TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11–0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient ( Corneal neovascularization and lymphangiogenesis can be induced by various triggers including limbal insufficiency, inflammation, trauma or surgical manipulations ,2. This TNFα-/-mice than in wild-type mice Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: YesReviewer #3: NoReviewer #4: No**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: NoReviewer #4: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this study, Maier et al. investigate the potential role of TNFRs 1 and 2 in corneal neovascularization and lymphangiogenesis. The authors find a decrease in the vascularized area in TNFR-deficient animals as well as well as downregulation of TNF itself in their model. However, they conclude that TNF as well as its receptors do not play a significant role in the pathogenesis of neovascularization and lymphangiogenesis. The authors should consider to moderate this statement and their interpretation.FIGURE 1The data in figure 1 would be more convincing if a negative control was included. Since the authors have access to the TNFR-deficient mice, these samples should be included to demonstrate specificity of their antibodies.What was the reason for using two different antibodies (CD68 and F4/80) in these samples?It is not clear why the TNFR-deficient mice would show TNFR gene expression. The authors should validate their RT primers and repeat these experiments.Also, the western blot in figure 1F is inconclusive. Why is their no difference between wild type and knock out? Immunoblots for TNFR1 should also be included.FIGURE 2CD31 positive/blood vessel area should be highlighted/indicated in figures 2B and 3CFIGURE 4The data in figure 4 is inconclusive and is hard to interpret as is. The quality of the immunoblot is rather low. The authors could also consider evaluating the function of TNF using an in vitro system. This would further strengthen interpretation and conclusion.Minor comments:The title is confusing and should be revised. The authors study the effects of deficiency rather than malfunction.The overall quality of the figure-PDF file is low. High resolution images and unified labels (font + size) should be provided with the revised manuscript.Reviewer #2: It is pleasing to read the report by Dr. Maier and colleagues in which the group tested the relative efficacy of two TNF receptor deletions on vascularization following corneal injury; the reporting of minimal or negative results is not done often enough and this finding herein is important. The mouse experiments and data are sound, with no problems in their presentation, interpretation, or subsequent discussion. There is some concern with the human data: it is very hard from the way the imaging studies were performed to claim co-localization or lack thereof from any particular cell type (the imaging problem exists with the mouse tissue as well). Since this paper is not presenting a tissue-specific deletion of either Rp55 or Rp75, it's relative localization is not critically important to the findings. Being purposefully vague might be the safest route, claiming that protein is expressed and 'does not appear' to localize with macrophages... the epithelium is usually 'hot' for fluorescence so that claim is questionable and S3 and S4 are not convincing (and lack proper labels and legends).Reviewer #3: In this manuscript, the authors characterized corneal neovascularization and lymphangiogenesis in the TNF-Rp55 or TNF-Rp75 KO mice. They concluded that in the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75do not play a significant role in corneal neovascularization and lymphangiogenesis. However, the data showed that a reduction of neovascularization in these KO mice compared with WT at day 14 and reduced lymphangiogenesis in TNF-Rp75 KO mice. This suggests that TNF signaling has a role in these two processes.Other comments:1. In most experiments, how many mice were used is unclear.2. The authors only explored the expression of 3 genes that are related to neovascularization and lymphangiogenesis. Since many genes involve in regulation of neovascularization and lymphangiogenesis, the authors can't make a conclusion that TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role.3. TNF-a is an important cytokine in regulating corneal inflammation, neovascularization and lymphangiogenesis. In this study, a suture-induced corneal neovascularization model was used to identify the effect of TNF-a in corneal neovascularization and lymphangiogenesis. The model is so mild, many of the immunological cells and cytokines were not found significantly changed in this model. So that’s possible that you can’t observe much effects of TNF-a in this situation. Also, the suture-induced corneal neovascularization is not suitable to mimic the clinical scenario of corneal graft rejection. As we know, the corneal graft rejection is reasoned by exogenous antigen stimulation, which is not similar to the suture induced corneal neovascularization.4. A group of non-injured mice need to be added in each experiment. The authors should identify the expression of TNF-a has been increased in your models. Otherwise, there is no point to investigate the roles of TNF-a and its receptors in this model.Reviewer #4: The authors show that TNFα and its receptors are not required for neovascularization and lymphangiogenesis in corneal suture model in mice. The data presented in Fig1 E, F and G indicates that the authors are working with mice where the TNFR receptors are not knocked and thus cannot make any claim on the role of TNFR in angiogenesis and lymphangiogenesis. I have to recommend rejecting the paper in its current form.The following issues need to be addressed.1. The quality of tissue in immunofluorescence images Fig1A and S3 and resolution of image is very poor and cannot be interpreted. It is unclear what region of the eye is shown in Fig1A although from result section it appears to be cornea. There is no indication of where TNFα receptor are localizing in the cornea. Please indicate where the receptors are localizing.2. Fig 1B suffers from the same issue as Fig1 A. The tissue section is of extremely poor quality. The limbus region is suspect. Please show a phase image so one might see the iridiocorneal angle. The labels in B and C are confusing is label for C on left makes no sense to the reader. Should it not be cornea? If B and C are from the suture experiment (says Mouse+Suture on the right of panels) where are the controls or sham eyes?3. Why is TNFR2 75KDa not reduced in the TNFR2 knockout mouse in Fig 1D? Please reword Line 223-227 “The mRNA expression of TNFRp55 in the TNF-RP55d mice, however, is significantly reduced  and the functionality of the receptors is disturbed. There is no evidence for differential regulation ofthe other receptor in the TNF-Rpd animals, neither on mRNA nor on protein level  as isis very confusing.4. Fig 1F and G leads to question the entire premise of the paper- it seems TNFR1 protein is present in mutant hence the authors are working with essentially wt mice?? What is the state of the TNFR2 mutant mice? Does it still express TNFR2 protein? Please validate mice before doing the suture experiments. Please show a western blot showing the protein levels of TNFR1 and TNFR2 in wt and respective mutant mice.5. Also need to this experiment with the double knockout mice given TNFα knockout seems to exacerbate neovascularization (Fujita 2007).6. The figures all seem to be of poor resolution making it really hard to zoom in on panels to see detail.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  26 Sep 2020We thank the editorial board to give us the opportunity to improve our paper substantially according to the academic editor and reviewers’ comments. We answer the academic editor and reviewer’s concerns point by point as follows.Journal requirements:https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfAnswer: We revised the manuscript according to the PLOS ONE`s style requirements. 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (a) methods of sacrifice, (b) methods of anesthesia and/or analgesia, and (c) efforts to alleviate suffering.Answer: We included the details on (a) methods of sacrifice, (b) methods of anesthesia and/or analgesia, and (c) efforts to alleviate suffering. “After suture placement, the mice were regularly examined by animal keepers. All efforts were made to minimize discomfort of the animals.”“In brief, mice were deeply anesthetized by injection of ketamine/xylazine.”“3, 8, 14 or 21 days after suture, mice were sacrificed by injection of ketamine/xylazine and subsequent cervical dislocation, and eyes were processed for further experiments.” 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.https://doi.org/10.5281/zenodo.4049178)Answer: All data are available at the open access database Zenodo using the following link .Answer: We added Figure S3 Fig depicting 2 representative uncropped blots. Furthermore, all blot data are available at a public data repository  and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.https://doi.org/10.5281/zenodo.4049178).Answer: Data of melting curve analysis are available at a public data repository . The differences, however, were very subtle and only detectable at 1 time point (D14). The animal model should show that TNFα -signaling might play a role by increasing TNFα -sensitivity of participating cells at constant TNFα levels. Again, these data cannot provide evidence for a significant role of TNFα- signaling in the suture model. Fig1Q2: The data in figure 1 would be more convincing if a negative control was included. Since the authors have access to the TNFR-deficient mice, these samples should be included to demonstrate specificity of their antibodies.Answer: We agree with the reviewer that we did not show a control for immunohistochemistry by using specimen from TNFR deficient tissue. For that we would have to use TNFR KO mice, lacking the expression of the respective gene by transgenic knocked down. In our case, however, we use KI models leading to lack of function of the respective proteins. Here, TNF-Rp55 and TNF-Rp75 are produced but their functional activity was switched off by the transgene. For more details how the KI mutations were created see answer to Q4. An isotype control, depicting the background staining of the secondary antibody, is included to the supplement . We added arrows and text to ease the understanding of the figureQ3: What was the reason for using two different antibodies (CD68 and F4/80) in these samples?Answer: We used CD68 predominantly for human samples to detect macrophages and F4/80 in murine samples since they are more convincing to identify all macrophages in mice [1]. Unfortunately, F4/80 is only detectable in rodents.Q4: It is not clear why the TNFR-deficient mice would show TNFR gene expression. The authors should validate their RT primers and repeat these experiments.Answer: This is a misunderstanding of our model. We have to apologize that the current description was not clear enough. Thus, we now clarify the nature of the mutant mice, their genetic background and the functional consequences more precise. As already stated above, the TNFR deficiency is a functional one. The mice harbor a null mutation of the respective TNFR leading to an altered gene product that lacks the molecular function of the wild-type gene. The non-functional proteins themselves are expressed, however. The TNF-Rp55 null mutations were created by disruption of the coding sequence of the TNF-Rp55 gene at base pair 535 (corresponding to the cDNA sequence) by insertion of a neo gene [2]. This resulted in the transcription and transduction of a non-functional TNF-Rp55 protein. Pfeffer et al validated the mutation by an experiment that showed the inability of spleen cells of the TNF-Rp55-/- mice to bind TNF-aA similar strategy was pursued by Erickson et al creating the TNF-Rp75-/- mice [3]. In TNF-Rp75-/- mice challenged by LPS (that leads to shedding of the extracellular domain of TNF-Rp75), no soluble TNF-Rp75 could be detected in the serum. We added the following sentence in the methods part to explain precisely the KI Model . This resulted in the transcription and transduction of a non-functional TNF-Rp55 protein. A similar strategy was pursued by Erickson et al creating the TNF-Rp75d mice. A neomycin-resistance gene under control of the Pgk promoter20 was inserted in the second exon, which contains the signal peptide region of TNF-R2, resulting in the transcription and transduction of a non-functional TNF-Rp75 protein (22)”Q5: Also, the western blot in figure 1F is inconclusive. Why is there no difference between wild type and knock out? Immunoblots for TNFR1 should also be included.We validated the role of the TNFα signaling at the level of the TNFα -receptors. Figure 1F is an immunoblot showing the protein expression of TNF-Rp55 in TNF-Rp55d, TNF-Rp75d mice and wild-type littermates. As shown in bar chart 1G, there is no difference in protein expression of TNF-Rp55 among the groups. Especially TNF-Rp55 is not upregulated in the TNFrp-55d mice. Thus, we have convincing evidence, for the lack of compensatory upregulation of the TNFα-receptors expression in the KI-animals.Fig2Q6: CD31 positive/blood vessel area should be highlighted/indicated in figures 2B and 3CAnswer: We indicated the CD31/Lyve-1 covered area by arrows Fig2 and Fig3Q7: The data in figure 4 is inconclusive and is hard to interpret as is. The quality of the immunoblot is rather low. The authors could also consider evaluating the function of TNF using an in vitro system. This would further strengthen interpretation and conclusion.Answer: We tried to evaluate the role of TNFα signaling in suture induced neovascularization and lymphangiogenesis at several levels. We analyzed gene expression and protein expression of TNFα and its receptors in wild-type and TNF-Rp55d or TNF-Rp75d mice respectively and could not find reliable evidence. Thus, taken our results from animal experiments, in vitro experiments would not add valuable data to the manuscript. More complex signaling cascades that involve many different cell types and structures, an environment that is impossible to mimic in vitro might help to explain the pathogenesis of suture induced alterations in the animals.The protein concentration of TNF-α in the cornea is very low, also compared to other, mainly structural proteins that are expressed abundantly in the cornea. We provide an uncropped, unedited version of the original blot . Unfortunately we are not able to substantially improve the quality of the signal. Even precipitation of TNFα did not improve the visibility of the TNF-α band.Minor comments:Q8: The title is confusing and should be revised. The authors study the effects of deficiency rather than malfunction.Answer: We changed the title to “Effects of TNFα receptors TNF-Rp55 or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse.Q9: The overall quality of the figure-PDF file is low. High resolution images and unified labels (font + size) should be provided with the revised manuscript.Answer: The reviewer is right; the images in the current version are of poor quality/resolution. We significantly improved the resolution of the images and unified all the labels. We think that the current version is more appropriate now.Reviewer #2: It is pleasing to read the report by Dr. Maier and colleagues in which the group tested the relative efficacy of two TNF receptor deletions on vascularization following corneal injury; the reporting of minimal or negative results is not done often enough and this finding herein is important. The mouse experiments and data are sound, with no problems in their presentation, interpretation, or subsequent discussion. There is some concern with the human data: it is very hard from the way the imaging studies were performed to claim co-localization or lack thereof from any particular cell type (the imaging problem exists with the mouse tissue as well). Since this paper is not presenting a tissue-specific deletion of either Rp55 or Rp75, it's relative localization is not critically important to the findings. Being purposefully vague might be the safest route, claiming that protein is expressed and 'does not appear' to localize with macrophages... the epithelium is usually 'hot' for fluorescence so that claim is questionable and S3 and S4 are not convincing (and lack proper labels and legends).Answer: We thank the reviewer for his interest in the manuscript and we are thankful for the comments to improve the manuscript. We know that the results of co-localization analysis on sagittal sections have to be handled carefully. We agree with the Reviewer to state our conclusion more vaguely. Thus, we rephrased the sentence on p.9 line 228f. It is less speculative now by only stating “We also detected macrophages by CD68 staining, the receptors and macrophages, however, do not appear to co-localize.” We renewed the negative controls by more convincing ones and added labels to S1 Fig (S3)/S2 Fig (S4) to ease the understanding at a glance.Reviewer #3: In this manuscript, the authors characterized corneal neovascularization and lymphangiogenesis in the TNF-Rp55 or TNF-Rp75 KO mice. They concluded that in the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75do not play a significant role in corneal neovascularization and lymphangiogenesis. However, the data showed that a reduction of neovascularization in these KO mice compared with WT at day 14 and reduced lymphangiogenesis in TNF-Rp75 KO mice. This suggests that TNF signaling has a role in these two processes.Answer: Indeed, the reviewer is right that we found differences in vascularization and lymphangiogenesis (determined by CD31 or LYVE-1 coverage). The differences, however, were very subtle and only detectable at 1 time point (D14). These subtle differences might be statistically significant but not biologically relevant: The exact values for CD31 coverage on day 14 are: WT 15.6% ± 2.6%, TNF-Rp55d 12.4% ± 2.6% and TNF-Rp75d 9.6% ± 0.8%; for LYVE-1 coverage WT 9.7% ± 1.3%, TNF-Rp55d 8.4% ± 1.9%, TNF-Rp75d 7.2% ± 2.2%. The animal model should show that TNFα -signaling might play a role by increasing TNFα -sensitivity of participating cells at constant TNFα levels. Again, these data cannot provide evidence for a significant role of TNFα- signaling in the suture model. Other comments:Q1: In most experiments, how many mice were used is unclear.Answer: we provide numbers for all individual figures now in the figure legend and the supplement (S1 Table/S2 Table for WB and PCR) as well as an overview of the total amount of animals in the methods part. We added this sentence (p5. Lines 127-128): “A total number of 47 TNF-Rp55d mice, and 47 TNF-Rp75d mice and 48 wild-type mice were used for this study.”Q2: The authors only explored the expression of 3 genes that are related to neovascularization and lymphangiogenesis. Since many genes involve in regulation of neovascularization and lymphangiogenesis, the authors can't make a conclusion that TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role. Answer: The reviewer is right that we only focused on few markers of neovascularization and lymphangiogenesis. The scope of the study, however, was to assess the differences in standard markers for neovascularization and lymphangiogenesis comparing to different KI models and WT before and after suture placement. We consider VEGF-A/VEGF-C and LYVE-1 as very robust and established markers for these 2 events. A detailed analysis of plenty of different angiogenesis markers like angiopoietin-1 angiopoietin-2, Tie-1, or endothelial cell adhesion molecules like VE-cadherin, PECAM-1, or several integrins would be out of the scope of the manuscript [4]. Furthermore, the take home message of the paper might not benefit from this extension of the analysis due to several problems, e.g. different time-points of regulation and low expression levels that we already suffered from with our rather robust read-out. Taken together, we did not find differential expression of TNFα after suture placement nor any conclusive effects at the level of the TNF-receptors, respectively. Q3: TNF-a is an important cytokine in regulating corneal inflammation, neovascularization and lymphangiogenesis. In this study, a suture-induced corneal neovascularization model was used to identify the effect of TNF-a in corneal neovascularization and lymphangiogenesis. The model is so mild, many of the immunological cells and cytokines were not found significantly changed in this model. So that’s possible that you can’t observe much effects of TNF-a in this situation. Also, the suture-induced corneal neovascularization is not suitable to mimic the clinical scenario of corneal graft rejection. As we know, the corneal graft rejection is reasoned by exogenous antigen stimulation, which is not similar to the suture induced corneal neovascularization.Answer: Indeed the reviewer is right that the suture-induced corneal neovascularization is not the ideal mouse-model to analyze corneal graft rejection, but it mimics the clinical scenario of a high-risk situation for a corneal graft rejection after keratoplasty. Compared to the alkali burn induced corneal neovascularization, the suture-induced model has a number of advantages especially studying the corneal lymphangiogenesis and associated prolymphangiogenic cytokines [5]. First, the sutures are more reproducible than the alkali burns on the cornea. Suturing only causes localized epithelial loss and inflammatory infiltration between the suture and the limbus, but chemical burns deplete the whole epithelial layer of the central cornea and cause strong cellular infiltration of the whole cornea [6]. Therefore, the suture-induced model mimics the clinical scenario of mild corneal lesions better than the alkali burn induced model. Fortunately, chemical burns in patients have become less common.We clarified this in the discussion part, deleted the chapter about corneal graft rejection and added the following paragraph . “Second, it is more suitable to analyse lymphangiogenesis and associated secretion of prolymphangiogenic cytokines [5]. The induced neovascularisation and lymphangiogenesis mimic the clinical scenario of high-risk situation for a corneal graft rejection after keratoplasty and thus it has more translational impact than the models with severe damage and reduced stimulation of the lymphangiogenesis. Suturing only cause localized epithelial loss and inflammatory infiltration between the suture and the limbus, but chemical burns deplete the whole epithelial layer of the central cornea and cause strong cellular infiltration of the whole cornea [6].” Q4: A group of non-injured mice need to be added in each experiment. The authors should identify the expression of TNF-a has been increased in your models. Otherwise, there is no point to investigate the roles of TNF-a and its receptors in this model.Answer: The reviewer rises two important questions: the lack of an appropriate control and the absence of a significant TNFα increase upon suture placement. We have an inert control: only one eye was sutured, the counter-eye served as control. We added a sentence in the method part to highlight the presence of an appropriate control (p6 lines 133f.). The fact that TNFα was not significantly increased in our model does not question our conclusions. First, it might be an issue of the perfect time point that we did not find increased TNFα protein expression. We only checked Day 14 after suture placement. Theoretically, the receptor could also become more sensitive to TNFα at the same concentration by time. We checked not only for TNFα expression, but also TNFα receptor expression as well as function. Thus, we can safely state that TNFα does not play a role in this model. The TNFα increase alone is not necessary or sufficient.Reviewer #4: The authors show that TNF-α and its receptors are not required for neovascularization and lymphangiogenesis in corneal suture model in mice. The data presented in Fig1 E, F and G indicates that the authors are working with mice where the TNFR receptors are not knocked and thus cannot make any claim on the role of TNFR in angiogenesis and lymphangiogenesis. I have to recommend rejecting the paper in its current form. The following issues need to be addressed.Q1: The quality of tissue in immunofluorescence images Fig1A and S3 and resolution of image is very poor and cannot be interpreted. It is unclear what region of the eye is shown in Fig1A although from result section it appears to be cornea. There is no indication of where TNFα receptor are localizing in the cornea. Please indicate where the receptors are localizing.Answer: We agree with the reviewer that the resolution of the images was very bad in the draft of our manuscript. We addressed this issue and we now provide significantly better images in S1 Fig (S3) and S2 Fig (S4). We added arrows to depict structures/ staining and we added several remarks in the figure legends to improve understanding. Q2: Fig 1B suffers from the same issue as Fig1 A. The tissue section is of extremely poor quality. The limbus region is suspect. Please show a phase image so one might see the iridiocorneal angle. The labels in B and C are confusing is label for C on left makes no sense to the reader. Should it not be cornea? If B and C are from the suture experiment (says Mouse + Suture on the right of panels) where are the controls or sham eyes?Answer: We agree with the reviewer that the images in Fig1B are lacking the iridiocorneal angle. We display a bigger detail of the images now showing the iridiocorneal to ensure the localization of the section (at the limbus corneae). Furthermore, we are very sorry for the confusion induced by the labelling. All images in Fig1B and C are from WT mice that were sutured. Fig1B illustrates expression of F4/80 and TNFRp55 (left) and TNFrp75 (right) in the limbus area whereas Fig1C shows the expression of F4/80 and TNFRp55 (left) and TNFRp75 (right) in the suture area. We improved the labelling and the figure legends to ease the understanding for the reader here.Q3: Why is TNFR2 75KDa not reduced in the TNFR2 knockout mouse in Fig 1D? Please reword Line 223-227 “The mRNA expression of TNFRp55 in the TNF-RP55d mice, however, is significantly reduced  and the functionality of the receptors is disturbed. There is no evidence for differential regulation of the other receptor in the TNF-Rpd animals, neither on mRNA nor on protein level  as is very confusing.Answer: As explained above the mutation in TNFR deficient mice does not lead to a lack of expression, thus, mRNA expression of TNFR2 does not have to be reduced in TNF-Rp75d mice. The explanation is as follows: As already stated above, the TNFR deficiency is a functional one. The mice harbor a null mutation of the respective TNFR leading to an altered gene product that lacks the molecular function of the wild-type gene. The non-functional proteins themselves, however, are expressed. The TNF-Rp55 null mutations were created by disruption of the coding sequence of the TNF-Rp55 gene at base pair 535 (corresponding to the cDNA sequence) by insertion of a neo gene [2]. This resulted in the transcription and transduction of a non-functional TNF-Rp55 protein. Pfeffer et al validated the mutation by an experiment that showed the inability of spleen cells of the TNF-Rp55-/- mice to bind TNFα. Erickson et al, creating the TNF-Rp75-/- mice, pursued a similar strategy [3]. In TNF-Rp75-/- mice challenged by LPS (that leads to shedding of the extracellular domain of TNF-Rp75), no soluble TNF-Rp75 could be detected in the serum. Furthermore, we rephrased the sentence p.9 lines 234-237 accordingly:” The mRNA expression of TNFRp55 in the TNF-RP55d mice, however, is significantly reduced . There is no evidence for differential regulation of TNF-Rp75 in TNF-Rp55d animals or vice versa, neither on mRNA nor on protein level Q4: Fig 1F and G leads to question the entire premise of the paper- it seems TNFR1 protein is present in mutant hence the authors are working with essentially wt mice?? What is the state of the TNFR2 mutant mice? Does it still express TNFR2 protein? Please validate mice before doing the suture experiments. Please show a western blot showing the protein levels of TNFR1 and TNFR2 in wt and respective mutant mice.Answer: The reviewer rises several important questions. First, the expression of TNF-Rp55 in TNF-Rp55d mice. As already stated in the answer to Q4 (Reviewer1), the mice harbor null-mutations of the respective genes leading to a malfunction of the receptors and not a lack of expression. Second, the reviewer asks for WB analysis of TNFR1 and TNFR2 expression in the different KI-models/WT mice. Figure 1F depicts an immunoblot showing the protein expression of TNF-Rp55 in TNF-Rp55d mice, TNF-Rp75d mice and wild-type littermates. As shown in bar chart 1G there is no difference in protein expression of TNF-Rp55 among the groups. Unfortunately, we cannot provide a WB of TNF-Rp75 since the antibody did not show a proper signal in WB in several trials.Q5: Also need to this experiment with the double knockout mice given TNFα knockout seems to exacerbate neovascularization (Fujita 2007).Answer: Thank you for the recommendation. The TNFα knockout mouse model is a completely different approach compared to our KI models. To evaluate our hypothesis, we however, decided to investigate initially whether one receptor is upregulated when the other is deficient. However, TNF-Rp55 is not upregulated in the TNF-Rp75d mice. Thus, we have convincing evidence, for the lack of compensatory upregulation of the TNFα-receptors expression in the KI-animals on both gene expression and protein expression level. Additionally, TNFα expression remained unchanged after suture placement. Suture placement led to differential regulation of TNFα at the mRNA level, no significant changes were detectable in protein levels, though. No differences were detectable in TNFα comparing WT to mutants. Therefore, we do not expect any influence of TNFα in a double KI or KO model. Q6: The figures all seem to be of poor resolution making it hard to zoom in on panels to see detail.Answer: As already stated above, we substantially improved the resolution of all images and headings.REFERENCES1. Khazen W, M'Bika J P, Tomkiewicz C, Benelli C, Chany C, Achour A, et al. Expression of macrophage-selective markers in human and rodent adipocytes. FEBS Lett. 2005;579(25):5631-4. Epub 2005/10/11. doi: 10.1016/j.febslet.2005.09.032. PubMed PMID: 16213494.2. Pfeffer K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, Shahinian A, et al. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell. 1993;73(3):457-67. Epub 1993/05/07. doi: 10.1016/0092-8674(93)90134-c. PubMed PMID: 8387893.3. Erickson SL, de Sauvage FJ, Kikly K, Carver-Moore K, Pitts-Meek S, Gillett N, et al. Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature. 1994;372(6506):560-3. Epub 1994/12/08. doi: 10.1038/372560a0. PubMed PMID: 7990930.4. Shih SC, Robinson GS, Perruzzi CA, Calvo A, Desai K, Green JE, et al. Molecular profiling of angiogenesis markers. The American journal of pathology. 2002;161(1):35-41. Epub 2002/07/11. doi: 10.1016/S0002-9440(10)64154-5. PubMed PMID: 12107087; PubMed Central PMCID: PMCPMC1850687.5. Giacomini C, Ferrari G, Bignami F, Rama P. Alkali burn versus suture-induced corneal neovascularization in C57BL/6 mice: an overview of two common animal models of corneal neovascularization. Exp Eye Res. 2014;121:1-4. Epub 2014/02/25. doi: 10.1016/j.exer.2014.02.005. PubMed PMID: 24560796.6. Jia C, Zhu W, Ren S, Xi H, Li S, Wang Y. Comparison of genome-wide gene expression in suture- and alkali burn-induced murine corneal neovascularization. Molecular vision. 2011;17:2386-99. Epub 2011/09/17. PubMed PMID: 21921991; PubMed Central PMCID: PMCPMC3171500.AttachmentRevision_TNFa_AnswerRevisionNRfinal.docxSubmitted filename: Click here for additional data file. 23 Oct 2020PONE-D-20-16932R1Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mousePLOS ONEDear Dr. MaierThank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please pay particular attention to the quality of images and results presented as requested by reviewers. 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You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Christina L Addison, Ph.D.Academic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: (No Response)Reviewer #3: All comments have been addressedReviewer #4: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: YesReviewer #3: YesReviewer #4: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: (No Response)Reviewer #4: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: (No Response)Reviewer #4: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: (No Response)Reviewer #4: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have improved the manuscript and addressed some of my concerns. However, there are fundamental discrepancies with previous literature that need to be explained prior to publication in PLOS ONE.B6.129-Tnfrsf1atm1Mak/J, Jackson lab stock 002818 and B6.129S2-Tnfrsf1btm1Mwm/J, Jackson lab stock 002620 are targeted knockout animal strains and do not express TNFRp55 or TNFRp75 respectively.These are conventional/“traditional” knockout mouse models that were generated in the Mid 90’s using a neomycin resistance gene flanked by homology arms to disrupt gene expression. Although the authors are correct that, in these studies, specific functional assays were used to confirm that the respective genes were no longer functional, this approach leads to a knockout of the targeted gene and not to a mutant form of the targeted gene.In particular, while the authors indicate that the targeted approach by Pfeffer et al. leads to “the transcription and transduction of a non-functional TNF-Rp55 protein”, Pfeffer et al. generated mice that lack expression of TNFRp55. Similarly, Erickson et al. created mice that do not express TNFRp75. These 2 mouse lines are well-established null/knockout/deficient mouse models that have been utilized extensively. In addition, the knockout strategy is well-described in these two published studies.With this in mind, the following questions still need to be addressed in a revised manuscript.Q2: The data in figure 1 would be more convincing if a negative control was included. Sincethe authors have access to the TNFR-deficient mice, these samples should be included todemonstrate specificity of their antibodies.Q4: It is not clear why the TNFR-deficient mice would show TNFR gene expression. Theauthors should validate their RT primers and repeat these experiments.Q5: Also, the western blot in figure 1F is inconclusive. Why is there no difference between wild type and knock out? Immunoblots for TNFR1 should also be included.Reviewer #2: Not sure why the labels on the images indicate the fluorphore as opposed to the antigen targeting - please correct.Adding the negative control to Figure 1, instead of only in a supplement, would still be advisable, but not absolutely necessary.Reviewer #3: (No Response)Reviewer #4: The authors have tried to address all the comments to the best of their abilities. However I still have several concerns:1. My primary issue is the quality of the immunofluorescence data. The quality of the mouse eye sections (they look battered) are still quite terrible and in good conscience cannot allow this to be published . The VEGFA stain is quite peculiar. A schematic to show the location of the suture would be helpful with the limbus and parts of the eye clearly labeled so one may interpret authors images.2. Please show data points for all graphs.3. The images on the pdf are still terrible. The quality of the fig page improves when dowloaded. It is quite onerous for the reviewer to download each image . I don't know why the supplementary images are not part of the document. Please do so in the next iteration.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  20 Dec 2020We thank the editorial board to give us the opportunity to improve our paper substantially according to the reviewers’ comments. We answer the reviewer’s concerns point by point as follows.Reviewer #1: The authors have improved the manuscript and addressed some of my concerns. However, there are fundamental discrepancies with previous literature that need to be explained prior to publication in PLOS ONE.Q1: B6.129-Tnfrsf1atm1Mak/J, Jackson lab stock 002818 and B6.129S2-Tnfrsf1btm1Mwm/J, Jackson lab stock 002620 are targeted knockout animal strains and do not express TNFRp55 or TNFRp75 respectively.These are conventional/“traditional” knockout mouse models that were generated in the Mid 90’s using a neomycin resistance gene flanked by homology arms to disrupt gene expression. Although the authors are correct that, in these studies, specific functional assays were used to confirm that the respective genes were no longer functional, this approach leads to a knockout of the targeted gene and not to a mutant form of the targeted gene.In particular, while the authors indicate that the targeted approach by Pfeffer et al. leads to “the transcription and transduction of a non-functional TNF-Rp55 protein”, Pfeffer et al. generated mice that lack expression of TNFRp55. Similarly, Erickson et al. created mice that do not express TNFRp75. These 2 mouse lines are well-established null/knockout/deficient mouse models that have been utilized extensively. In addition, the knockout strategy is well-described in these two published studies.With this in mind, the following questions still need to be addressed in a revised manuscript.We thank the reviewer for his thoroughness concerning the knock-in models. We still think there is a massive misunderstanding. We want to emphasize that B6.129-Tnfrsf1atm1Mak/J, Jackson lab stock 002818 and B6.129S2-Tnfrsf1btm1Mwm/J, Jackson lab stock 002620 lead to the transcription /translation of a truncated protein without proper function and are not KO-mutations in the sense that no mRNA and no protein at all are produced.It is well known that the classic knockout models produced in the 90s are sometimes not full knockouts in a sense that the mRNA is truncated behind the neomycin cassette and the protein’s length corresponds with nucleotide sequence before the neomycin cassette. In some cases, mRNAS from more distal parts of the gene are produced and lead to a protein that represents a C-terminal part of the protein. Normally in the papers that introduce the model demonstrate the knockout most of the time by mRNA techniques for nucleotide sequences close to neomycin cassette. Thus, we checked this possibility in our study.As the referee brought up this point in the sense to use TNFRp55d mice as antibody control for the anti-TNFR1 antibody we focus here in our response onto this mouse strain. The evidence comes from our PCR experiments where we show TNFRp55 expression in TNFRp55d mice, when using a primer that binds to nucleotide position 934-957 (F)/1085-1065 (R).According to the information from Jackson about this strain, the neo cassette is inserted at nucleotide position 535. Using a primer pair that binds to nucleotide 500-520 (F) and 714-733 R or 481-501 (F) and 695-715 R, respectively did not lead to a specific amplification product in TNFRp55d mice, whereas in WT and TNFRp75d it did.Figure1: mRNA expression of TNFRp55 in WT, TNFrp55d and TNFRp75d. Amplification product size: 234bp (left), 235bp (middle). GAPDH  Served as housekeeping gene. WT P17 O2 retina served as a murine control.Along with this the immunogen for generating the antibody, we used for IHC and WB (CAT#: AP06465PU-N), Origene is a synthetic peptide, corresponding to the amino acids 370-420 of the human TNF-R1, and corresponding to the C-terminal end of the antibody. Therefore, we cannot use the TNFRp55d mice as an antibody control. Thus, we conclude, that it is very likely that TNFRp55 is expressed as a truncated and non-functional protein in B6.129-Tnfrsf1atm1Mak/J.We base this conclusion both on our experimental evidence and the data from Pfeffer and Erickson.Our results presented in the manuscript are further substantiated by the new evidence derived from specific mRNA expression in TNFRp55d using primers beyond the neo-cassette and the use of C-terminal amino acids as immunogen.All the following questions (Q2-Q5) address the same issue: the expression of TNFRp55 in TNFRp55d mice. Thus, for answering the questions we refer to the answer to Q1.Q2: The data in figure 1 would be more convincing if a negative control was included. Sincethe authors have access to the TNFR-deficient mice, these samples should be included todemonstrate specificity of their antibodies.Not feasible due to truncated protein and antibody binding at the C-terminal end.Q4: It is not clear why the TNFR-deficient mice would show TNFR gene expression. Theauthors should validate their RT primers and repeat these experiments.TNF primers validated see above.Q5: Also, the western blot in figure 1F is inconclusive. Why is there no difference between wild type and knock out? Immunoblots for TNFR1 should also be included.See above; immunoblot for TNFR1. The western blot shows therefore the truncated protein in the TNFR1-KO, the full protein in the TNFR2 und WT probe. It shows that the TNFR2 knockout does not lead to a compensatory upregulation of the TNFR1.Reviewer #2: Not sure why the labels on the images indicate the fluorophore as opposed to the antigen targeting - please correct.Adding the negative control to Figure 1, instead of only in a supplement, would still be advisable, but not absolutely necessary.Answer: We thank the reviewer for his suggestion. However, we think this is a misunderstanding. Negative control, in this case, means that we incubated the section only with the secondary antibody, omitting the primary antibody, in order to show false positive staining of the secondary antibody. This is the reason why we indicated the fluorophore and not the antigen.As can be seen in the answer to Reviewer 1, it is impossible to have a TNFR1 negative control using the TNFRp55d mice, since they still express a truncated and non-functional TNFR1 protein, that can be detected by the antibody that binds to an amino acid sequence at the C-terminus. Reviewer #3: (No Response)Reviewer #4: The authors have tried to address all the comments to the best of their abilities. However, I still have several concerns:Q1. My primary issue is the quality of the immunofluorescence data. The quality of the mouse eye sections (they look battered) are still quite terrible and in good conscience cannot allow this to be published. The VEGFA stain is quite peculiar. A schematic to show the location of the suture would be helpful with the limbus and parts of the eye clearly labeled so one may interpret authors images.Answer: The quality of the mouse section depends on the processing of the sections. Embedding in paraffin, sectioning, rehydrating and especially the heat mediated antigen retrieval can lead to separations between the different corneal layers, which are very thin in the mouse cornea. Nonetheless, the various corneal layers, epithelium, stroma and endothelium, can be differentiated and are displayed very well in our figures. Indeed, the labeling is not sufficient; therefore we named the anatomic structures and marked the position of the sutures in our Figure 1, Supp. Figure 1 and 4. The limbus region is of particular interest due to the outgrowth of blood and lymph vessels into the cornea to the sutures. Q2. Please show data points for all graphs.https://doi.org/10.5281/zenodo.4049178). If Reviewer 3 is still convinced that the manuscript might profit from adding all data points, we can change the graphs accordingly.Answer: We agree with the reviewer about the importance of showing all data that is included in the experiments to ensure and improve scientific transparency and reproducibility. In our opinion, however, including all data points in the individual graphs would lead to graphs that are very confusing and difficult to interpret. All data points and all raw data analyzed for this manuscript are available at the open access database Zenodo using the following link . I don't know why the supplementary images are not part of the document. Please do so in the next iteration.Answer: We are very sorry for the bad quality of the pdf generated by the submission website. Unfortunately, we are not able to change this. When you download the images, they are in an adequate resolution and quality.The journal`s guidelines for submissions said to submit the supplement separately. We followed this suggestion. We add the images to the rebuttal letter to ease the review process.AttachmentTNFa_rebuttal2_finalNR.docxSubmitted filename: Click here for additional data file. 23 Dec 2020Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mousePONE-D-20-16932R2Dear Dr. MaierThank you very much for your careful consideration of the reviewer's concerns. We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.I would however advise you to carefully scrutinize image quality in the PLoS One system after uploading as indeed the quality of the labels of certain figures render them illegible, and the fluorescent images of those you attached are of much greater quality than those generated in the PDF of the manuscript following upload to the PLoS One Editorial Management system. As such I would contact the journal directly to ensure the image quality in your final manuscript that is to be uploaded online meets your standards and is not impaired by document transfers or resolution issues. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Christina L Addison, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 20 Jan 2021PONE-D-20-16932R2 Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse Dear Dr. Maier:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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Treatment for periodontitis can be performed starting from initial therapy and usually accompanied by additional therapy such as local drug delivery. VCO can be used as an alternative to antibiotics.  Virgin coconut oil is a natural product from coconut that has many benefits such as antibacterial, anti-inflammatory, and antioxidant. Inflammatory disease of the periodontal tissues has a high prevalence worldwide. The main etiology of periodontitis plaque of biofilm contains colonies of pathogenic microorganisms. The occurrence of inflammation in the periodontal tissue stimulates the release of inflammatory mediators, such as TNF-α and TGF-β1.  To determine the effectiveness of VCO gel on periodontal tissue regeneration materials through the expression of TNF-Porphyromonas gingivalis into the gingival sulcus. Wistar rats were then divided into 3 groups. On the 7th and 14th days, the rats were sacrificed and the jaw was sampled to determine the amount of TNF-α and TGF-β1 expression in the regenerative process of periodontal tissue.  This is an experimental laboratory with a posttest-only control group design. VCO was made from grated fresh coconut and then mixed with NaCMC to obtain gel viscosity. The subjects of this study were 30 male periodontitis-induced Wistar rats by injecting α and TGF-β1 increased significantly in the treatment group, but not as much as the increase in the positive control and negative control groups.  The amount of TNF-α and TGF-β1 in the regeneration process of periodontal tissue in periodontitis-induced rats. VCO gel can affect the expression of TNF- Increasing public awareness in utilizing natural ingredients as medicines has led to an increasingly rapid increase in the production of medicinal plants. This is because drugs derived from natural ingredients have relatively small side effects . CoconutA. actinomycetemcomitans and P. gingivalis which are the main bacteria that cause periodontal disease [VCO contains saturated fatty acids including Medium-Chain Fatty Acids (MCFAs) and Medium-Chain Triglycerides (MCTs). MCFA is a lauric acid which has antiprotozoal, antiviral, and antibacterial properties. MCT in VCO can increase immunity against disease and accelerate healing from illness and can prevent obesity , 7, 8. TPeriodontal disease is a disease caused by damage to the supporting tissues of the teeth that starts with reversible inflammation of the gingiva, which then gets worse until bone destruction occurs known as periodontitis. According to the FDI World Dental Federation in 2015, several countries in the world in 2010 had high prevalence of periodontitis, including Australia with a prevalence of more than 15%, while in Indonesia, almost all regions had a prevalence of more than 15%. The prevalence of periodontitis in people aged 15 years according to Riskesdas 2018 data is 67.8%, and this means that, out of ten people in Indonesia, 7 people suffer periodontitis , 10.Porpyromonas gingivalis , Tanerella forsythia, Provotella intermedia, and Treponema denticola which are often found in destructive periodontitis with deep pockets. One of the systemic is synthetic hormones found in hormonal contraceptives containing progesterone and estrogen [The etiology of periodontal disease can be divided into two groups, local factors and systemic factors. Local factors are usually caused by bacterial plaque, especially estrogen –13. Periestrogen , 15.α, IL-1β, IL-6, IL-8, and superoxide anion in human monocyte cell culture. Tumor Necrosis Factor-α (TNF-α) is a cytokine that stimulates cell proliferation and differentiation. Cardoso et al. [α, which causes tissue disruption. Increased levels of TNF-α, which are part of the pathogenesis of infection through the mechanism of phagocytic cells, such as polymorphonuclear, neutrophil, monocytes, and macrophages, will trigger the release of chemical mediators such as TNF-α that play a role in the occurrence of periodontal disease [α as proinflammatory cytokines in periodontal disease caused by a periapical lesion and TGF-β as a anti-inflammatory cytokines after endodontic treatment which shows that two cytokines play a role in the periodontal regeneration process [When inflammation occurs in the body, the immune response will release cytokines, chemical messengers, or intermediaries in intercellular communication. These inflammatory cytokines include TNF-o et al. , 16 deri disease –19. Neto process .α and TGF-β, which are synthesized by various normal cells and platelets. They affect the activation of macrophages, fibroblast proliferation, synthesis of connective tissue fibers and their matrix, local angiogenesis, healing, and also, regulation of lymphocytes [β1 is synthesized by various normal cells and platelets and affects macrophage activation, fibroblast proliferation, synthesis of connective tissue fibers and their matrix, local angiogenesis, healing, and regulation of T lymphocytes [β1 plays a very important role in the inflammatory response. The aim of this study was to determine the effectiveness of VCO gel on the expression of TNF-α and TGF-β1 in the tissue regeneration process after periodontitis.Growth factors regulate growth and differentiation of cells . Transfophocytes –24. TGF-phocytes , 26. TGF. gingivalis ATCC 33277 bacteria which were prepared in per liter culture media: 5 g yeast extract, 5 g peptone, and 200 ml goat blood filled up to 1 liter. Then, it was autoclaved for 15 minutes and put into the gingival sulcus of Wistar rats. After 5 days, periodontitis will form on the gingiva of Wistar rats which is characterized by a change in the colour of the gingiva and an increase in pocket depth.This is an experimental laboratory with a posttest-only control group design. VCO was made from grated fresh coconut and left for up to 24 hours to obtain colorless and odorless pure coconut oil. Then, the VCO was mixed with NaCMC to form gel for easy application during the research treatment. The subjects of this study were male Wistar rats weighing 150–200 grams which were induced by periodontitis by inserting a silk ligature on the lower anterior teeth and injecting Pα and TGF-β1 was then determined by immunohistochemical staining using TNF-α and TGF-β1 antibodies and viewed under a light microscope with 1000x magnification for further calculation of the number of expressions. The data obtained were tested for normality using the Kolmogorov–Smirnov test, and then, the data were tested for homogeneity using Levene's test. To analyze the differences between the research groups, one-way ANOVA was used. The results of the analysis were declared significant or having the difference if the p value <0.05.Wistar rats were divided into 3 groups, the treatment group receiving initial SRP therapy and VCO gel, the positive control group receiving SRP and metronidazole gel, and the negative control group only receiving SRP. Furthermore, euthanasia was carried out on the 7th and 14th days after treatment, with 5 Wistar rats from each treatment group. Then, the Wistar rats were fixed on the workbench for decapitation and separation of the cranium from the mandible. Each jaw piece was fixed in 10% buffered formalin, which would then be made into HPA preparations. The expression of TNF-P. gingivalis bacteria into the mandibular anterior gingival sulcus, periodontitis occurred in Wistar rats, characterized by changes in gingival colour and increased pocket depth. SRP as initial therapy for periodontitis was combined with VCO gel in the first group, SRP was combined with metronidazole gel in the positive control group, and only SRP was performed in the negative control group. The histopathological results in all research groups showed lymphocyte cells expressing TNF-α and TGF-β1 in the mandibular gingival sulcus that had been induced by periodontitis.On the fifth day after the binding of silk ligature and administration of α expression, were observed on the 7th and 14th days in the three treatment groups. α expression in male Wistar rats with periodontitis. In the treatment group with initial SRP therapy combined with VCO gel and the negative control group with SRP treatment alone, a significant increase was seen in the amount of TNF-α expression (p < 0.05) between day 7 and day 14, while in the positive control group, the combination of SRP and metronidazole gel showed the amount of TNF-α expression on the 7th and 14th days was not significant (p > 0.05).The results of immunohistochemical examination, TNF-α expression in the three groups, on the 7th and 14th day. On the 7th day, TNF-α expression in all groups increased, indicating an inflammatory response due to periodontitis. Combination of SRP and VCO gel showed an increase in TNF-α expression that was not as high as that of the positive control group and the negative control group on the 14th day, which indicated that VCO gel administration could suppress inflammation.The bar chart in α expression (α antibody (1000x magnification) showed an increased TNF-α expression indicated by brownish colour on the 7th and 14th days after treatment (p < 0.05). These results are shown as a bar chart in Immunohistochemical examination of TNF-pression  performeβ1 expression on the 7th and 14th days. It can be seen that all groups, the treatment group, positive control, and negative control, experienced a significant increase in TGF-β1 expression between the 7th and 14th days.β1 expression in the three groups, it can be seen that the treatment group experienced a significant increase amount of TGF-β1 (p < 0.05), compared to the other groups. The increasing amount of TGF-β1 expression was also smaller when compared to the that of positive control group and the negative control group on the 7th and 14th day. β1 antibody (1000x magnification). The increase of TGF-β1 expression on the 7th and 14th day in the three groups is shown in bar charts in Based on the bar chart , from thP. gingivalis bacteria were induced in the gingiva of Wistar rats, where the major causative agent in periodontitis is this bacterium which has been identified by the researchers and is associated with the severity of periodontal disease [α expression, compared to other groups.Periodontitis is defined as an inflammatory disease of the tooth-supporting tissue, caused by certain microorganisms. In this study,  disease . The treα is a proinflammatory cytokine whose sources are neutrophils, monocytes, and macrophages. One of the possible infection mechanisms is activation that stimulates monocytes and neutrophils to produce TNF-α, which causes tissue disruption. Increased levels of TNF-α are part of the pathogenesis of infection through the mechanism of phagocytic cells, as polymorphonuclear, neutrophil, monocytes, and macrophages will trigger the release of chemical mediators such as TNF-α cytokines that play a role in periodontal disease [Periodontitis TNF- disease –30.α will upregulate the receptor activator of RANKL NF-κβ ligand (RANKL). The upregulation of TNF-α and RANKL will increase the production of receptor activator of RANKL NF-κβ (RANK). This results in a binding between RANK and RANKL which then causes the uptake of the TNF-Receptor Associated Factor-6 (TRAF-6) adapter protein. TRAF-6 will induce c Fos and activator protein 1 (AP-1), which in turn induces Nuclear Factor of Activated T cells c1 (NFAT C1). RANK-RANKL binding that causes the uptake of the adapter protein TNF-Receptor Associated Factor-6 (TRAF-6) will cause a signal transduction sequence that causes increased activation of NFATC1, which functions as a transcription factor for osteoclast formation and enhances the number of mature osteoclasts. These mature osteoclasts then form active osteoclasts, which stimulate periapical alveolar bone resorption [An increase in TNF-sorption , 30, 31.α and TGF-β1 on the 7th day increased in small amounts on the 14th day when the repair phase had begun. TNF-α and TGF-β1 increased significantly when compared with those of the positive control group and the negative control group. The significant increase in TNF-α expression (p < 0.05) in this study was probably due to the lauric acid content in the VCO gel which functions in the inflammatory process and also has antibacterial properties, so it can accelerate the tissue regeneration and healing process.Cytokine production is an immune response. The expression of TNF-α-expressing cells in periodontal tissues that were inflamed due to the induction of P. gingivalis bacteria by administration of VCO gel, not as much as TNF-α expression in the positive control group and negative control group. This proves that the VCO gel helps in suppressing the inflammatory process so that it can accelerate tissue regeneration.In this study, it was proven that there was a significant increase in the number of TNF-β1-expressing cells in the periodontal tissue that experienced inflammation due to the induction of P. gingivalis bacteria was higher than that in the periapical tissue in the negative control group but lower when compared to that in the positive control group considering that TGF-β1 is a multifunctional regulator of cell growth and differentiation during formation and repair. Moreover, TGF-β1 has various (pleiotropic) effects, including as a growth inhibitor for most epithelial cells and for leukocytes. Since inflammation is still occurring in the periodontal tissue, the release of TGF-β1 is blocked.It was also found that the number of TGF-Rattus norvegicus applied VCO gel after tooth extraction. The results obtained were an increase in the number of fibroblasts 0.4 times that in the group that was applied to VCO compared to povidone iodine. VCO also affects dermal and epidermal healing and provides strength to epithelial tissue [This research conducted by Nevin et al. on 18 Sprague Dawley with excision wounds proved that VCO was able to increase the proliferation of fibroblast cells so that the density of collagen fibers increased, whereas wounds treated with VCO healed faster, indicated by a decrease in complete epithelialization time and a higher rate of reepithelialization of various skin components. Pepsin-soluble collagen also showed a significant increase in VCO-treated wounds , 33. Anol tissue .The VCO gel which is produced from coconut flesh is known to have the ability to accelerate cell metabolism and has anti-inflammatory and anti-infection properties, with minimal possibility of resistance to chemical medicament. Further research with various concentrations of VCO is needed to determine the best levels in the therapy tissue regeneration.α and TGF-β1 in periodontitis-induced Wistar rats with SRP treatment and VCO gel administration, which is a marker of periodontal tissue regeneration. Further studies are needed with a larger study population to analyze the VCO gel on periodontal regeneration, both in soft and hard tissues, with various levels of VCO concentrations.Based on the results of this study, it can be concluded that there was a significant increase in the expression of TNF-


Promoter region SNPs in TNF-α have been studied in association with Recurrent Pregnancy Loss (RPL) occurrence in various populations. Among them, −238G > A, −308G > A and − 376G > A have been frequently investigated for their potential role in recurrent abortions. The aim of the present study is to evaluate the correlation among TNF-α 238, TNF-α 308 and TNF-α 376 polymorphisms and recurrent pregnancy loss risk in Greek women.This study included 94 Caucasian women with at least two miscarriages of unexplained aetiology, before the 20th week of gestation. The control group consisted of 89 Caucasian women of proven fertility, with no history of pregnancy loss. DNA samples were subjected to PCR amplification using specific primers. Sanger sequencing was applied to investigate the presence of TNF-α 238, TNF-α 308, TNF-α 376 polymorphisms in all samples.p-value 0.396 and 0.374, respectively). The TNF-α 376 variant was not detected at all in both control and RPL groups. When TNF-α 238 and TNF-α 308 genotypes were combined no association with RPL was detected . In subgroup analysis by parity, RPL patients carrying the A allele reported less previous births.The TNF-α 238 and TNF-α 308 variants were both detected in RPL and control groups , but with no statistically significant association (This is the first study demonstrating TNF-α 238 and TNF-α 308 gene expression and the absence of TNF-α 376 variant in Greek women with RPL. However, no association emerged between each polymorphism studied and the occurrence of recurrent pregnancy loss. Accordingly, TNF-α -308G > A, −238G > A and -376G > A variants are not considered genetic markers for identifying women at increased risk of recurrent pregnancy loss in the Greek population. Recurrent pregnancy loss (RPL) is defined as three or more consecutive spontaneous abortions before the 20th week of gestation, as proposed by the European Society of Human Reproduction and Embryology, and occurs in approximately 1–2% of reproductively active couples . RPL is Emerging evidence indicates that endometrial immune dysregulation could be responsible for several, if not many, cases of RPL of unknown origin. Among the immune factors, cytokines seem to play a pivotal role in reproductive dysfunctions –11. TumoSeveral single nucleotide polymorphisms (SNPs) have been reported to regulate the transcription and production of TNF-α. In particular, promoter region SNPs in TNF-α have been studied in association with RPL occurrence. Among them, −376G > A rs1800750), −308G > A (rs1800629) and − 238G > A (rs361525) have been frequently investigated for their potential role in recurrent abortions , 17. Alt00750, −3Given the controversial data upon TNF-α variants and RPL, the aim of the present study is to investigate the possible correlations between TNF-α 308, TNF-α 238, and TNF-α 376 polymorphisms and recurrent pregnancy loss risk separately and combined, thus revealing the genetic profile probably related to recurrent abortions occurrence in the Greek population. These three above-mentioned TNF-α variants were selected to be studied herein because they are among the most frequently reported TNF-α variants in association with RPL occurrence in various populations.The study group included 94 Caucasian women with at least two miscarriages of unexplained aetiology, before the 20th week of gestation who visited the Recurrent Miscarriage Outpatient Clinic of Alexandra Hospital. Women with a history of thromboembolic, infectious, autoimmune, endocrine or chromosomal disorders, and cervical anatomical abnormalities were excluded from the study. The control group included 89 Caucasian women of proven fertility with no history of pregnancy loss. The study protocol was approved by the Ethics Committee of Alexandra Hospital and a signed informed consent was obtained from each participant of the present study.2+, 10 mM dNTP mixture, 1 μl 50 mM MgCl2, 1 μl Primer Sense mix, 1 μl Primer Antisense mix, 1 μl Template DNA, 0.3 μl Taq DNA polymerase and 17.2 μl of distilled water. The PCR conditions were 95 °C for 10 min and 95 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min for 29 cycles, with a final extension step at 72 °C for 10 min. The tubes were then incubated at 72 °C for 10 min. PCR products were subjected to agarose electrophoresis to verify for the expected length. Subsequently, Sanger sequencing was applied in an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems™) to determine the presence of TNF-α 238, TNF-α 308, TNF-α 376 polymorphisms in all samples. BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™) was used in the sequencing reactions.Peripheral blood (2-3 ml) was collected from all the recruited women. DNA isolation was performed using the PureLink Genomic DNA kit provided by Invitrogen Life Technologies. DNA samples were subjected to PCR amplification using specific primers for TNF-α 376, TNF-α 238, and TNF-α 308 polymorphisms. All primers were designed by TIB MolBiol and their sequences were as follows: for TNF-α -238G > A, forward primer: 5′-TTCCTGCATCCTGTCTGGAA, reverse primer: 5′-CAGCGGAAAACTTCCTTGGT-3′; for TNF-α -308G > A, forward primer: 5′-AGGCAATAGGTTTTGAGGGCCAT-3′, reverse primer: 5′-TCCTCCCTGCTCCGATTCCG-3′; for TNF-α -376G > A, forward primer: 5′-GCTGCACTCGATGTACCAC-3′, reverse primer: 5′-CAAGTTCTACAGAGCGAAGG-3′. The PCR protocol included adding 10X PCR Buffer minus Mgp-value < 0.05 was considered as statistically significant. Statistical analysis was performed using STATA version 13.1 .The differences between polymorphism frequencies were assessed by the chi-square test, while unpaired t-test was performed to evaluate the differences for age and body mass index (BMI) between groups. A TNF-α gene were studied in 94 cases and 89 controls. In particular, the TNF-α -238G > A variant was detected in heterozygosity in 7 out of the 94 patients , while it was found to be present in 4 women of the control group . However, no statistically significant difference was observed between patient and control groups indicating that GA genotype can be equally detected either in women with RPL or controls  and control groups , whereas no statistically significant association emerged between GA genotype and RPL patients  and heterozygous (GA) for TNF-α -308G > A polymorphism have an increased risk of developing RPL compared to women who carry the wild-type allele. In addition, when stratifying the results according to the geographical origin of the participants, Asian populations show a stronger association between TNF-α -308G > A polymorphism and RPL occurrence than European populations. In contrast, for TNF-α -238G > A polymorphism, no statistically significant difference was observed in the incidence of homozygous (AA), heterozygous (GA) and homozygous for the wild-type allele (GG) genotypes between women who have experienced recurrent pregnancy loss and fertile women [The association between TNF-α polymorphisms and RPLs has been investigated in many studies; however, the results remain contradictory and unreliable. Population diversity could explain such opposing conclusions. In particular, previous meta-analyses have shown that the risk of RPLs is significantly associated with the incidence of TNF-α −308G > A and − 238G > A polymorphisms in the overall population , while ile women .In a prospective cohort study the frequency of TNF-α -308G > A variant was investigated in 1652 pregnant women with intrauterine fetal death, preeclampsia, preterm labor before 34 gestational weeks, and small-for-gestational-age (SGA) infants. The researchers concluded that there was no statistically significant difference in the incidence of TNF-α -308G > A between the group with the above-mentioned pregnancy complications and the control group . SimilarP = 0.021) and miscarriage (P = 0.023) [P = 0.011) risk for recurrent miscarriages. Additionally, there were significant differences in the frequency distribution of -376G > A genotype (P = 0.002) between patients and control group [Regarding TNF-α -376G > A polymorphism, Palmirrotta and his colleagues showed no association with recurrent miscarriages in Caucasian Italian women, but TNF-α -376G/−308A/−238G haplotype was associated with low TNF-α levels (= 0.023) . Howeverol group . All theAn interesting observation emerged upon association of TNF-α 238 and 308 polymorphisms and parity status in RPL patients, since parity was less reported in patients carrying the A allele. A similar disclosure has been published for susceptibility to pre-eclampsia, wherein an analysis by parity revealed that carriers of A allele and AA genotype frequency of maternal TNF-α -308G > A polymorphism were associated with an increased risk of pre-eclampsia .TNF-α gene polymorphism spectrum and the levels of the respective protein and therefore its function, may elucidate the potential link to recurrent abortions. Additionally, the establishment of a panel of cytokine gene polymorphisms associated with recurrent abortions would serve as an important biomarker for the prediction of miscarriages, promoting the individualized treatment of women with a history of recurrent pregnancy loss, thus increasing successful pregnancy potential. It is a common observation that patients with the same diagnosis may respond differently to therapies and it is becoming increasingly evident that this differential responsiveness may be due to specific DNA variations at the genomic level. New technologies such as next-generation sequencing (NGS), and bioinformatics, will enable a better understanding of the role of various genetic polymorphisms that will facilitate the next breakthrough toward further improving the clinical success for each individual RPL patient. Consequently, the challenge for future medicine is to move from a population-based view to an individually-based one through the implementation of novel technologies in the daily medical practice, establishing the Precision Medicine in human reproduction [Remarkably, elevated levels of TNF-α at the maternal-fetal interface induce activation of natural killer cells (NK cells) and subsequent damage to placental growth , 31. Invoduction , 33.TNF-α gene polymorphisms in Greek women with recurrent pregnancy loss, while revealing lack of association between the above-mentioned variants and the development of RPL in the Greek population. Such novel findings could help to further elucidate the pathophysiology of RPL in the Greek population, highlighting the importance of genetic profiling of each RPL patient.To our knowledge, the present work firstly reports the distribution of In conclusion, the present study demonstrates TNF-α 238 and TNF-α 308 gene expression in the Greek women population and the absence of expression of TNF-α 376 variant. Moreover, it is disclosed that parity was less reported in RPL patients carrying the A allele. On the other hand, TNF-α 238 and TNF-α 308 variants were not correlated with the occurrence of recurrent pregnancy loss. Additionally, no association emerged between TNF-α 238 and TNF-α 308 genotype combination and recurrent abortions. Ultimately, TNF-α -308G > A, −238G > A and -376G > A variants are not considered genetic markers for identifying women at increased risk of recurrent pregnancy loss in the Greek population.


However, the contribution of SCARF1 to hepatocellular carcinoma (HCC) is currently unknown. We utilized publically-available RNA-sequencing data from The Cancer Genome Atlas (TGCA) to explore SCARF1 expression in HCC and correlated it with a number of clinicopathological features. Flow adhesion assays were used to determine the role of SCARF1 in CD4+ T cell subset recruitment. SCARF1 expression was downregulated in HCC tumor tissues, compared to non-tumoral tissues, and loss of SCARF1 expression was associated with poorly differentiated/aggressive tumors. Additionally, higher SCARF1 expression in HCC tumor tissues was highly prognostic of better overall, disease-free and progression-free survival. SCARF1 within HCC was largely associated with tumor endothelial cells and adhesion studies suggested that it played a role in the specific recruitment of proinflammatory CD4+ T cells (CD4+CD25−) to HCC tumor tissues. Endothelial SCARF1 expression in tumor biopsies may provide critical prognostic information. Additionally, SCARF1 may also be a novel endothelial target that could help re-programme the microenvironment of HCC by promoting effector T cell tumor infiltration.Scavenger receptor class F member 1 (SCARF1) is thought to play an important role in the selective recruitment of CD4 Due to in vitro –16. We hin vitro , 14; howScavenger receptors are a large super-family of proteins which are defined by their ability to bind and endocytose a vast range of endogenous and exogenous ligands, eliciting the “scavenging” of unwanted macromolecules from the bloodstream . Functio+ T cells to the sinusoidal endothelium under physiological shear stress , also known as scavenger receptor expressed by endothelial cells (SREC)-I, was first identified in cDNA libraries from human umbilical vein endothelial cells (HUVEC) . SCARF1 r stress . In thisr stress .http://cancergenome.nih.gov), we describe the differential regulation of scavenger receptors in HCC tumor tissues, compared to non-tumorous control tissues, and specifically focussed on the downregulation of SCARF1 expression. We corroborated these findings with immunohistochemical staining, which also showed reduced protein expression in HCC tumor tissues, and next explored the relationship of SCARF1 expression with tumor progression. Consequently, we found an association with loss of SCARF1 expression with aggressive tumor biology. Following this, we evaluated the prognostic value of SCARF1 expression in HCC tumors by generating survival curve data, via KM Plotter . In support of the pathological findings, high SCARF1 expression in HCC tumor tissues was found to correlate with a better overall survival, disease-free survival and progression-free survival. Next, via a combination of TGCA data analysis and immunofluorescent staining, we determined that SCARF1 within HCC was largely associated with tumor endothelial cells. Finally, we extended our previous findings with primary human liver endothelial cells by studying subsets of CD4+ T cells. Using flow-based adhesion assays under physiological levels of shear stress our findings suggested that SCARF1 could play a role in the recruitment of proinflammatory CD4+ T cells (CD4+CD25−), rather than immunosuppressive T cell subsets, to the HCC tissue microenvironment. Our results demonstrate that SCARF1 could be a prognostic biomarker in HCC. Furthermore, SCARF1 expression could potentially be targeted to alter the inflammatory status of the tumor microenvironment, shifting it toward an anti-tumoral immune response and supporting immunotherapy regimes for HCC.Here, through the utilization of the publically-available TGCA (The Cancer Genome Atlas) RNA-sequencing datasets (https://xenabrowser.net/) was used. Correlation of SCARF1 expression with tumor progression/aggression and cell-specific markers was performed via the cBioPortal website  (accessed 25th Feb 2020). With the use of the publically-accessible tool KM Plotter , survival data was generated from the TGCA dataset over a 60-month time period, with the data being split into two groups (“High” and “Low”) by the median of SCARF1 expression. Resultant data was exported to Prism® 6 software (GraphPad Software Inc.) and survival curves were produced. The Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/) was used to generate a list of the top 25 genes regulated in conjunction with SCARF1 in HCC tumor tissues. Level of CD4+ T cell infiltration of HCC tumors was correlated with SCARF1 expression via the Tumor IMmune Estimation Resource .Publically-available data from the The Cancer Genome Atlas (TGCA) was utilized throughout this study. To explore scavenger receptor family expression in tumor and relevant non-tumorous tissue controls from the TGCA dataset, the University of California Santa Cruz (UCSC) Xena tool (All liver tissue samples were collected from patients undergoing transplantation for chronic liver disease or primary hepatocellular carcinoma (HCC) at the University Hospitals Birmingham NHS Foundation Trust, with written informed consent and local ethics committee approval. All experiments were performed in accordance with the regulations and guidelines sanctioned by the West Midlands—South Birmingham Research Ethics Committee, Birmingham, UK (LREC reference 06/Q2702/61 and 04/Q2708/41).2O. Nuclei were then counterstained with Mayer's Hematoxylin  for 30 s and slides were washed in warm H2O for 2 min. Sections were subsequently dehydrated in sequential washes of alcohol (3×) and xylene (3×) and mounted using DPX  mounting medium (CellPath). Images were taken using an Axio ScanZ1 microscope (ZEISS). Surface area coverage of SCARF1 staining was performed by via threshold analysis using ImageJ software. Five random high-power fields of view were analyzed per section, with the average value taken for each matched pair of tumor and non-tumorous tissues.Immunohistochemical staining was performed on 7 μm thick acetone-fixed cryosections, stored at −20°C. Prior to staining, sections were thawed to room temperature (RT) and hydrated with PBS/0.1% Tween® 20 (PBST) for 5 min. Endogenous peroxidase activity was then blocked with 0.3% hydrogen peroxide in methanol and blocking of non-specific binding was performed by incubation with 2X Casein Solution . Sections were incubated with anti-SCARF-1 primary antibody  diluted in PBS for 1 h at RT and then washed twice in PBST for 5 min. Isotype matched controls at appropriate concentrations were performed in all experiments. Subsequently, sections were incubated with the anti-rabbit ImmPRESS™ HRP for 30 min at RT. Excess secondary antibody was washed off with PBST for 5 min (twice) and sections were then incubated with DAB chromogen (Vector Laboratories Inc.) for 2 min; the reaction was stopped with the addition of distilled HFor immunofluorescent staining, 7 μm acetone-fixed cryosections were thawed and then blocked for non-specific binding by incubation in PBS with 10% goat serum and casein solution, for 30 min at RT. This was followed by 1 h incubation with primary antibodies for SCARF-1  and CD31 . Samples were washed three times in PBS followed by 30 min incubation with Alexa Fluor® conjugated secondary antibodies . Nuclei were stained with 300 nM DAPI (Invitrogen) and slides were subsequently mounted with ProLong™ Gold Antifade Mountant (Invitrogen). Fluorescence images were acquired using a Zeiss 780 Zen confocal fluorescence microscope (ZEISS).2.Liver sinusoidal endothelial cells (LSEC) were isolated from ~30 g human liver tissue as described previously . Briefly+CD25+ T lymphocytes were subsequently isolated from PBMCs by Dynabeads™ Regulatory CD4+/CD25+ T Cell Kit, and in accordance with manufacturer's instructions. The CD4+/CD25− fraction obtained via this isolation protocol was kept and used as an “effector” population in flow-based adhesion assays, as previously described  were isolated from whole blood via density gradient centrifugation; briefly, whole blood was layered on Lympholyte®-H (Cedarlane) and centrifuged at 800 × g for 25 min. The PBMC layer was removed and washed in PBS with 2% FCS and 1 mM EDTA (Gibco™ by Thermo Fisher Scientific) and centrifuged at 800 × g for 5 min. A platelet depletion step was then performed by a second wash in PBS with 2% FCS and 1 mM EDTA and centrifugation at 350 × g for 10 min. CD4escribed .in vitro, under conditions of physiological flow. Briefly, approx. 7.5 × 105 LSEC were seeded in rat tail collagen-coated μ-slide VI 0.4 and grown to confluence overnight. Cells were then stimulated with 10 ng/ml TNFα for 24 h to induce endothelial activation. CD4+CD25+ or CD4+CD25− T lymphocytes were isolated (see ‘Primary Lymphocyte Isolation’ above) and resuspended at a cell density of 1 × 106 cells/ml in a flow medium of Endothelial SFM with 0.1% BSA. Lymphocytes were then perfused over the LSEC at a physiological shear of 0.05 Pa, with each channel of the μ-slide perfused for 5 min. Subsequently, channels were washed though for 3 min with flow media alone, after which video recordings were taken. All flow assays were imaged via phase-contrast microscopy on an Olympus IX50 Inverted Microscope (Olympus) and 12 frames from each channel were analyzed. The number of adherent lymphocytes was firstly counted and then normalized to cells/mm2/106 cells perfused using the following equation: adherent cells/flow rate (0.28 ml/min) × bolus (5 min) × field of view area (0.154 mm2) × (1/ concentration of lymphocytes 1 × 106 cells/ml). The addition of SCARF-1 blocking antibody  or rabbit polyclonal negative control  was performed immediately preceding each assay and incubated for 30 min .All data were tested for normal distribution by the D'Agostino-Pearson omnibus test. All data were found to be non-parametric and so were expressed as median ± interquartile range (IQR), with the number of experimental repeats (n) specified in each case. For single comparisons, statistical significance was determined by Mann–Whitney MARCO, SCARA5, CLEC7A, MRC1, SCARF1, SCARF2, STAB1, STAB2, CD163, AGER) demonstrated significantly decreased expression in HCC tumor, compared to non-tumorous liver tissues  did not exhibit any regulation  lower in HCC tumor tissues in comparison to non-tumorous tissues  reduction in SCARF1 expression in tumor tissues, when compared to distal, non-tumorous tissues  and Grade 4 (p ≤ 0.01) tumors, when compared to Grade 1 tumors  through to highly developed and metastatic disease (Stage IV). When compared to patients with Stage I disease, cohorts of patients with Stages II, III and IV disease all demonstrated a trend for decreased SCARF1 expression; however, only the data for the Stage II cohort was calculated to be statistically significant (p ≤ 0.01)  . We furtdy Score  and Buffdy Score . In both tissues , thus prSCARF1 expression correlates with more advanced and aggressive tumors, we next sought to investigate its prognostic value in HCC. With regards to overall survival, high expression of SCARF1 was highly indicative of a better prognosis , disease-free survival  and progression-free survival  in patients with non-viral HCC, but exhibited no prognostic value in viral HCC patients. Furthermore, expression of SCARF1 showed no prognostic value with regards to cancer staging or in the presence/absence of vascular invasion  to generate a list of genes commonly regulated in conjunction with SCARF1 within HCC tumor tissues  exhibited a highly significant positive correlation with SCARF1 expression  to correlate SCARF1 expression with the level of CD4+ T cell infiltration of HCC tumors. Using TIMER, we confirmed that SCARF1 expression is absent from tumor cells, as indicated by a negative “purity” correlation  inhibitory effect on the adhesion of proinflammatory (CD4+CD25−) T cells (effectors), but a negligible (~10%) effect on regulatory (CD4+CD25+) T cells (Tregs)  .in vivo on the endothelium of human HCC, thus suggesting that they may also contribute to immune cell recruitment to the tumor microenvironment (Hepatocellular carcinoma (HCC) predominantly manifests on a background of cirrhosis and, consequently, in conjunction with the global rise in chronic liver diseases, incidence of HCC is also set to rise. Novel medical therapies are urgently required as patients often present to clinic with advanced tumors without curative options . Recentlironment , 14.Scavenger receptors represent a major subset of innate pattern recognition receptors able to bind a number of cancer-relevant ligands, such as heat shock proteins (HSPs) , 53 and SCARF1 within HCC tumor tissues were endothelial-specific  T cells (effectors), but had little effect the adhesion of regulatory (CD4+CD25+) T cells (Tregs). This is in direct contrast to our previous work on another endothelial-expressed scavenger receptor, Stabilin-1, which is also present in HCC tumors, but plays a role in the specific recruitment of anti-inflammatory regulatory (CD4+CD25+) T cells . This is particularly pertinent as previous studies have specifically shown that an increased prevalence of Tregs is an independent prognostic factor in HCC; therefore, shifting this balance could have a significant impact on patient outcome  T cells to HCC tumors, leading to decreased tumoral progression and, ultimately, a better overall outcome  of the drug “Clevergen” in patients with HCC and also reports consulting for Faron Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.