Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs. They were used for all experiments after 5 to 10 passages. Recombinant human TNF-α was obtained from R&D systems , and Akt inhibitor X, a specific pharmacological inhibitor of Akt, was obtained from Merck Millipore  Western blotting was performed as described previously TF activity was analyzed at the luminal surface of adherent endothelial cells and in whole cell lysates of ECs using a colorimetric assay , as previously described MT1-MMP activity was performed as described previously MT1-MMP and Akt expression was silenced by siRNA as previously described Quantitative RT-PCR was performed as previously described Akt and FoxO1 activation was determined by Western blotting using an anti-phospho-Akt antibody and an anti-phospho-FoxO1 antibody . The anti-phospho-Akt antibody detects endogenous levels of Akt1 only when phosphorylated at Ser473, and also recognizes Akt2 and Akt3. Anti-Akt antibody  was used as a loading control. We used an anti-phospho-NF-kB p65 antibody  and an anti-active/cleaved caspase-3 antibody . Anti-caspase-3 antibody  was used as a loading control.Membrane and cytosol fractions of ECs were performed as described previously A mouse monoclonal antibody available to phospho-Akt (Biolabs) was used for immunostaining. We cultured the ECs on chamber slides, and then treated them with or without TNF-α. The ECs were fixed with 4% paraformaldehyde followed by permeabilization with 0.1% Triton in PBS, and then incubated with the antibodies to phospho-Akt and MT1-MMP (Chemicon) diluted 1∶100 in PBS with 5% BSA at room temperature for 60 min. After washing, goat-anti-rabbit Alexa 594 and donkey-anti-mouse Alexa 488 (Life Technologies) were reacted for 60 min. The stained cells in the field were analyzed using a fluorescence microscope and x20 and x10 magnification with scale bars representing 50 µm .2O2 in methanol for 15 minutes at room temperature. After incubation with 5% BSA in PBS for 1 hour at room temperature to block non-specific binding, the cells were incubated with HRP-labelled goat anti-biotin (1∶100) diluted with 5% BSA in PBS for 2 hours at room temperature, and then washed in PBS. The sites of HRP were visualized by H2O2 and DAB for 10 minutes. The cells were visualized under a microscope and x100 magnification with scale bars indicating 50 µm . TUNEL assay images were stored in a computer and the intensity of EC apoptosis staining was quantitatively analyzed by the NIH Image Program (ImageJ).TUNEL assay was performed to analyze DNA fragmentation in ECs undergoing apoptosis as described previously P<0.05 was considered significant. Data are expressed as means ±SD.Statistical analyses were performed using ANOVA with Scheffé's post hoc test if appropriate. A value of We first performed Western blot analysis for TF and endothelial anticoagulant TM expression in human aortic ECs in TNF-α stimulation. TM is an endothelial cell membrane glycoprotein that forms a complex with thrombin, converting thrombin from a procoagulant to an anticoagulant enzyme Next, we transfected ECs with MT1-MMP siRNA and then added TNF-α to ECs. To selectively inhibit Akt, we used Akt siRNA as well as Akt inhibitor X, a specific pharmacological inhibitor of Akt Akt phosphorylation was reduced by approximately 36% within 60 minutes in the ECs treated with 10 ng/mL TNF-α . TIMP-2 To identify molecular interaction between MT1-MMP and Akt in TNF-α stimulation, we performed immunoprecipitation studies with an initial antibody to MT1-MMP in ECs. As shown in To investigate the relationship between MT1-MMP and Akt in the mechanism(s) of TNF-α-induced endothelial apoptosis, we performed terminal deoxy (d)-UTP nick end labelling (TUNEL) staining. TUNEL staining showed that 48 hours' treatment with TNF-α induced endothelial cell apoptosis, which was blocked by the silencing of MT1-MMP with siRNA . QuantitIn the present study, we demonstrated that MT1-MMP/Akt signaling axis modulates FoxO1 and NF-κB phosphorylation in TNF-α-stimulated procoagulant activity and endothelial apoptosis in ECs.Endothelial cells, which maintain anti-inflammatory and anti-thrombotic surfaces, orchestrate inflammatory and thrombotic responses in the vascular wall The membrane-associated MMP, MT1-MMP regulates various cellular functions not only as a pericellular protease but also as a signaling molecule in both physiological and pathological settings . A chemical inhibitor of MT1-MMP, TIMP-2 inhibited TNF-α-increased MT1-MMP activity in ECs . TIMP-2 The Akt-dependent pathway has been reported to play an important role in vascular hemostasis, including the expression and activity of TF in vascular cells Our previous studies reported that a complex formed with MT1-MMP and LOX-1 plays an integral role in ox-LDL-mediated signaling pathways in ECs, and that MT1-MMP regulates the activation signaling pathways via a receptor for advanced glycation end products in cultured smooth muscle cells MT1-MMP possesses transmembrane and cytoplasmic domains in addition to extracellular domains, and the cytoplasmic domain of MT1-MMP has an important role in cell invasion and proliferation, indicating that MT1-MMP functions as a signaling molecule We further investigated the integral role of MT1-MMP for the mechanism(s) of TNF-α-induced endothelial apoptosis through Akt-mediated a forehead transcription factor FoxO1 signaling. Our TUNEL staining data show that 48 hours' treatment with TNF-α induced endothelial cell apoptosis, which was blocked by siRNA-mediated suppression of MT1-MMP . TNF-α iThe present study offers new insights into the action of MT1-MMP on Akt-dependent regulation of FoxO1 activation in TNF-α-stimulated ECs. We provide the first evidence that MT1-MMP may play an integral role in TNF-α-induced signaling pathways in cooperation with Akt for the modulation of procoagulant activity and apoptosis of ECs. TNF-α-induced inflammatory responses and procoagulant activity have been implicated in the pathogenesis of vascular diseases including atherosclerosis and myocardial infarction Our study suggests that inhibition of the MT1-MMP/Akt signaling pathway may be an attractive strategy for treating endothelial disordered hemostasis in the development of vascular diseases linked to TNF-α-induced inflammation.


Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin KDOI:http://dx.doi.org/10.7554/eLife.05560.001 2, but it is not clear how this protein also helps to control cholesterol levels in the cornea.People with a rare genetic disorder called ‘Schnyder corneal dystrophy’ gradually lose their vision, because their corneas become increasingly cloudy. This cloudiness is caused by a build-up of excessive amounts of cholesterol, and the disorder itself is caused by mutations in a gene that encodes a protein called UBIAD1. Researchers have previously discovered that the UBIAD1 protein is involved in making vitamin KAn enzyme called HMG CoA reductase makes a molecule that is used to make cholesterol and many other similar sterol molecules. A ‘feedback loop’ operates in cells to control the amount of the reductase and prevent cholesterol from becoming too high or too low. Sterol molecules, together with another molecule called geranylgeraniol, participate in this feedback loop by promoting the destruction of the reductase enzyme. Here, Schumacher et al. reveal a link between UBIAD1 and the reductase that may explain how UBIAD1 contributes to the production of excess cholesterol in patients with Schnyder corneal dystrophy.The experiments show that, in the presence of sterol molecules, UBIAD1 can bind to HMG CoA reductase to protect the reductase from being destroyed by other proteins. Geranylgeraniol—which stops the UBIAD1 protein from binding to the enzyme—is required to completely destroy the reductase enzyme. However, when UBIAD1 is missing, the reductase enzyme is destroyed even in the absence of geranylgeraniol.Furthermore, the experiments show that the genetic mutations linked to Schnyder corneal dystrophy lead to the production of versions of the UBIAD1 protein that bind to the reductase enzyme even when geranylgeraniol molecules are present. This prevents the normal breakdown of the reductase enzyme, which could lead to the build up of cholesterol in the cornea of individuals with the disorder.Schumacher et al.'s findings show that the UBIAD1 protein helps to control the levels of cholesterol in cells by protecting the HMG CoA reductase enzyme from destruction. These findings may aid the development of new therapies to lower cholesterol levels in cells, which may help patients with Schnyder's corneal dystrophy and other conditions caused by high cholesterol levels.DOI:http://dx.doi.org/10.7554/eLife.05560.002 SCD  is a rare autosomal dominant eye disease that is characterized by bilateral opacification of the cornea . The cli3) derived from plant-derived phylloquinone (vitamin K1), generating MK-4   . UBIAD1 amin K2)  (Nakagawamin K2) . It has amin K2) .10.7554/To date, 24 UBIAD1 mutations have been identified in ∼50 SCD families . SeveralIn mammalian cells, the ER (endoplasmic reticulum)-localized reductase catalyzes reduction of HMG CoA to mevalonate . This reThe ERAD of reductase is initiated by intracellular accumulation of sterols, which causes the enzyme to bind to ER membrane proteins called Insig-1 and Insig-2 , 2003b . Insig bIn the current studies, we use proximity-dependent biotinylation  to identTo identify proteins that associate with reductase, we employed a novel chimeric protein composed of the membrane domain of reductase (amino acids 1–346) fused to the promiscuous biotin ligase BirA*, which features proximity-dependent biotinylation of the chimera's near-neighbor proteins . Cells sUBIAD1 belongs to the UbiA superfamily of prenyltransferases . SimilarWe next used RNA interference (RNAi) to examine a role for Insigs in the 25-HC-induced binding of reductase to UBIAD1. SV-589 cells were transfected with duplexes of small interfering RNAs (siRNAs) against mRNAs encoding green fluorescent protein (GFP), which is not expressed in the cells, or Insig-1 and Insig-2. Following transfection, the cells were depleted of sterols, incubated in the absence or presence of 25-HC, and lysed for subsequent immunoprecipitation with polyclonal anti-reductase. Immunoblotting of precipitated proteins revealed that 25-HC stimulated co-precipitation of both UBIAD1 and Insig-1 with reductase in SV-589 cells transfected with control GFP siRNA  (lanes 5–8). − cells  were briefly incubated with 25-HC in the absence or presence of geranylgeraniol. The cells were then harvested, lysed, and subjected to anti-reductase immunoprecipitation. The results show that wild type UBIAD1 co-precipitated with reductase in the 25-HC-treated cells ; horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit IgGs (affinity-purified) were from Jackson ImmunoResearch Laboratories ; digitonin was from Calbiochem ; geranylgeraniol and farnesol from Sigma–Aldrich  and Santa Cruz Biotechnology ; biotin was obtained from Sigma–Aldrich ; and 25-hydroxycholesterol from Steraloids . Apomine was synthesized by the Core Medicinal Chemistry laboratory at the University of Texas Southwestern Medical Center. Other reagents, including new born calf lipoprotein-poor serum , sodium compactin, sodium mevalonate, and stock solutions of digitonin were prepared or obtained from previously described sources .Escherichia coli BirA  that exhibits promiscuous biotin ligase activity (http://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf) Guide RNA sequences unique to human UBIAD1 were selected from a published list (http://arep.med.harvard.edu/human_crispr).The previously described expression plasmids pCMV-HMG-Red(TM1-8)-T7 and pCMV-HMG-Red-T7 encode the membrane domain (amino acids 1–346) and full-length (amino acids 1–887) forms, respectively, of hamster reductase followed by three tandem copies of the T7 epitope tag under transcriptional control of the cytomegalovirus (CMV) promoter , 2003b. activity . The cDNactivity . (See hthed list . (See ht2. Human embryonic kidney (HEK)-293S/pHMG-Red(TM1-8)-BirA* cells were generated as follows: on day 0, HEK-293S cells were set up at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 6 µg/dish of pCMV-HSV-HMG-Red(TM1-8)-BirA* using FuGENE6 transfection reagent  as previously described  were generated as follows: on day 0, SV589 cells were set up at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 5 µg/dish each of hCas9, hUBIAD1-gRNA12 and hUBIAD1-gRNA19 using FuGENE6 transfection reagent as described above. On day 2 and 3 the transfection above was repeated. On day 4 cell clones were isolated using serial dilution in 96-well plates. Clones were screened for the absence of UBIAD1 by immunoblot analysis using mouse monoclonal IgG-H8 and rabbit polyclonal antibodies against human UBIAD1 . A homozygous 113 bp deletion/frameshift mutation (starting at codon 60) of UBIAD1 was identified by PCR and sequencing of the PCR products by standard techniques. UBIAD1−/pcDNA3.1, UBIAD1−/pMyc-UBIAD1, and UBIAD1−/pMyc-UBIAD1 (N102S) are UBIAD1− cells stably transfected with pcDNA3.1, pCMV-Myc-UBIAD1, and pCMV-Myc-UBIAD1 (N102S), respectively. These cells were generated as follows: on day 0, UBIAD1−cells were set up at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 6 µg/dish of pcDNA3.1, pCMV-Myc-UBIAD1, or pCMV-Myc-UBIAD1 (N102S) using FuGENE6 transfection reagent as described above. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium was added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Cells from single colonies of cells expressing moderate levels of Myc-UBIAD1 were selected and monolayers were maintained in medium B (medium A supplemented with 10% FCS and 700 µg/ml G418) at 37°C, 5% CO2.UBIAD1-deficient cells  NC-LPPS, 10 µM compactin, and 50 µM mevalonate. After 16 hr at 37°C, the cells were treated for an additional 6 hr in sterol-depleting medium containing 50 µM biotin and 1 µg/ml 25-HC. Cells were subsequently harvested, washed with phosphate-buffered saline (PBS), and resuspended in 0.3 ml/plate of buffer containing 10 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 5 mM sodium EDTA, 5 mM sodium EGTA, and 250 mM sucrose supplemented with a protease inhibitor cocktail consisting of 5 mM dithiothreitol, 0.1 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Pefabloc, 5 µg/ml pepstatin A, 25 µg/ml ALLN, and 10 µg/ml aprotinin. The cell suspension was passed through a 22.5 gauge needle 25 times and centrifuged at 1000×g for 10 min at 4°C. The resultant post-nuclear supernatant was subjected to an additional round of centrifugation at 100,000×g for 30 min at 4°C. The membrane pellet of this spin was resuspended in 0.3 ml/10 plates of solubilization buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 8 M urea, and 2% SDS and subjected to dounce homogenization. The solubilized membrane pellet was then precleared with 1 ml/10 plate of agarose beads  by rotation for 6–16 hr at 4°C. After centrifugation at 300×g for 5 min at 4°C, the supernatants were mixed with 0.8 ml of streptavidin-coupled agarose beads  and rotated overnight at 4°C. The beads were collected by centrifugation at 2500×g for 1.5 min at 4°C, and washed three times with three volumes of solubilization buffer diluted 10-fold in Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 5 mM EGTA. The streptavidin-coupled beads were then eluted three times through incubation for 10 min at 98°C in 1 volume of buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 5 mM biotin, 10% (vol/vol) dimethylsulfoxide, 2% SDS, and 0.4 M urea. The elutions were pooled and precipitated with 4 vol of ice-cold acetone at −20°C for 1 hr. Precipitated material was collected by centrifugation at 16,000 at 4°C and dissolved in a mixture consisting of 10 µl of buffer containing 10 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1% SDS, 1 mM EDTA, and 1 mM EGTA; 20 µl of buffer containing 62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M urea, 10% glycerol, and 100 mM dithiothreitol; 10 µl 4× SDS-PAGE loading buffer. The samples were incubated for 20 min at 37°C prior to SDS-PAGE. The gels were subjected to staining with colloidal coomassie as described (HEK-293S/pHMG-Red(TM1-8)-BirA* cells were set up on day 0 at a density of 3 × 10escribed  and threescribed  were excRNA interference (RNAi) was performed as previously described with minor modifications . Duplexeg for 10 min at 4°C. The detergent-solubilized material was then subjected to immunoprecipitation as described previously . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase: Implications for Schnyder Corneal Dystrophy” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.eLife and open up several future areas for study. Nevertheless, some aspects of the study require clarification and a modest amount of new experimentation to fully support the authors' conclusions. The following three points were deemed essential to the central conclusions, while the 'minor comments' below are more peripheral.This study presents a series of experiments that identifies UBIAD1 as an HMG CoA reductase interactor that regulates its degradation in a geranylgeraniol (GGOH)-dependent manner. Dominant mutations in UBIAD1 that cause Schnyder corneal dystrophy (SCD) disrupt this regulation. The work resolves a long-standing question of how GGOH enhances the sterol-dependent degradation of mammalian HMGR, which has been a consistent yet mysterious feature of HMGR degradation as studied by this group for a number of years. The results make sense, and lead to a number of hypotheses and perhaps distinct therapeutic approaches for Schnyder Corneal Dystrophy. The topic and findings should be of interest to the readers of 1) 2) In 3) Due perhaps to the relatively modest effects of UBIAD on reductase, there appears to be a fair amount of sample-to-sample variability . While consistent trends from multiple different approaches make an overall convincing case, certain individual experiments were less convincing to some reviewers. In particular, the key result of Minor comments:1) The reductase-BirA* strategy for identifying interacting partners should be documented. The approach is clearly useful for finding membrane protein interactors, and it is likely that readers will want to apply this to other systems if it is robust. Unless there is a compelling reason otherwise, I recommend including a figure and appropriate supplemental table documenting the interactors identified and where among these UBIAD1 ranked. It would be worth noting if known interactors  were observed as expected.2) In 3) In many of the figures, relative reductase levels in the starting lysate and after the IP are different. As one example, lanes 1-3 in 4) The authors discuss a role for UBIAD1 after Insig binding and ubiquitination. However, UBIAD1 knockdown seems to substantially reduce reudctase-Insig interaction . Please  1)is the central result, and it is worth making it completely convincing by presenting a time course of reductase degradation in wild type versus UBIAD knockout cells. Ideally, this would be done by radiolabeled pulse-chase, but even a western blot at different times after 25-HC or Apomine treatment would provide a clear picture of what is happening. This is important because the steady state levels of reductase are different in wild type and knockout cells, complicating the comparison. Thus, a carefully controlled and quantified time course would help to directly show different rates of degradation.We thank the reviewers for this suggestion. A new experiment  is now i2) In, the authors analyze the levels of transfected HMG membrane domain (residues 1-346) and not the endogenous protein. This is important since the effect of WT and mutant UBIAD1 on this truncated form  and on endogenous reductase  appears quite different. While the steady state levels of the truncated protein significantly increase upon UBIAD1 transfection, the endogenous protein does not seems to change much. Can the authors please explain the rationale for using the truncated membrane domain, or if possible, present data with the full-length protein?The experiment of 3) Due perhaps to the relatively modest effects of UBIAD on reductase, there appears to be a fair amount of sample-to-sample variability . While consistent trends from multiple different approaches make an overall convincing case, certain individual experiments were less convincing to some reviewers. In particular, the key result ofis not totally convincing and quantification of independent experiments would help to validate the conclusion that N102S is GGOH-insensitive.We agree with the reviewers’ assessment that the effects of geranylgeraniol on displacement of N102S UBIAD1 from reductase are modest. Thus, we have included in the revised manuscript an additional two independent experiments that demonstrate UBIAD1 (N102S) is more resistant to geranylgeraniol-mediated displacement from reductase than its wild type counterpart see . We prevMinor comments:1) The reductase-BirA* strategy for identifying interacting partners should be documented. The approach is clearly useful for finding membrane protein interactors, and it is likely that readers will want to apply this to other systems if it is robust. Unless there is a compelling reason otherwise, I recommend including a figure and appropriate supplemental table documenting the interactors identified and where among these UBIAD1 ranked. It would be worth noting if known interactors  were observed as expected.We thank the reviewers for this suggestion. We have now included in the revised manuscript the documentation of proteins identified as associated proteins of the reductase-BirA* chimera. As shown in 2) In, reductase levels increase substantially during the time course, even with 25-HC. It eventually seems to be degraded in a 25-HC-dependent manner, although the level at 240 min seems higher (in the IP sample) or slightly lower (in the lysate) relative to starting amount. Yet, in later experiments , 4 hours with 25-HC results in what appears to be ∼90% degradation. Please clarify this apparent discrepancy.In the revised manuscript, we have replaced the previous time-course experiment with a new one that measures the association of UBIAD1 with reductase. The results show that UBIAD1 appears in the reductase immunoprecipitates after 30 min of treatment and continues to co-precipitate with reductase at later times, even though total reductase is reduced by accelerated degradation.3) In many of the figures, relative reductase levels in the starting lysate and after the IP are different. As one example, lanes 1-3 inshow substantial reduction in reductase with 25-HC in lysate, but an apparent increase in the IP samples. Is this simply experimental variability or is there some other reason ? Please comment on this in the text or figure legend so a reader is not confused.We believe some of these differences may result from experimental variability or by slight differences in rates of degradation among different experiments. We attempted to eliminate this variability by blocking degradation of reductase using the proteasome inhibitor MG-132; however, as shown in 4) The authors discuss a role for UBIAD1 after Insig binding and ubiquitination. However, UBIAD1 knockdown seems to substantially reduce reudctase-Insig interaction . Please clarify why they propose UBIAD1 acting late, and if the reason(s) are not convincing, some caveat is warranted.


The purpose of this retrospective study was to develop a two- and three-dimensional analysis of the airway using cone-beam computed tomography (CBCT) and to determine whether the airway space would be changed in mandibular prognathism after bimaxillary surgery involving maxillary posterior impaction.Patients requiring orthognathic surgery from 2012 to 2014 were recruited for this study. CBCT scans were obtained at three points: preoperatively (T0), immediate postoperatively (T1), and after 6 months postoperatively (T2). The nasopharynx, oropharynx, and hypopharynx were measured on the CBCT scan for each patient in a repeatable manner. With the midsagittal plane, linear measurements in the middle of each were obtained. For the CBCT, volumetric measurements of each and total airway were obtained.p < .001). However, the change of the diameter and volume of the nasopharynx was not statistically significant . In the oropharynx, the change of both the diameter and volume showed statistical significance between preoperatively and immediate postoperatively  and also preoperatively and after 6 months postoperatively . In the hypopharynx, the change of both the diameter and volume showed statistical significance between preoperatively and immediate postoperatively  and also preoperatively and after 6 months postoperatively .A total of 22 consecutive patients (11 men and 11 women) were included in the present study. The total volume was significantly reduced (The bimaxillary surgery involving maxillary posterior impaction can reduce the volume of airway in the patients of mandibular prognathism. Although total airway volume was reduced significantly, the changes in the volume and diameter of the nasopharynx were not statistically significant. The maxillary posterior impaction affects on the nasopharyngeal airway minimally. Typically, class III malocclusion patients represent a combination of facial malformations in the skeletal and dental alveolar bone . Most ofBut this surgical movement of the maxilla may reduce the volume of airway space . In the In these patients, cone-beam computed tomography (CBCT) has been attracting attention for the three-dimensional assessment of the airway, an important role in the process of diagnosis in place of the conventional CT , 9. CBCTThe purpose of this retrospective study was to develop a two and three-dimensional analysis of the airway using CBCT and to determine whether the difference in the airway volume would be changed with posterior impaction of maxilla in orthognathic surgery.From January 2012 to January 2014, a selected group of 22 patients with skeletal class III malocclusion who have complete records were chosen  which was used in the Gangneung-Wonju National University dental hospital, Department of Oral and Maxillofacial Radiology for patient radiographic evaluation. The subjects were positioned with the Frankfort horizontal plane parallel to the floor and instructed to maintain maximum intercuspation with the tongue touching the palate and to avoid swallowing during the scanning period. The three-dimensional image reconstruction was performed to use of the software .CBCT scans were obtained at three points: preoperatively (T0), immediate postoperatively (T1), and after 6 months postoperatively (T2). The nasopharynx, oropharynx, and hypopharynx were measured on the CBCT scan for each patient in a repeatable manner at each point Table .. But inThree linear measurements and volumetric measurements were made for each image, respectively. Initially, the examiner measured the airway’s anteroposterior length in the midsagittal plane slice at three levels Fig. : anteriot test was used for tests of significance in patients preoperatively (T0), immediate postoperatively (T1), and after 6 months postoperatively (T2). Results are expressed as mean ± SD, and statistical significance was accepted at P <.05. Statistical analyses were performed with SPSS for Windows, version 19.0 .The paired The patient information data are summarized in Tables p = .160). It did not show a statistically significant difference in immediate postoperatively and after 6 months postoperatively (p = .556). Also, there was no statistical significance in the difference between preoperatively and after 6 months postoperatively (p = .339). The change of diameter in the oropharynx was statistically significant between preoperatively and immediate postoperatively (p < .001). It did not show a statistically significant difference in immediate postoperatively and after 6 months postoperatively (p = .253). However, it presented statistically significant in the difference between preoperatively and after 6 months postoperatively (p = .001). The change of diameter in the hypopharynx was statistically significant between preoperatively and immediate postoperatively (p = .001). It did not show a statistically significant difference in the immediate postoperatively and after 6 months postoperatively (p = .932). However, there was statistical significance in the difference between preoperatively and after 6 months postoperatively (p = .001).The linear measurements are summarized in Table p = .137). It did not show a statistically significant difference in immediate postoperatively and after 6 months postoperatively (p = .405). Also, there was no statistical significance in the difference between preoperatively and after 6 months postoperatively (p = .358). The change of volume in the oropharynx was statistically significant between preoperatively and immediate postoperatively (p < .001). It did not show a statistically significant difference in immediate postoperatively and after 6 months postoperatively (p = .361). However, it presented statistical significance in the difference between preoperatively and after 6 months postoperatively (p < .001). The change of volume in the hypopharynx was statistically significant between preoperatively and immediate postoperatively (p = .010). It did not show a statistically significant difference in immediate postoperatively and after 6 months postoperatively (p = .836). However, there was statistical significance in the difference between preoperatively and after 6 months postoperatively (p = .007). Total volume decreased significantly preoperatively and after 6 months postoperatively compared preoperatively .The volumetric measurements are summarized in Table p = .021).Comparisons of the change in total volume between male and female are summarized in Table In the patients of skeletal class III malocclusion who were treated with the surgical procedure, it causes a change in the position of the tongue and hyoid bone and the tongue base moved to the posterior , 13. As Evaluation of the airway through the CBCT is reliable. In the airway analysis, many studies have used CBCT than conventional CT , 18. CBCIn this study, we compared the changes in airway volume and diameter of the patients who underwent maxillary posterior impaction surgery. All patients were accompanied mandibular set back surgery via SSRO or VRO. Although total airway volume was reduced significantly, the changes in volume and diameter of nasopharynx were not statistically significant. The maxillary posterior impaction affected minimally on the nasopharyngeal airway. The average movement of 4 mm is very small so that it could not result in airway change. However, oropharynx and hypopharynx showed statistically significant differences between preoperatively and postoperatively. In the author’s opinion, this was due to mandibular set back movement. The amount of movement of the mandible is usually larger than that of the maxilla. And this mandibular movement is granted a direct effect on the position of the tongue and hyoid bone. Therefore, the diameter and volume of the oropharynx and the hypopharynx are reduced, and total airway volume was also decreased.Female patients showed the narrower airway after surgery compared to male patients. The reduction of the airway was related not only to the position of the tongue and hyoid bone but also to the edema of the respiratory mucosa. Articles which analyzed the changes in the edema by gender are rare. It was believed that this was the result of the difference in the hormone between the male and female. More research is needed.Snoring occurred in one patient who underwent both SSRO and maxillary posterior impaction. She had narrow airway before the surgery. Comparing to preoperative CBCT measurements, we could find reduction of the airway, and in the postoperative 6-month CBCT evaluation, the airway did not returned to the preoperative. However, in the postoperative 6-month CBCT evaluation, the patient did not complain about her snoring anymore Fig. . AnotherThere are many reports of airway reduction after orthognathic surgery , and somThere are many articles dealing with airway change after orthognathic surgery. Pereira-Filho VA et al. evaluated the correlation of maxillary advancement surgery and airway change. In this study, maxillary advancement surgery could dilate the volumes of nasopharynx and oropharynx but not hypopharynx . R. FoltThe bimaxillary surgery involving maxillary posterior impaction can reduce the volume of airway of mandibular prognathism patients. Although total airway volume was reduced significantly, the changes in volume and diameter of nasopharynx were not statistically significant. The maxillary posterior impaction affects the nasopharyngeal airway, minimally.


We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of “cellular sipping” changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6hrs after TNF-α stimulation, but this was lost by 18hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms. Malignant neoplasms arise from acquisition of somatic mutations during initiation, expansion of clones of initiated cells through the action of proliferative signals in promotion, and emergence of increasingly malignant sub-clones to result in disease progression Of particular relevance to the current work is our recent paper describing the exchange of membrane and cytoplasm between cultured parenchymal malignant cells and human gingival fibroblasts (h-GF), a process we have termed ‘cellular sipping’ With regard to the biological significance of cellular sipping, we observed that the morphology of neoplastic cells that have imbibed fibroblast material is intermediate to that of isolated fibroblasts and neoplastic cells cultured alone While cell adhesion would seem to be a minimal and essential requirement for cellular sipping, with a view to better understanding the exchange mechanism in cellular sipping it becomes interesting to consider whether or not the increased cellular sipping upon h-GF stimulation with TNF-α Further, Intercellular adhesion molecule 1 (ICAM-1) and Vascular cell adhesion molecule (VCAM-1) are both increased in h-GF stimulated by TNF-α As outlined above, we aimed to determine whether or not TNF-α increases binding of SAOS-2 to h-GF, as well as the possible role of ICAM-1 and VCAM-1 in any such binding, with a view to better understanding the role of the cytokine in cellular sipping.2, 75 cm2 and 225 cm2) and all other cell culture plastic ware used were supplied by Costar . TNF-α was purchased from Chemicon .Hank’s balanced salt solution (HBSS), cell culture medium 199 (M199), gelatine, Naphthol AS-MX phosphate, fast red violet, Tris HCl, haematoxylin and Polymyxin B solution were obtained from Sigma-Aldrich . Iron fortified bovine calf serum was from Bovogen . Trypsin (0.25%)/EDTA (1 mM) was from JRH Biosciences . The antibiotics penicillin and streptomycin were from CSL Biosciences . Bovine serum albumin (BSA) fraction V and amphotericin B and were from ICN Biomedicals Inc. . Phosphate buffered saline (PBS) tablets were obtained from Oxoid , dimethyl sulfoxide (DMSO) used in freezing cells for storage was purchased from Ajax Chemicals . Anti-Human ICAM-1 antibody and FITC labelled IgG were obtained from Abcam . Anti-Human VCAM-1 antibody was obtained from BD . Falcon FACS tubes were obtained from Becton Dickinson Labware . Centrifuge tubes were purchased from Iwaki, Scitech Division . Disposable membrane filters were obtained from Sartorius, Ministart . Cell culture flasks  comprising M199 with antibiotics and a lower BCS concentration (10%). Explant fragments were removed by aspiration once appreciable h-GF were observed migrating onto the culture surface. Established h-GF monolayers were released with trypsin/EDTA and cultured up to 8th passage at a split ratio or 1∶3. Throughout all culture procedures and experiments, M199 contained the antibiotics penicillin (100 U/ml), streptomycin (100 µg/ml) and amphotericin B (0.25 µg/ml), while all experiments were with h-GF from 6th to 8th passage. Human umbilical vein endothelial cells (HUVEC) were obtained by collagenase perfusion and cultured in M199 supplemented with BCS (20%), Endothelial Cell Growth Supplement (50 µg/ml), heparin (30U/ml) and antibiotics as indicated for h-GF h-GF were isolated by explant culture from human gingival biopsies which were obtained with written informed consent from the Oral surgery unit, Westmead Centre for Oral Health, Westmead Hospital, under a protocol approved by the Sydney West Area Health Service Human Research Ethics Committee as outlined earlier 2 as outlined previously The human osteosarcoma cell line SAOS-2 was obtained from the American Type Culture Collection  and propagated at a split ratio of 1∶3 in CM at 37°C under 5% CO2. h-GF were treated with TNF-α at concentrations of 0 nM, 0.029 nM, 0.039 nM, 0.058 nM, 0.12 nM, 0.58 nM or 1.16 nM from 0 to 24 hrs before washing twice with M199 alone and immediate evaluation of SAOS-2 binding. In some experiments, adhesion of SAOS-2 to h-GF was evaluated after an initial 24 hr period of h-GF stimulation with TNF-α (1.16 nM), and from 0 to 24 hrs of further h-GF culture either with or without continuing TNF-α stimulation at (1.16 nM).Confluent h-GF were harvested with trypsin/EDTA and pelleted cells seeded at confluence into gelatinised 12 well tissue culture plates and allowed to attach overnight at 37°C under 5% CO5 cells/ml in CM before application of 1 ml/well of SAOS-2 cell suspension to washed h-GF in wells. After allowing 5 minutes for attachment, the non-adherent SAOS-2 were removed from wells by gentle washing with M199 and monolayers fixed with 10% neutral buffered formalin for 5 minutes followed by three washes with PBS and one last wash with Mili-Q water.Confluent SAOS-2 were harvested using a method identical to that for h-GF, with the exception that SAOS-2 were adjusted to a final concentration of 1.5×10In some experiments, specificity of responses to TNF-α was probed by addition of Polymyxin B (1g/ml) known to bind and inactivate trace amounts of potentially contaminant lipopolysaccharide Endogenous alkaline phosphatase activity in SAOS-2 was exploited to aid identification of these cells in adhesion assays, using a histochemical approach with Naphthol AS-MX phosphate and fast red violet as described elsewhere Confluent h-GF or HUVEC in gelatine coated 6 well tissue culture plates were either stimulated with TNF-α (1.16 nM) or provided with fresh CM. After from 0 to 24 hrs, cells were washed with PBS and released with trypsin/EDTA before peletting by centrifugation and resuspension in PBS with BCS (1%) and a further centrifugation. Supernatants were discarded and pellets resuspended in PBS with BCS (1%) for 20 min incubation on ice before addition of anti ICAM-1 (1 µg/ml) or VCAM-1 (5 µg/ml) and a further 30 mins incubation on ice. Cells were then washed with PBS with BCS (1%) and following centrifugation, resuspended in 100 µl of FITC labelled anti-mouse IgG (1∶100 dilution) for 30 min in ice. Following this, cells were further washed with PBS containing BCS (1%) and fixed with paraformaldehyde (2% in PBS) for 15 min at RT. Following fixation, cells were again washed with PBS containing BCS (1%), resuspended in 400 µl volumes of PBS with BCS (1%) and analysed with a Becton Dickinson LSRII Flow Cytometer using BD FACS Diva software, counting 10,000 cells per sample studied. Controls consisted of unstained cells, h-GF untreated with TNF-α, or labelled with only the secondary antibody. Data was analysed using Cyflogic 1.2.1 software and expressed graphically as fluorescence distribution profiles proportionate to the peak incidence of fluorescence.When BCS (10%) was present in the adhesion assay there appeared to be slightly higher binding relative to when BSA (4%) was used instead, but this was not statistically significant. Polymyxin B did not affect results, while TNF-α was inactivated by boiling . SimilarFACS analysis revealed that background levels of ICAM-1 in h-GF and HUVEC were elevated by 24 hr of treatment with TNF-α . The cytIn a parallel SAOS-2 binding experiment performed at the same time and with h-GF from same donor as shown in To further examine the role of ICAM-1 expression in SAOS-2 binding to h-GF, three experiments were performed with h-GF from additional separate donors in which a blocking antibody against ICAM-1 was seen to reduce binding of SAOS-2 to untreated h-GF (p<0.04), while similar low levels of SAOS-2 binding were seen when blocking antibody was applied to TNF-α stimulated h-GF (p<0.005) .To determine if TNF-α induced binding of SAOS-2 to h-GF declined after removal of the stimulus, an experiment was performed in which h-GF were first stimulated with TNF-α for 24 hrs, before being washed and further cultured with CM either with or without cytokine for increasing times, and then testing for SAOS-2 binding. To examine a potential direct response of SAOS-2 to TNF-α with regard to h-GF binding, SAOS-2 were pre-treated with TNF-α for 24hrs, and subsequent binding to culture plastic, h-GF and h-GF pre-treated with TNF-α studied . Pre-treTNF-α increased SAOS-2 binding to h-GF, and we conclude this was mediated at least in part by increased ICAM-1 expression. Abrogation of cytokine activity by boiling supports the activity as dependent on native TNF-α conformation, and independence from Polymyxin-B is consistent with the absence of any effect by potentially contaminant lipopolysaccharide. While TNF-α and other ligands of the TNF family receptors can induce apoptosis in fibroblasts and other cells, this is typically only when protein and or mRNA synthesis are inhibited Increased SAOS-2 binding to cytokine treated h-GF was rapid and consistent with changes in ICAM-1 expression, but nonetheless reversible with prolonged culture following removal of the TNF-α stimulus. No clear role for VCAM-1 was observed, but our observations are consistent with the work of others demonstrating increased binding of a range of cells, often via ICAM-1, following cytokine stimulation Had SAOS-2 binding not been increased by pre-treatment of h-GF with TNF-α, we would have concluded that earlier reported increased cellular sipping between SAOS-2 and h-GF with TNF-α Our data also support the idea that by increasing cell adhesion, TNF-α may encourage separate complex tumour parenchymal-stromal cell interactions, consistent with a pro-tumour activity for TNF-α noted by others


P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x106) and provided 80–100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.In order to design  P. falciparum, P. vivax, P. ovale and P. malariae. Of these, P. falciparum (Pf) is the most lethal. The vast majority of deaths occur among young children in Africa. P. vivax is the most prevalent species outside of sub-Saharan Africa and responsible for approximately 50% of all malaria cases worldwide  for 30 min. Samples were then washed and green-fluorescent sporozoites were visualized using a Nikon Eclipse 90i fluorescent microscope. IFAs using air-dried sporozoites were performed as previously described .5) were cultured with varying concentrations of WHcAg, CS or synthetic peptides derived from WHcAg or CS protein. For cytokine assays, culture supernatants were harvested at 48 h for IL-2 determination and at 96 h for interferon-gamma (IFNγ) determination by ELISA. IFNγ production was measured by a two-site ELISA using mAb 170 and a polyclonal goat anti-mouse IFNγ .Spleen cells from groups of 3 each of (B10xB10.s) F1 mice were harvested and pooled 4–6 weeks after immunization with the various WHcAg hybrid VLPs. Spleen cells , synthesized to comprise the P. falciparum CSP C-terminal region. Thus, the CSP gene in the resulting plasmid, pIC-CSPPfCT, consists of the P. berghei N-terminal and repeat regions (base pairs 1 to 786) and the remainder of the P. falciparum CSP (base pairs 787 to 1026). We then excised the hybrid CSP gene from pIC-CSPfCT as a KpnI-XhoI fragment and inserted it into the transfection plasmid, pR-CSPfCT. KpnI and SacI were used to release the inserted fragment from pR-CSPfCT prior to transfection of WT P. berghei (ANKA strain) parasites, as previously described  that weV12.PF3.1. In thispathogen . This ispathogen ; incorpopathogen –43. Typipathogen , whereaspathogen ,42 contapathogen .In the absence of head-to-head comparative studies to date, the WHc(C61S)-Mal-78-3T candidate embodies a number of unique characteristics that may be advantageous in comparison to other CS-vaccine candidates. The enhanced immunogenicity and protective efficacy of WHc(C61S)-Mal-78-3T suggests that the suboptimal performance of a preerythrocytic vaccine candidate is not likely due to the selection of the CS repeat region as a target or to a paucity of B cell epitopes, but rather to insufficient production of protective Abs. For example, the RTS,S vaccine shares similar CS-specific B and T cell epitopes with WHc(C61S)-Mal-78-3T but the carrier moieties are markedly different. WHc(C61S)-Mal-78-3T efficiently self-assembles into hybrid VLPs, which are stable even at 65°C, whereas the HBsAg-based RTS,S requires the addition of excess native HBsAg particles. Compared to the HBsAg, hepadnavirus nucleocapsids are inherently more immunogenic in mice and humans during natural infection or after immunization –48, are P. vivax CS repeat B cell epitopes demonstrates the power and flexibility of the WHcAg VLP combinatorial technology, especially when combined with the hybrid Pb/Pv sporozoite technology [P. vivax immunogen capable of eliciting sterile immunity to blood stage infection in this hybrid Pb/Pv challenge model after active immunization. It is notable that passive transfer of 400 μg of the P. vivax CS-specific Mab 2F2 was not able to confer sterile immunitiy against a 5 min exposure to the bites of 4 Pb/Pv-infected mosquitoes [P. vivax CS-targeted immunogens such as WHc-Pv-78. The CS repeat epitopes and variants from other P. vivax strains can also be inserted onto WHcAg-CS hybrid VLPs. Further development of P. falciparum and P. vivax-specific WHcAg-CS hybrid VLPs would allow their use either separately or combined in a bivalent malaria vaccine, depending on the regional malaria threat.The immunogenicity and protective efficacy of WHcAg hybrid VLPs carrying chnology  used forsquitoes . The faiS1 FigA) Scheme representing the strategy used for replacing the CSP gene of P. berghei (ANKA) with the P. falciparum (3D7) C-terminal region. The annealing sites of the primers used to verify recombination by PCR are indicated below. Restriction sites shown are K—KpnI; Se-SexAI; P-PacI; Xh—XhoI; S—SacI. (B) The resulting amino acid sequence of the hybrid CS protein. The sequence derived from the P. falciparum (3D7) sequence is depicted in red text.((TIF)Click here for additional data file.S2 FigA) Amino acid sequences of each epitope engineered onto the WHc VLPs (see B) Amino acid sequences of the synthetic peptides used for antibody titer determination by ELISA.((TIF)Click here for additional data file.S3 FigMice were immunized with rCS and sera either unadsorbed or adsorbed with NANP/NVDP-containing VLPs  were incubated with 10,000 sporozoites prior to challenge. NMS, normal mouse sera.(TIF)Click here for additional data file.S4 FigGroups of 3 mice were immunized (2 doses: 20 μg and 10 μg in IFAd) with the indicated WHcAg hybrid VLPs. Secondary antisera were pooled and serially diluted and analyzed by ELISA for binding to solid-phase WHcAg, NANP and NVDP. End-point titers of pooled sera are shown.(TIF)Click here for additional data file.S5 FigP. berghei UTC, TH.3R and CS.T3 T cell domains with the P. falciparum T cell domains incorporated into the WHc-Mal-78-3T VLP. The percentage represents homologies between the two sequences.Alignment of the (TIF)Click here for additional data file.S1 TableMean endpoint dilution titers from 9–10 mice in each group are shown.(PDF)Click here for additional data file.S2 TableMean endpoint dilution Ab titers shown. 1°, primary; 2°, secondary antisera. IFA assay used Pb/Pv dry sporozoites.(PDF)Click here for additional data file.


The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. This single-cell-thick epithelium prevents the entry of harmful microbes, toxins, and antigens from the intestinal lumen into the subjacent tissue, lymphatics, and vasculature. To permit efficient absorption, the small intestine has a thin and freely moveable nonadherent unstirred mucus layer to inhibit bacterial-epithelial interactions and diffusion of large molecules.40 is that newly generated intestinal epithelial cells (IECs) within the crypt migrate toward the villus tip region, where loss of senescent epithelial cells occurs in the extrusion zone ion zone . IECs ha49 and the “stem cell zone” model originally suggested by Cheng and Leblond.13 The “+4 model” hypothesizes that definitive stem cells occupy the fourth cell position when counting from the crypt base immediately above the Paneth cell zone. These putative stem cells positively label for B-cell lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), homeodomain-only protein (Hopx), telomerase reverse transcriptase (Tert), and leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1).68 In the “stem cell zone” model, it is postulated that the crypt base columnar (CBC) cells that are interspersed between Paneth cells are the true stem cells. In mice, in which intestinal cell dynamics have been best studied, 6 or more crypts63 provide each villus with new epithelial cells by asymmetric cell division of 14 to 16 CBCs at the crypt base.65 This CBC population, expressing leucine-rich repeat-containing G protein–coupled receptor 5 (Lgr5),4 produces transit-amplifying daughter cells that undergo migration along the crypt-villus axis and differentiate into absorptive enterocytes, goblet cells, or neuroendocrine cells. In vivo confocal imaging in mice has shown that central Lgr5-positive CBC cells maintain a position within the crypt base, while CBC cells in the upper part of the stem cell niche may be passively displaced into the transit-amplifying cell population.65 This conveyor belt from the crypt results in a continuous supply of epithelial cells, which on reaching the extrusion zone undergo apoptosis and are shed into the lumen. Paneth cells are an exception and either remain at the crypt base or migrate downward, depending on which hypothesis is true. Paneth cells possess prominent eosinophilic granules containing antimicrobial substances, including α-defensins and lysozyme,8 and have been hypothesized to protect and potentially regulate the stem cell niche. However, they are variably abundant among species, and while exceptionally abundant in some, such as the giant anteater,79 and prominent in primates, rodents, and the horse,28 they are absent in dogs, cats, and pigs,8 which raises questions about the role of these cells in this regard.Cell loss at the villus tip is, in normal circumstances, compensated for by stem cell mitosis within the crypts of Lieberkühn. The definitive identity of the intestinal stem cell, however, has been controversial for many years. Proposed stem cell models include the “+4 model” put forward by Marshman and colleagues8 which participate in the membranous phase of digestion. Individual epithelial cells are anchored at their basolateral pole to the basement membrane by hemidesmosomes and attached to their neighbors by a narrow continuous belt of tight junctions , which are generated within the intravillus mesenchyme.78 Many other factors also influence the rate of physiological cell loss at the villus tip and the rate of cell replacement from the crypts, including luminal nutrition,10 growth factors such as transforming growth factor β,8 hormones such as glucagon-like peptide 2,10 neural influences,41 circadian rhythms,37 and, importantly, the intestinal microbiota.71 In neonatal pigs, for example, prior to achieving climax flora, an IEC turnover of 7 to 10 days compares to a 2- to 3-day turnover when pigs are 3 weeks of age.28 This slower turnover rate occurs in germ-free animals, resulting in longer villi.71 Germ-free conditions also result in perturbation of nutrient absorption; mice reared in these conditions do not absorb monosaccharides or develop adipose reserves as efficiently as conventionally reared mice.3Uncompensated enterocyte loss results in a decrease in the villus/crypt ratio. Major questions surround the control of migration and maturation of IECs and how and why apoptosis and shedding are restricted to the villus tip extrusion zone. Differences in the interaction between IECs and the basement membrane along the crypt-villus axis have been hypothesized, via gradients in expression of some basement membrane components9 Fixation by definition means that the dynamic process of extrusion cannot be fully appreciated; rather, various stages of the process may be observed without an obvious picture of the sequence of events.IEC shedding as a process represents a very challenging phenomenon to study and understand. The small intestine undergoes extremely rapid postbiopsy and postmortem autolysis. Despite the high rate of physiological IEC loss from individual villi, the very short time it takes for individual cells to be extruded dictates that shedding events are observed relatively rarely in fixed specimens, even when fixation has been optimal. In fact, when hematoxylin and eosin (HE)–stained sections of the human small intestine were systematically examined, a shedding cell was observed in less than 6% of villus sections.31 In reindeer and seals, fragments of shedding enterocytes are lost either by extrusion or by phagocytosis by underlying macrophages.55 In cattle, γδ T cells are closely associated with apoptotic intestinal epithelial cells.76 Histologic studies in humans have shown shedding to be of the whole-cell extrusion type and that shed enterocytes are not associated with lymphocytes or macrophages.9 Therefore, the mechanism of epithelial shedding in humans appears morphologically similar to the process observed in mice, rats, and hamsters, suggesting that these species are appropriate models of human cell shedding and regulation.Nonetheless, the morphological features associated with intestinal epithelial cell death, extrusion, shedding, and detachment have been studied in fixed tissues in several species and have revealed notable interspecies differences. In the guinea pig, apoptotic fragments are pinched off effete enterocytes, leaving junctional complexes intact between neighboring cells.47 Enterocytes undergoing extrusion exhibit basolateral movement of tight junctions down the plasma membrane shared with neighboring enterocytes ,38 in mice following systemic tumor necrosis factor (TNF) administration,39 and during our own light microscopy studies of acute murine endotoxic shock ,84 indomethacin,70 the synthetic double-stranded viral RNA analogue (and Toll-like receptor 3 agonist) polyinosinic/polycytidylic acid,51 ischemia,8 ischemia-reperfusion injury,33 burn injury,74 trauma,72 increased lymphatic pressure,44 cocaine- or atropine-induced villus contraction,44Cryptosporidium parvum infection,22 and toxins, such as that from Bacteroides fragilis.85A diverse range of stimuli and disease processes have been demonstrated to cause pathological epithelial cell shedding. These include TNF,29 These studies have shown that one of the first events observed in cells destined to undergo shedding is the redistribution of ZO-1 to form a “funnel” around the apical cytoplasmic border of shedding cells. Along with other tight junction proteins, including claudins, the process extends to the basolateral border of the shedding cell completing extrusion.29TNF, used extensively to induce pathological intestinal epithelial cell shedding, has provided valuable insights into the highly orchestrated events that lead to IEC extrusion and shedding in C57BL/6-Tg(Vil1-mRFP1/TJP1)#Tjr fluorescent-tagged ZO-1 transgenic mice.29 Both ZO-1 redistribution and MLCK activation have been observed in neighboring enterocytes in a histologic study of cell shedding in humans.9 Marchiando et al48 showed that caspase activity, myosin motor activity (dynamin), and microtubule rearrangement are all required for shedding, thus refining the “zipper” model.47E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) are redistributed during this highly dynamic process.82 This suggests that forces generated in the cell undergoing extrusion may be sufficient to complete the process and that the help of neighboring cells may not be necessary.It is unclear whether neighboring cells cooperate in aiding the shedding cell to exit the monolayer by forming an actin-myosin “purse string.” It has been suggested through in vitro observations in isolated villi that actin rearrangement takes place only in the enterocytes undergoing shedding and not in neighboring cells.69 Membrane-bound pro-TNF is capable of juxtacrine signaling but can become solubilized by action of ADAM17,6 otherwise known as TACE (TNF-converting enzyme).TNF is probably a critical mediator for many stimuli of pathological epithelial cell shedding and acts via two known receptors: p55/TNFR1 or p75/TNFR2. TNF is synthesized as a pro-hormone; both secreted (17-kDa) and membrane-bound (18.5-kDa) forms exert biological effects.73The cellular response to TNF is highly context dependent and varies by cell type, the relative expression of the 2 receptors, and downstream signaling responses. TNFR1 is expressed in a much wider variety of cell types than TNFR2; TNFR1 and TNFR2 are both present in the small intestinal epithelium.62 In histologic studies, intraperitoneal administration of TNF caused significant duodenal villus atrophy by 60 minutes postinjection (PI).84 Our studies of LPS-induced IEC shedding84 demonstrated that TNFR1 is a critical receptor for IEC apoptosis and cell shedding. Other studies have shown that TNFR1-deficient mice are resistant to the lethal effects of LPS and superantigen,61 potentially pointing to the importance of gut damage compared with other organ systems in the most acute phases of sepsis or LPS-induced mortality. The intestinal epithelium has been shown to be more sensitive to acute cytopathic changes and increased permeability than the pulmonary epithelium in an in vivo model of acute septic shock in cats and in human epithelia in response to inflammatory cytokines in vitro.36 These morphological alterations in intestinal epithelium may therefore reflect one of the earliest observable injuries in this type of inflammatory response, which may coincide with early gut barrier dysfunction.TNF is a potent stimulus of IEC shedding in mice.62 Following acute TNF administration, epithelial-specific expression of TNFR1 is necessary to induce apoptosis and shedding,66 rather than indirect vascular dysfunction causing ischemia/ischemia-reperfusion injury. This was shown by analyzing small intestinal IEC loss and apoptosis in a mouse model of acute TNF administration in which there is a conditional gain of function of the Tnfr1 allele.66 These mice express TNFR1 within the intestinal epithelium rather than ubiquitously and have similar TNF-induced cell shedding as in wild-type mice. It is not clear if TNF delivered via the bloodstream or TNF generated by the epithelium is the most significant contributor to IEC apoptosis and shedding. It has however been shown that direct instillation of TNF into the duodenal lumen can cause intestinal damage in rats34 and that epithelial-specific dysregulated TNF production is highly important in driving epithelial damage.30TNF-induced IEC apoptosis and detachment in mice is mediated through TNFR1 and is independent of TNFR2 and p53.tm1ImxTnfrsf1b homozygous mice,84 suggesting that this receptor plays a role in mediating apoptosis. Alternatively, the TNFR2 receptor may be responsible for suppressing TNF production, since markedly elevated amounts of TNF and increased pulmonary inflammation occur in these mice in response to LPS administration.60We reported that IEC apoptosis and cell shedding in response to LPS was lessened in TNFR2-deficient 84 However, the frequency of cell shedding still increases toward the villus tip. Hypoperfusion of the villus tip, particularly in pathological IEC shedding, has been hypothesized to be highly important in this effect.7 The vasculature of the villus is organized in such a way that venules run parallel and in very close proximity to the central arteriole, allowing countercurrent exchange of oxygen. This has important implications during hypoperfusion since when flow velocity decreases, more oxygen is transferred from the arteriole to the adjacent venules in the basal portion of the villus, potentially exacerbating the sensitivity of the villus tip to perfusion deficits.81 Interestingly, even in hypoxic conditions, there is increased production of TNF by intestinal epithelial cells,77 lending support to the notion that a diverse range of stimuli may converge on the TNF-TNFR1 pathway.In pathological epithelial cell apoptosis and cell shedding, the shedding process is not confined to the extrusion zone of the villus tip but extends further down the villus.32 Following LPS administration to cats, Crouser and coworkers16 reported intestinal epithelial necrosis at 2 hours PI; increased iNOS levels and apoptosis were observed only at 4 hours PI.16 During this study, ileal blood flow and oxygen saturation over the course of the experiment were relatively unchanged. The concept of gut mucosal damage being due to gut ischemia has also been challenged by comparing fluid resuscitation and superior mesenteric artery ligation in anaesthetized endotoxemic rabbits.46 Considered together with the resistance of TNFR1-deficient tm1ImxTnfrsf1a homozygous mice to LPS-induced IEC apoptosis and shedding,84 and given that this process still occurs when functional TNFR1 is present only in intestinal epithelium,66 these findings suggest that gut injury in endotoxic shock is very dependent on epithelial responses rather than purely on blood flow alterations and blood oxygenation. Indeed, increased villus epithelial apoptosis occurs even when dysregulated TNF production is confined to the intestinal epithelium.30Although the mechanisms of gut injury that occur in septic or endotoxic shock have not been fully established, it is thought to be initiated by the hypoxic and ischemic conditions brought about by blood maldistribution. In reality, it is likely to be a multifactorial process. Inducible nitric oxide synthase (iNOS), an important mediator of vasodilation and hemodynamics, has been strongly implicated in endotoxic shock-induced intestinal injury. It has been demonstrated that iNOS is responsible for rearrangement of tight junctions at later time points after LPS administration.83 These gaps contain residual ZO-1 left by the departing cell and are subsequently sealed by the process of neighboring epithelial cells re-forming tight junctions within 20 minutes.29 It has also been demonstrated that systemic TNF caused 20% of gaps to result in increased permeability and loss of barrier function.39The use of in vivo confocal microscopy has shown that after enterocytes are shed from the apex of the villus, epithelial discontinuities or “gaps” develop.38 It has also been shown that epithelial gap density correlates with disease severity in IBD.45 Additionally, there is a strong association of increased intestinal permeability in patients at high risk of developing intestinal disease.52 Although a cause-and-effect relationship is not established, this association suggests that increased gut permeability is important in the pathogenesis and progression of these conditions. Although increased intestinal permeability has been reported in intestinal diseases such as IBD,12 probably the best evidence that gut permeability can be directly implicated in the pathogenesis of intestinal disease has been described by Arrieta et al.1 They showed that interleukin 10 (IL-10)–deficient 129(B6)-Il10tm1Cgn mice developed increased small intestinal permeability prior to developing spontaneous colitis. When these mice were administered AT-1001, a synthetic peptide that blocks the zonulin receptor leading to reduced small intestinal permeability, the mice had a significant reduction in the severity of colitis.In the context of pathological epithelial cell shedding in the intestine of patients with IBD, it has been shown by confocal endomicroscopy that shedding events correlate with permeability defects and can aid in the prediction of disease relapse.84 Although these data imply fluid loss into the gut lumen, it is not until later time points, after the discontinuation of IEC shedding, that increased gut to circulation permeability occurs. The temporal change in fluid and large molecular movement may, however, reflect changing tonicity of the gut content as it has been demonstrated that hypotonicity of the luminal content favors the inward permeation of solutes.38Our studies of LPS-induced apoptosis and cell shedding in mice suggest that increased gut permeability and clinical diarrhea temporally correlate with IEC shedding.27  is the umbrella term for a family of highly evolutionarily conserved cytosolic proteins that are important in preserving intestinal epithelial integrity in response to inflammatory stimuli.30 These proteins, when activated, are released from their specific inhibitor of κB (IκB) protein, dimerize in various combinations, and oscillate into and from the nucleus in an energy-dependent manner.56 In the nucleus, NF-κB proteins act as transient transcription factors controlling the expression of a large array of genes, including those involved in regulating inflammation, cell stress, cell adhesion, and crucially in the context of intestinal epithelial cells, cell death, or survival.75 Nuclear translocation of p65/RelA occurs in villus IECs that resist shedding in a Cryptosporidium parvum model of IEC shedding in piglets.22Mice lacking the p65/RelA NF-κB subunit in IECs exhibit profoundly increased IEC apoptosis, have dysregulated IEC proliferation, and suffer spontaneous intestinal failure with loss of the crypt-villus architecture.84 and other studies have shown that transgenic mice with specific NF-κB family member deletion24 or alterations in NF-κB activation pathways30 have dramatically altered susceptibility to acute stimuli of small intestinal injury. NF-κB appears to be important in dictating the fate of the epithelial cell in response to TNF or LPS, with p105/p50 signaling favoring cell survival and p100/p52 favoring apoptosis  and death, it is difficult to separate cause and effect. However, in less complex organisms, such as the fruit fly Drosophila melanogaster, with its simpler organ and circulatory systems, failure of the gut barrier is a predictor of imminent death of the organism.64Many questions remain regarding how pathological IEC apoptosis and shedding affect gut permeability and what the immediate and longer term consequences of increased gut permeability may be. Acute epithelial defects appear to be associated with villus contraction and initial fluid exudation into the gut lumen, followed later by the movement of large molecules out of the gut lumen and into the bloodstream. The most obvious detrimental outcome in acute inflammatory responses appears to be the serious derangement of fluid and electrolyte balance, with loss of albumen into the gut lumen.58 with low-grade inflammation.11 Chronic metabolic endotoxemia, when artificially induced in mice on a normal diet, has been shown to initiate obesity and insulin resistance,11 similar to if these animals are fed a high-fat diet.11 A high-fat diet in mice also increases the amount of Gram-negative bacteria within the gut and promotes LPS translocation across the intestinal epithelium.11 Mounting evidence suggests that epithelial responses and intestinal permeability may be highly modulated by the gut microbiota.21 Host-microbial interactions likely play a significant role in regulating intestinal epithelial apoptosis and cell shedding.In more chronic situations, increased gut permeability and exposure of subepithelial tissues to gut luminal antigens may trigger inappropriate immune and inflammatory responses. Diet also seems to play an important role in regulating intestinal permeability. High-fat diets cause increased gut permeability and metabolic endotoxemia, as well as increased circulating LPS, originating from the gut,17 Chronic progressive idiopathic intestinal diseases in animals may also share a TNF-based pathogenesis. TNF is integral to effective leukocyte-mediated immune responses, and preventing its signaling, although an improvement on the more nonselective immune suppression therapy with corticosteroids, results in defective immune function. A better understanding of how TNF production affects the epithelial as opposed to the immune cell compartments is required to allow more targeted treatment approaches. Targeting regulatory elements downstream of TNF receptors, such as NF-κB signaling, may also allow for more sophisticated therapeutic strategies to be developed that prevent pathological intestinal epithelial cell apoptosis and cell shedding, thereby enhancing barrier integrity.IEC apoptosis and cell shedding probably occur in a variety of clinical scenarios in which there are surges of TNF. Many patients with IBD are given anti-TNF antibody treatment, but this is extremely costly, and unfortunately, a proportion of patients either fail to respond or become refractory.30 Modulating this proinflammatory cytokine via TACE, mitogen-activated protein kinase,30 or matrix metalloproteinase 1380 may therefore represent alternative strategies for preventing pathological intestinal epithelial cell shedding.Intriguingly, the epithelial cell compartment appears to be an important source of TNF production initiating enterocyte apoptosis and cell shedding. Transcriptional and posttranscriptional control is critical in determining the outcome of excessive TNF production.Investigating the process of intestinal epithelial cell death and the key initiating and regulatory factors may further our understanding of the pathogenesis of idiopathic inflammatory intestinal diseases and possibly intestinal cancers. It may also allow prevention of gut barrier dysfunction in intestinal and systemic disease states. Addressing and understanding the mechanisms of gut barrier dysfunction may allow future development of therapeutic and prophylactic strategies for the prevention of the serious sequelae of bacterial translocation, sepsis, and endotoxemia in human and animal medicine.


The evolution of the tumor necrosis factor/tumor necrosis factor receptor superfamily (TNF/TNFR) is complicated and not well understood. To date, most TNFR studies have focused on vertebrate models leaving the role of TNFRs in invertebrates largely unexplored. The evolution of important cellular processes including stress response, apoptosis, development, and inflammation will be better understood by examining the TNF/TNFR superfamily in ancient invertebrate phyla. How widespread is this gene family within the evolutionary tree of life and is there evidence for similar function in invertebrates? A first step is to identify the presence or absence of these genes within basal metazoan taxa using the signature cysteine-rich domain (CRD) of the TNFR superfamily. In this perspective, we will start by examining what is currently known about the function of TNFRs in invertebrates. Then, we will assess the role of TNFRs in apoptosis and explore the origins of the domains found in TNFRs including the death domain (DD) and CRD. Finally, we will examine the phylogenetic relationship between TNFRs containing DDs identified to date. From these data, we propose a model for a Precambrian origin of TNFRs and their functional role in apoptosis. The TNFR superfamily is remarkably diverse across animal phyla.The role of TNFRs in apoptosis is ancient.The cysteine-rich domain (CRD) of TNFRs is present across all domains of life.Why is the TNFR repertoire of choanoflagelletes so diverse?What is of the functional role of invertebrate TNFRs that lack death domains?When did the TNF-ligand and its role in apoptosis emerge?What are the ligands of invertebrate TNFRs?1 These results led to the treatment of human cancers using bacterial extracts termed ‘Coley’s toxins’, named after the oncologist W. Coley.2 More than 50 years later, lipopolysaccharide was isolated from these extracts and shown to induce tumor regression; however, it was determined that it was a factor in the serum, termed tumor-necrotizing factor that actually caused the tumor regression. The cellular receptors for TNF were first described in 1984 leading to the discovery of a novel protein domain termed death domain (DD) that was located on the intracellular portion of TNFRs and was responsible for initiating programmed cell death (apoptosis).3–5 These ‘Death Receptors’ were shown to be central regulators of apoptosis serving as both internal and external sensors that maintain organismal integrity.5 In humans, a total of 19 different TNF ligands and 29 TNFRs have been described to date.6 In addition to apoptosis, the TNF/TNFR superfamily is involved in cellular differentiation and proliferation through the activation of a variety of signaling pathways. For a more detailed description of these pathways and the many cellular processes that the TNF/TNFR superfamily is involved in, the authors point the reader to the review by Gaur and Aggarwal.7 TNFRs have also been extensively studied in other vertebrate model systems such as zebrafish8 and mice,9 however the focus of this study will be exclusively on invertebrate TNFRs.Although the focus of this study will be on the function and origin of invertebrate TNFRs, a brief history of the TNF/TNFR superfamily is required to place these studies in proper context. The path towards discovery can be traced back to the late nineteenth century when the regression of human tumors was observed following bacterial infection.10 For example, in the tunicates, Ciona savignyi and Ciona intestinalis, TNF-α was upregulated in hemocytes in response to a lipopolysaccharide challenge and was primarily expressed in inflammatory granulocytes that occurred at the area of inflammation.11 The lancelet fish, Amphioxus, has an extensive repertoire of TNF/TNFRs present in its genome.12 In response to an lipopolysaccharide immune challenge, moderate upregulation of TNF and TNFR occurred and it was a dynamic response depending on the homolog. From these data, it was concluded that TNF and TNFR could be important in the immune response to mucosal pathogens in Amphioxus.13 Overall, the TNFR superfamily has been demonstrated to have a dynamic role in immune function and is activated in response to immune challenges from lipopolysaccharide in multiple chordate species.11–13The phyla Chordata is defined by the presence of a notochord during development and includes both invertebrate and vertebrate species. Invertebrate members of the phyla chordata  are considered to be the most closely related to humans, and have been the focus of many studies on diversity of the innate immune system and its divergence from the adaptive immune system.α homolog was characterized in Disk Abalone  and was constitutively expressed in infected and non-infected tissues. However, it was significantly upregulated in the gills that were exposed to multiple pathogens.14Members of the TNF/TNFR superfamily have been widely described in the phylum Mollusca  and most of the evidence points to its role in the immune response to Gram-negative bacteria. For example, the first TNF-Crassostrea gigas), TNFRs has an important role in biotic and abiotic stress response, specifically six of the Pacific Oyster TNFRs were differentially expressed in response to bacteria exposure.15 In addition, many of the Pacific Oyster TNF ligands exhibit a dynamic gene expression response pattern, where more than half of the TNFs are responsive to biotic challenges. Pacific Oysters infected with Vibrio sp. also had several different TNFs that responded differently; four TNFs are late responders, three are repressed, and one is a very early responder.15 Although the complete picture of the mechanisms controlling this TNF expression is not completely understood, it is apparent that the expression is sensitive to timing during an infection, and is dynamic in what does and does not respond.In the Pacific Oyster (Ostrea edulis), TNF expression was upregulated in hemolymph cells during a Bonamiosis infection.16 This upregulation appears to be correlated with the severity of the infection, and again presents the nature of TNF/TNFR expression as dynamic.16 Finally, in the scallop (Chlamys farreri), TNFR was upregulated in the gill tissues and mantle in response to a Listonella anguillarum challenge.17In the Flat Oyster , which has a TNF homolog named Eiger and a TNFR homolog called Wegen.18 Although there only appears to be one TNF and one TNFR in Drosophila, the work to date has been the most extensive on any invertebrate. The role of the Eiger/Wegen system in immunity is very intricate, where tolerance phenotypes for intracellular Gram-positive bacteria have been observed, and faster death is observed in Eiger mutants exposed to extracellular microbes.19 In addition, Eiger mutants in Drosophila appear to affect many components of the immune system including melanization, Imd signaling, and the response to genotoxic stress.20 Finally, Eiger signaling has other important functions in the sensory system of Drosophila such as its involvement in the sensitization of nociceptive sensory neurons during thermal allodynia and UV exposure.21Information on the role of the TNF/TNFR signaling system in arthropods is limited to the fruit fly  consisting of six cysteines that form three disulfide bridges.36 Choanoflagellates,37 sponges,38 Ctenophores,40 bacteria (PRJNA177747), and plants41  have the highest number of CRD-containing proteins with 44 and 84, respectively . Within Group 1, A. digitifera proteins 06604Acd, 02522Acd, and 06605Acd are clustered together with mammalian TNFR1A. Although this TNFR is involved in apoptosis, its main function is in the regulation of pro-inflammatory signaling pathways.45 In Group 5, 7 of the 13 coral TNFRs cluster with the TNFR EDAR proteins. These proteins have previously been shown to be important for epidermal morphogenesis.46 Further testing would need to be done in corals to understand whether this is occurring, but it is possible that this role in morphogenesis could be a very primitive role for this extensive gene family.To infer phylogenetic relationships between DD containing TNFRs neighbor-joining analysis was preformed on 62 amino acid sequences of the TNFR protein of representative vertebrates and invertebrates . Our neiTaken together, the DD-associated TNFRs from corals cluster into groups that contain TNFR members functioning in inflammation, morphogenesis, and apoptosis. The inflammation response of TNFRs has been widely studied in other invertebrates, but warrants more study in basal invertebrates . Although not yet functionally confirmed, this same function could exist in corals, and could therefore represent the most ancestral function of DD TNFRs. This would expand the functional repertoire of ancestral DD TNFRs to include not only apoptosis, but also inflammation and morphogenesis.Nematostella vectensis (sea anemone) and Hydra magnipapillata, have well-developed methods to investigate protein function.48 Apoptosis in general has been examined in N. vectensis50 and H. magnipapillata,51 but TNFRs have yet to be directly investigated. An integrative approach that involves multiple Cnidarian species combined with additional basal metazoan phyla is required to better understand the origin of evolution of TNFR-mediated apoptosis. Finally, although this review has shown that the function of apoptosis is likely a very old, there are other possible functional avenues to explore, including a role in inflammation and morphogenesis.To investigate these hypotheses, the DD-binding partners of TNFRs should be determined. If a single fusion event occurred in the basal metazoan ancestor resulting in the fixation of TNFR-induced apoptosis, we would expect similar proteins to associate with the DD across phyla. In corals, a key unknown is the localization of specific TNFRs within coral tissue. Characterizing which cell types express TNFRs containing DDs will provide insight into what the ancestral state of TNFR-mediated apoptosis may have been. Whereas corals currently lack robust molecular tools for mechanistic investigations, two fellow Cnidarian model systems,