteins CryD1, CryD2, CryD3, CryD1+CryD2, Cry D2+CryD3, CryD1+CryD3, CONS and POLY were added to deplete region-specific antibodies against domains-1, 2, 3, 1+2, 2+3, 1+3, conserved face and polymorphic face, respectively . The extComparing anti-3D7 and anti-QV GIA reversal showed increased levels of cross-reactive antibodies in anti-QV correlated with increased GIA reversal by chimera combinations that contained domain-3  and the overall reversal for D1+3 chimera was the highest for anti-QV . ConversA mAb competition ELISA was performed to determine the ability of anti-3D7 and anti-QV serum pool to inhibit the binding of labelled cross-reactive mAbs  to a non-vaccine strain 102-1 AMA1 . A loweret al. have suggested that overlapping strain-specific antibodies can achieve broad inhibition in vitroGiven the failure of AMA1 vaccines to protect broadly, this study investigates the molecular basis of anti-AMA1 mediated cross-strain invasion inhibition. Using a mixture of antibodies against monovalent vaccines, Drew While we know of no mechanisms or precedents that would definitively explain our finding that co-administering polymorphic allelic AMA1 proteins increases the immunogenicity of subdominant conserved epitopes, a simple hypothesis based on the observations in influenza can be advanced. During serial influenza virus infections, memory B cells specific for the more abundant strain-specific epitopes on the head of hemagglutinin (HA) are readily expanded while fewer B cells specific for the conserved stem epitopes are crowded out. When a pandemic strain infects that contains a very different HA head, the strain-specific epitopes are replaced by new epitopes but exposure to the conserved stem epitopes continue 228, K230; ∼70% of alleles). It is currently unclear why 1e-loop was the most susceptible target. As compared to the relatively flexible 1b, 1f and domain-2 loop, the loops at the other end of the hydrophobic trough, 1c and 1d/C1L and 1e, are less flexible 2041 of RON2 Defining broadly inhibitory epitopes will be critical to apply structural vaccinology approaches to improve the breadth and GIA activity of all AMA1 vaccines. The 1e-loop region (residue 225–235) was identified as the most susceptible mAb target on AMA1. The four QV alleles display the most prevalent polymorphism combination within the 1e-loop  (USDA License No. 51-R-0051). SVL animal research facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACi). The vaccine protocol (SVP10-0059) was approved by the SVL Animal Care and Use Committee. The views mentioned are that of the authors and not of the U.S. Department of the Army or Department of Defense.http://ab.inf.uni-tuebingen.de/software/dendroscope/).Full-length AMA1 sequence of 175 field isolates  and 26 cP. falciparum FVO, M24, 7G8 and 102-1 AMA1 ectodomain (residues 83–531) were codon optimized and the proteins were expressed in E. coli essentially as described previously Genes encoding 449 amino acid P. falciparum AMA1 on the P. berghei AMA1 scaffold. Chimeras were based on AMA1 residues 83Gly to 531Glu of P. falciparum 3D7 sequence (accession number XP_001348015) and P. berghei AMA1 ANKA strain sequence (XP_678057 or CAH96497). The chimeras displaying the domain1, domain2, domain3, the hydrophobic trough, the polymorphic face, conserved face and the domain2+1e loop were termed as CryD1, CryD2, CryD3, HT, POLY, CONS and D2+1e respectively P. falciparum regions . Scaffold sequences were amplified from the native P. berghei AMA1 genomic DNA using primers that overlapped the P. falciparum DNA fragments. Full-length chimeric genes were then assembled by a PCR stitch reaction using external primers. Residue boundaries 83Gly–303Cys were treated as domain-1, residues 304Arg–418Cys as domain-2 and residues 419Leu–531Glu as domain-3. Five linear domain chimeras displaying the three domains: LinD1, LinD2, LinD3, LinD1+2 & LinD2+3 were produced. The genes for the chimeric proteins were cloned in pET32 based plasmid, expressed in E. coli Tuner strain, grown in superbroth and protein expression was induced by 0.5 mM IPTG for 2 hrs. The chimeric proteins were purified using Ni-NTA affinity, refolded and the purified over an anion exchange column. Mixtures of these chimeric proteins were tested in GIA reversal assays (for example CryD1+CryD2+CryD3 or POLY+CONS) to ensure that together these combinations covered all of the inhibitory epitopes of 3D7 AMA1protein, as was evidenced by ∼100% GIA reversal  were used to design continuous surface chimeric proteins that displayed the various three dimensional structural elements of  regions . Chimerireversal . P. bergThree rabbits per group each received three doses of 100 µg AMA1 vaccine per dose emulsified in Montanide ISA720 . The Quadrivalent vaccine (Quadvax) consisted of 25 µg each 3D7, FVO, HB3 and W2mef proteins; trivalent vaccines contained 33 µg of three allelic proteins and bi-allelic vaccine contained 50 µg of two allelic proteins. Emulsification was achieved by vigorous vortexing for 10–15 min and 1 ml vaccine was administered at multiple sites, subcutaneously, on the animal's back at four week intervals. Rabbits were bled out 2 weeks after the third vaccination. Sera were heat inactivated and stored at −70°C until used for invasion inhibition assays.280. Five mg of 3D7 or M24 AMA1 protein was covalently linked to 1 ml cyanogen bromide sepharose column according to the manufacturer's instructions (GE). Anti-AMA1 IgG was bound and eluted from this affinity column as described above.Pooled sera (1.33 ml from each of the 3 rabbits within a group) was diluted 5-fold in IgG binding buffer , and passed over a 1 ml Protein G column , the column was washed and bound IgG was eluted using IgG elution buffer (Pierce), neutralized with 1M Tris HCL pH 8.0, dialyzed against PBS and quantified by measuring OD405 = 0.5. Region-specific antibody titer was defined as: (End-point titer against a domain chimera/end-point titer against the full-length 3D7 AMA1 protein)×100.ELISA protocol has been previously described All inhibition assays in duplicate wells were performed by this method, unless stated otherwise P. berghei AMA1 protein showed no inhibition reversal, while the homologous 3D7 AMA1 showed complete reversal. GIA reversal = (inhibition in presence of 2.8 µM P. berghei AMA1−inhibition in the presence of 2.8 µM test protein)/inhibition in presence of P. berghei AMA1 at 2.8 µM.Serum pool of anti-QV or anti-3D7 AMA1 were diluted to give ∼60% inhibition of the 3D7 parasite. AMA1 allelic proteins derived from 3D7, FVO, HB3, W2mef, 102-1, 7G8, or M24 strains (∼150 µg/ml or 2.8 µM) were added to selectively deplete cross-reactive antibodies. Immunologically non-reactive P. berghei AMA1−inhibition in the presence of 4 µM test chimera)/inhibition in presence of 4 µM P. berghei AMA1.Region-specific inhibitory contribution of antibodies was determined using protein chimeras to reverse anti-QV or anti-3D7 AMA1 mediated inhibition of 3D7 parasites. To ∼60% inhibitory dose of serum pool, 4 µM chimeric proteins (∼200 µg/ml) were added. Percent GIA reversal = /×100].IgGs from rabbits were purified from pooled sera using protein G columns ; the eluted fractions were dialyzed against RPMI 1640  and concentrated with centrifugal filter devices . The purified IgGs were preadsorbed with uninfected human O+ erythrocytes, sterilized by filtration through a 0.22-µm filter and heat inactivated at 56°C for 20 min before use in the assay. Late trophozoite and schizont stages of in vivo using athymic nude mice and mAbs were purified from the ascetic fluid using a Protein G column . Other mAbs used in the study were: rat mAb 4G2dc1 that recognizes a cross-reactive conformational epitope Monoclonal antibodies were developed by immunizing 3 mice multiple times with QV using the Precision Antibody's immunization technology . Target specific antibody titers were determined by ELISA and a fusion was performed with B-cells from splenocytes and lymphocytes. The myeloma partner was derived from the cell line P3X63Ag8.653. Fused cells were selected in a HAT media and grown from a single cell. Hybridoma clone supernatants were screened by ELISA for reactivity to the four allelic proteins 3D7, FVO, HB3 and W2mef AMA1. Out of the total 38 clones obtained representative mAbs against all three domains were picked, preferably if they reacted to multiple allelic proteins. Selected mAbs were expanded 1 µg of the AMA1 proteins under non-reducing conditions was electrophoretically transferred to a nitrocellulose membrane immune-blots were performed essentially as described previously 450 was recorded.Two µg/ml of RON2 peptide labeled with biotin at the N-terminus was immobilized on streptavidin plates , followed incubation in BLOTTO Blocking Buffer  for 1 hr. An equal volume of 0.0015 µg/ml of 3D7 AMA1 and decreasing concentrations (150 µg/ml to 0.15 µg/ml) of mAbs  were added to the well. After 1 hr incubation the wells were washed and 1∶5000 dilution of rabbit anti-AMA1 polyclonal serum was used to detect bound AMA1. ABTS substrate was added to the well after 1 hr incubation OD230 to Ala was carried out by the technique of splice overlap extension. PCR was used to amplify overlapped DNA fragments from the 3D7 AMA1 ectodomain template in PHENH6 plasmid such that both PCR fragments contained the K230 mutation. The splice overlapped PCR was performed using PHENH6 forward and reverse primers that incorporate the flanking region from PHENH6. Preparation of phage clones and phage ELISA against the mAbs was essentially as described previously Mutagenesis of LysThe processing inhibition assay on 3D7 strain parasites was performed essentially as described previously at 200 µg/ml final mAb concentration 405, were added and incubated for 1 hr. Plates were washed and ABTS substrate was added. After 1 hr incubation, stop solution was added and plates were allowed to sit for 5 min before the OD405 was recorded.MAbs were labeled using Lightning-Link® Horseradish Peroxidase kit . AMA1 protein of 102-1 strain was coated on ELISA plates (100 ng/well). Wells were blocked with 1% casein blocker for 2 hrs, washed with PBS-Tween and then 50 µl individual rabbit serum dilutions were added to the wells for 1 hr. To the same well, 50 µl of HRP-labeled mAbs, diluted to yield 1–1.5 ODpost hoc testing, when comparing all groups to the QV group. Analysis of Variance (ANOVA) was used if the rabbit data were pooled and p values adjusted using either Dunnett's method  or Tukey's method . When two groups of data were to be compared a 2-sample t-test was used. ELISA data was log10 transformed to obtain close to normal distribution before statistical analysis. Group analysis was conducted using SAS software version 9.3. Correlation between sequence distance and GIA was analyzed by linear regression. For synergy analysis, GIA over a range of 1E10 concentrations (0–4 mg/ml) was measured against 3D7 parasites in the presence or absence of an IC30 concentration of mAb 4G2 (1.8 mg/ml). The observed inhibition by the mixture was compared to that predicted by an equation for Bliss independence as was applied to GIA by Williams et al.additive = [1−(1−% GIA1E10)*(1−%GIA4G2 at its IC30)]. GIA dose response curves were used to predict the concentration of antibody that would give either 50% or 30% inhibition using non-linear curve function within Graphpad PrismR software.To compare differences in GIA responses against different strains the data from individual rabbits in each of the vaccine group (explanatory variable) from 3 independent experiments (repeat component) was analyzed by Repeat Measures Multivariate Analysis of Variance (MANOVA). For ELISA the individual rabbit data was analyzed by MANOVA to discern differences between groups. Dunnett's method is used to adjust p-values for the Figure S1Inverse relationship between sequence distance and GIA. Mean invasion inhibition data from 3 rabbits was plotted against the amino acid difference between the vaccine and each of the 8 target strains (sequence distance), tested by the WRAIR GIA method. For anti-QV, the amino acid difference of a heterologous strain was against the most similar QV allele. Lines of best fit (solid black), error range (dotted black and pink), P value indicating if the slope is significantly non-zero, R2 and 95% CI of the slope (m) are shown.(TIF)Click here for additional data file.Figure S250.GIA with affinity purified antibodies used to calculate the IC Anti-3D7 and anti-Quadvax IgG were affinity purified over a 3D7 AMA1 column. Bound/eluted  or the flow-through fractions  were adjusted to equivalent IgG concentration and tested against 3D7, FVO and M24 parasite strains. Mean+s.e.m. of 3 independent experiments against 3D7 and FVO strains and one experiment in triplicate against the M24 strain are plotted.(TIF)Click here for additional data file.Figure S3Reversal of GIA activity using diverse allelic proteins. Anti-3D7 or anti-QV serum pools were diluted to yield ∼60% inhibition of 3D7 parasite strain. Seven AMA1 allelic proteins  were added to the invasion inhibition assay (2.8 µM or ∼150 µg/ml) to compete out the availability of cross-reacting antibodies. Bars are mean+s.e.m of three experiments. Percent reversal of inhibition = (inhibition in presence of P. berghei AMA1−inhibition in the presence of the test antigen)/inhibition in presence of P. berghei AMA1.(TIF)Click here for additional data file.Figure S4GIA conducted by the NIH reference laboratory using QV, trivalent and bivalent vaccine-induced IgG. (A) Total IgG at 2.5 mg/ml pooled from 3 rabbits vaccinated with QV or the two trivalent vaccines (3D7+FVO+W2mef and 3D7+FVO+HB3) or a bivalent vaccine (3D7+FVO). IgG against P. berghei AMA1 was used as the control. Lines are median inhibition across-strains. (B) Dose response of invasion inhibition by anti-QV IgG pools from two independently vaccinated groups of three rabbits. The concentration of total IgG that gave 50% invasion inhibition (IC50) against the 3D7 parasite strain was 0.16 and 0.19 mg/ml for QV pool-1 and QV pool-2 respectively.(TIF)Click here for additional data file.Figure S5Chimeras used in GIA reversal assays and mapping of conformational mAb epitopes. (A) Contiguous surface residues of P. falciparum 3D7 AMA1 (color) were grafted onto a scaffold of rodent malaria parasite P. berghei AMA1 (gray residues). P. falciparum AMA1 structural elements representing three domains as defined by the crystal structure , the polymorphic and conserved face (chimeras POLY and CONS), residues at the rim of the hydrophobic trough (HT) and the domain-2 loop together with the neighboring 1e-loop (chimera D2+1e) were displayed. Three linear domains as defined by the disulphide bonded pattern were also displayed . (B) The genes for the chimeras were expressed and proteins were purified as shown on the non-reduced coomasie blue gel. The P. falciparum 3D7 AMA1 and P. berghei AMA1 proteins (3D7 and PbAMA) were also run on this gel. (C) Reversal of 3D7 parasite invasion inhibition, mediated by a pool of three 3D7 AMA1 vaccinated rabbit sera using 3D7 AMA1 based chimeras , added individually or in combination, at 4 µM final concentration. Data are mean of three independent experiments.(TIF)Click here for additional data file.Figure S6Dose response GIA. Serial dilution of monoclonal antibodies were tested against the 3D7 parasite strain and 30% inhibitory mAb concentration (IC30) was calculated. Polyclonal anti-3D7 AMA1 IgG that was affinity purified over a 3D7 AMA1 affinity column was also tested. Results are from a single experiment.(TIF)Click here for additional data file.Figure S7Binding of 1e-loop mAbs to phage-displayed mutant AMA1. Residue 230 (within loop-1e) was switched from K to A, on a phage expressing the 3D7 AMA1 ectodomain. Binding of the mAbs against wild-type (wt) and mutant phage (K230A) was measured as OD450 (error bar is the range of duplicate wells). MAbs 5G8 , 4G2 and 1F9 bind to regions outside the 1e-loop were used as negative controls.(TIF)Click here for additional data file.Figure S8Region-specific ELISA. Polyclonal serum affinity purified over an M24 affinity column was tested in the chimera ELISA. Region-specific titers  were calculated as the ratio of end-point titers against a 3D7 chimera relative to the end-point titer against 3D7 AMA1 protein.(TIF)Click here for additional data file.Table S1The list of 201 isolates whose AMA1 sequences were used to create the dendrogram in . The strains highlighted in yellow were tested in invasion inhibition assays and found to be susceptible to QV antibodies. AMA1 field isolate sequences were obtained from Genbank (TIF)Click here for additional data file.Table S2Sequence of protein chimeras. An alignment of P. berghei ANKA strain AMA1 is shown along with the residues that were switched to P. falciparum 3D7 sequence (boxed in gray). The boundaries of loops and domains are shown.(DOCX)Click here for additional data file.