Soluble TNF superfamily (TNFSF) ligands are less stable and less active than their transmembrane (tm) analogues. This is a problem for the therapeutic use of recombinant TNFSF ligands in diverse diseases including cancer and autoimmunity. Creating TNFSF ligand analogues with improved targeting of their respective receptors is important for research and therapeutic purposes.Covalent internal cross-linking of TNF monomers by double mutations, S95C/G148C, results in stable trimers with improved TNFR2 function. The resulting mutein induced the selective death of autoreactive CD8 T cells in type-1 diabetic patients and demonstrates targeted proliferation and expansion of human CD4 Tregs.Stable TNF trimers, created by internal covalent cross-linking, show improved signaling. The high structural homology within the TNF superfamily provides an opportunity to extend internal cross-linking to other TNF superfamily proteins to produce active trimers with improved stability and receptor signaling, and with potential applications for cancer, autoimmunity, infections, and transplantation.The online version of this article (doi:10.1186/s40591-015-0044-4) contains supplementary material, which is available to authorized users. The tumor necrosis factor (TNF) superfamily, composed of both ligands and receptors, is one of the most successfully and frequently targeted protein families with clinical relevance to diverse diseases as cancer, autoimmunity, infections, graft rejection, inflammation and osteoporosis , 2. DepeTNF is a pleiotropic cytokine with diverse functions in cell proliferation, inflammation, apoptosis and morphogenesis . TNF is Additional file Building on our earlier research , 15, we Western blot analysis of the TNF07 mutein confirms a stable covalent trimer with an apparent molecular weight of 75 kDa compared to recombinant sTNF with an apparent molecular weight of 25 kDa     Fig. . The add56) Fig. . In mark03) Fig. . This prTo further test whether TNF07 was acting as a functional TNFR2 agonist, we sought evidence that the less robust proliferation of CD8 T cells with TNF or TNF07 in Fig. Here we show that the covalent cross-linking of sTNF at a strategic location between TNF monomers can generate a stable trimer with improved functional agonistic consequences on human CD4 or CD8 T cells. The results of the human functional T cell assays indicate that TNF07 has improved TNFR2 agonism, more potent than sTNF. Internal cross-linking at a different location did not result in improved TNFR2 activity. TNF07 not only induced the selective death of autoreactive CD8 T cells in type-1 diabetic patients, it also demonstrated impressive proliferation of normal CD8 T cells. TNF07 was also capable of expanding CD4-Treg cells, again a function of TNFR2 agonism. Various defects in TNF signaling pathways occur in both human and mouse models of several autoimmune disorders. sTNF, TNF inducers or TNFR2 agonism have shown promise for treating these subtypes of autoimmune diseases . Due to Due to the high structural homology, the concept of disulfide cross-linking is broadly applicable to all TNFSF ligands. It provides an alternative to other trimer stabilizing methods as a more naturally appearing, and less immunogenic structure upon human exposure. It is an expandable system that can be combined with receptor specific mutations to selectively target various TNFSF receptors, and also with secondary cross-linking methods of either the TNFSF ligands or their receptors , 7, 23.


Gammapapillomavirus. HPV175 and HPV180 were isolated from a condyloma. HPV178 was isolated from healthy skin adjacent to an actinic keratosis.We report the characterization of three novel human papillomavirus (HPV) types of the genus  Gammapapillomavirus. HPV175 (SE87) and HPV180 (FA69) were originally detected as complete genomes in a condyloma swab sample from a 30-year-old male (We report the complete genome sequences of three novel human papillomavirus (HPV) types of the genus Briefly, multiple-displacement amplification (MDA)-amplified HPV DNA  was reamAmplicons were cloned using a TOPO TA cloning kit and the pCR 2.1-TOPO vector  and sequenced using primer walking (Eurofins).Pairwise comparisons of the L1 open reading frames (ORF) of HPV175, HPV178, and HPV180 demonstrated closest nucleotide homology to HPV60 , HPV65 , and HPV121 , respectively.6-GGT), one putative TATA box (TATAA), and one putative polyadenylation site (ATATAA). In the URR of HPV178 (456 bp), three E2-binding sites were present, as well as one putative TATA box and one putative polyadenylation site. In the URR of HPV180 (544 bp), we identified four consensus E2-binding sites, three putative TATA boxes, and one putative polyadenylation site.HPV175, HPV178, and HPV180 all demonstrate a typical genome organization of cutaneous HPVs, with an upper regulatory region (URR), five early genes , and two late genes (L1 and L2). In the URR of HPV175 (488 bp) we identified five consensus E2-binding sites 29CxxC]  was obseSDTGKS and for HPV180 GKPNTGKS. An initiation codon of the putative start of the E4 ORF was present in HPV175 and HPV178, whereas the corresponding codon was absent in the E4 ORF of HPV180. This is in agreement with the HPV types of species 10 of the genus Gammapapillomavirus (Only the putative E1 protein of HPV178 had the conserved ATP-binding site (GPPDTGKS) , 5. For omavirus .KC108721), HPV178 (KJ130020), and HPV180 (KC108722).The complete genomic sequences are available in GenBank under these accession numbers: HPV175 (


Two papers ,2 were r


Objectives: Rheumatoid arthritis (RA) is characterized by defective bone repair and excessive destruction and ankylosing spondylitis (AS) by increased ectopic bone formation with syndesmophytes. Since TNF-α and IL-17A are involved in both diseases, this study investigated their effects on the osteogenic differentiation of isolated human bone marrow-derived mesenchymal stem cells (hMSCs).Methods: Differentiation of hMSCs into osteoblasts was induced in the presence or absence of IL-17A and/or TNF-α. Matrix mineralization (MM) was evaluated by alizarin red staining and alkaline phosphatase (ALP) activity. mRNA expression was measured by qRT-PCR for bone morphogenetic protein (BMP)-2 and Runx2, genes associated with osteogenesis, DKK-1, a negative regulator of osteogenesis, Schnurri-3 and receptor activator of nuclear factor kappa B ligand (RANKL), associated with the cross talk with osteoclasts, and TNF-α receptor type I and TNF-α receptor type II (TNFRII).Results: TNF-α alone increased both MM and ALP activity. IL-17A alone increased ALP but not MM. Their combination was more potent. TNF-α alone increased BMP2 mRNA expression at 6 and 12 h. These levels decreased in combination with IL-17A at 6 h only. DKK-1 mRNA expression was inhibited by TNF-α and IL-17A either alone or combined. Supporting an imbalance toward osteoblastogenesis, RANKL expression was inhibited by TNF-α and IL-17A. However, TNF-α but not IL-17 alone decreased Runx2 mRNA expression at 6 h. In parallel, TNF-α but not IL-17 alone increased Schnurri-3 expression with a synergistic effect with their combination. This may be related to an increase of TNFRII overexpression.Conclusion: IL-17 increased the effects of TNF-α on bone matrix formation by hMSCs. However, IL-17 decreased the TNF-α-induced BMP2 inhibition. Synergistic interactions between TNF-α and IL-17 were seen for RANKL inhibition and Schnurri-3 induction. Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA. Conversely, in the absence of osteoclasts, this could promote ectopic bone formation as in AS. In rheuel of RA .The mechanisms used by IL-17A to promote bone loss include activation of osteoclastogenesis, which occurs both directly and also through expression of receptor activator of nuclear factor kappa B ligand (RANKL) and M-CSF by stromal cells , 13. MorDue to these conflicting results, our objective was to examine whether IL-17A alone and/or TNF-α, positively or negatively modulate osteogenic differentiation in hMSCs. To study these aspects, we focused on key genes involved in bone turnover: BMP2, Runx2, DKK-1, RANKL, and Schnurri-3, a gene recently associated with bone resorption in mice –34, but l-glutamine, 100 U/ml penicillin, streptomycin. Cells were used between passage 3 and 6 at which cells were >99% stained negative for CD34 and CD45 and positive for CD73 and CD90 (antibodies obtained from PharMingen). For osteogenic differentiation, hMSCs were plated at a density of 5 × 103 cells/cm2 and cultured in stem Xvivo Osteogenic/adipogenic base Medium , supplemented with 100 nM dexamethasone , 10 mM β-glycerophosphate (Sigma), and 50 μM ascorbic acid (Sigma). hMSCs were differentiated for 21 days in the absence or presence of 1 ng/ml TNF-α  and/or 50 ng/ml IL-17A (R&D systems)  was used to optimize culture and expansion of hMSCs. Cells were cultured at 37°C in α-MEM  supplemented with 10% fetal bovine serum , 2 mM systems) , 24, 31.Cells were washed twice with PBS, fixed with 70% cooled ethanol for 1 h, and then washed with water. Cells were stained for 20 min at ambient temperature with alizarin red  and examined under light microscope. The red color obtained referred to calcium deposit.hMSCs seeded in 12-well plates were lysed with the assay buffer . The protein contents in the lysates were determined using the Bradford protein assay (Sigma). Ten microliters from the remaining lysate was mixed with 20 μl of MUP, used as a substrate (Abcam) in a 96-well plate, and incubated at room temperature for 30 min. Fluorescence intensity was measured at extension/emission of 360/440 nm. The alkaline phosphatase (ALP) activity was normalized to protein content and expressed as unit per microgram protein.RNA was purified using RNeasy kits . The concentration of RNA was quantified by spectrophotometry . Five hundred nanograms of total RNA was reverse transcribed with the Quanti Tec Reverse Transcription (Qiagen Kit) into cDNA. PCR amplification was performed on a Light Cycler  using Fast-Start™ DNA Master SYBR Green I real-time PCR kit . The expression of the genes was normalized to the expression of human cyclophilin B (CPB) . The list of primers (Qiagen) is as follows: BMP2 , RUNX2 , DKK-1 , RANKL , Shn 3 , TNF-α receptor type II (TNFRII) , and TNF-α receptor type I (TNFRI) .p-Values were determined for every analysis. p-Values <0.05 were considered significant.Analysis was performed using a Wilcoxon test from Graphpad Prism. p  < 0.05), which was further enhanced in the presence of the two cytokines . At day 21, maximum levels were observed in each condition except in the negative control. Thus, these results show that TNF-α but not IL-17A alone enhanced bone mineralization, which was further potentiated but without acceleration by IL-17A.A key characteristic of MSCs is their ability to differentiate into cells of different tissue lineages. Mineralization of the extracellular matrix is a marker of hMSCs differentiation into osteoblasts. To evaluate the effects of IL-17A and/or TNF-α on MM, hMSCs were cultured for 21 days in a medium supplemented with osteogenic factors with and without cytokines. Alizarin red staining was used to visualize mineralization. As shown in Figure 3 U/ml at day 3 vs. 7.4 × 103 U/ml at day 5, *p  < 0.05) or at day 7 in the presence of each cytokine alone . Day 14 shows almost the same results as day 7.Since TNF-α and IL-17A increased mineralization of the extracellular matrix, we next investigated their effects on ALP, an enzyme, which is essential for bone mineralization. ALP activity was measured at days 3, 5, 7, and 14 Figure . At day e Figure . MoreoveTherefore, these results support those of the alizarin red staining, by showing that the combined action of IL-17A and TNF-α resulted in synergistically increasing and accelerating osteoblast differentiation from hMSCs. However, ALP activity appeared more sensitive to the effects of IL-17A alone.p  < 0.05), with a kinetic curve demonstrating a decrease upon time . This inhibitory effect was not seen at 12 h . Overall, the combination of these two cytokines resulted in a significant increase of BMP2 mRNA expression as compared with controls . Therefore, these results showed that TNF-α increased the expression of BMP2 mRNA levels, an effect, which was inhibited by IL-17A.To better understand the effects of IL-17A and TNF-α on the differentiation of hMSCs, mRNA expression levels of BMP2 were measured at 6, 12, 24, and 72 h. BMP2 plays an important role in the development of bone and cartilage and induces osteoblast differentiation in a variety of cell types . BMP2 mRe Figure . IL-17A p  < 0.05) with a kinetic curve demonstrating a decrease upon time. IL-17A alone did not change Shn3-mRNA expression levels . No significant change of Shn3-mRNA expression levels was observed in the presence of either one or two cytokines between 12 and 24 h.Schnurri-3 (Shn3) is a zinc finger protein, which plays a key regulatory role in skeletal remodeling in mouse . In hMSCs Figure A. The coP  < 0.05). No significant change of Runx2 mRNA expression levels was observed in the presence of either one or two cytokines between 12 and 72 h . The combined action of the two cytokines resulted in a more profound decrease of RANKL mRNA levels . Moreover, this decrease was sustained upon time, since it was still observed at 24 h . This result shows that these two cytokines might enhance osteogenesis by reducing RANKL expression.The RANKL produced by osteoblasts plays a key role in osteoclast differentiation and activation . RANKL mp  < 0.05) . Therefore, these results show that the combined action of TNF-α and IL-17A on hMSCs may result from TNFRII but not TNFRI overexpression.TNF-α receptor type I and TNFRII play an important role in cell proliferation, survival, and death , 41. SinThe goal of this study was to evaluate the effects of cytokines on the differentiation of human MSCs into bone forming cells, focusing on TNFα classical target for treatment and on IL-17, an emerging one, these two cytokines being known to interact. The first conclusion is the effect of TNF-α to promote osteogenic differentiation of hMSCs by increased deposition of calcium into the extracellular matrix, enhanced ALP activity, enhanced expression BMP2 mRNA levels, and decreased expression of RANKL and DKK-1 mRNA levels with an increase of Schnurri-3. As it is often the case, the effects of IL-17A alone were absent or limited in similar assays. The combination of TNF and IL-17 showed complex interactions with a net effect on enhanced matrix formation, ALP activity, and Schnurri-3 expression with reduced RANKL expression. A key surprising effect was the inhibition of the TNF-enhanced BMP2 expression when IL-17 was added.Previous studies in human and murine systems have already shown an effect of IL-17A or TNF-α used alone on osteogenic induction , 31, 42.While TNF-α was shown to increase BMP2 expression in different cell types like chondrocytes, endothelial cells, and dental pulp cells –50, littAnother factor involved in the regulation of osteogenesis is DKK-1, a major inhibitor of the Wnt signaling pathway . DKK-1 pReceptor activation of NF-κB ligand, a type II membrane protein of the TNF superfamily, is expressed in osteoblasts, osteocytes, and some activated T cells, specifically the Th17 cells , 54. RANShn3 expression in mouse MSCs directly controls osteoblastic bone formation via Runx2, while indirectly regulating osteoclastic bone resorption , 33. ShnSince IL-17 increased the effects of TNF-α on hMSCs, we examined the possibility of an effect on TNF-R expression in order to explain the increased activity often observed with the combination of the two cytokines. TNFRI is ubiquitously expressed on nearly all cells but TNFRII has a more restricted expression, specifically on human lymphocytes –59 and Min vitro and in vivo results with TNF inhibitors are in line with the net effect of TNF on bone loss. Furthermore, the combined inhibition of TNF-α, IL-1, and IL-17 was more effective than the inhibition of a single cytokine to control inflammation and bone resorption in an ex vivo RA model (Our finding that TNFα and IL-17A activate osteogenesis, with IL-17A potentiating the effect of TNFα, may explain the mechanisms of ligament ossification with ectopic bone formation as observed in AS. It has been shown that the molecular pathways underlying AS recapitulate the presence of endochondral bone formation, where BMPs play a key role . SimilarRA model .Application to RA and AS pathogenesis and clinical manifestations indicates a key difference. In RA, the combination of TNF-α and IL-17 induces bone loss from massive osteoclast activation in sites, where osteoblasts and osteoclasts interact locally such as in juxta-articular sites . In contIn summary, this is the first study demonstrating that the two pro-inflammatory cytokines IL-17A and TNF-α can interact to induce osteogenic differentiation of human MSCs. Through complex interactions, IL-17A can potentiate the effects of TNF-α. An important new finding in the human context is the effect on Schunnri3, where the induction by TNF-α is further increased by IL-17A in a synergistic fashion. Such effect would provide an activation signal to osteoclasts. A close proximity to osteoclasts would be seen in whole bone as in juxta-articular sites of RA or vertebra in RA and AS. In tendons and ligaments where osteoclasts are not present, the lack of inter-cellular connection would lead to osteogenesis. At the site of tendon insertion to bone, the destructive pathway would be seen leading to erosion.The 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.


CRP) and the tumor necrosis factor-alpha (TNF-α) are considered markers of inflammation and have been shown to predict the risk of incident cardiovascular events. However, few studies have undertaken a comprehensive examination of SNPs (single nucleotide polymorphisms) of the CRP and TNF-α genes; due to this, we will present a protocol study to evaluate the role of the CRP and TNF-α genes in Mexican individuals. Methods/design: we will perform a case-control study to explore the CRP and TNF-α genotype distribution as well as the serum influence of rs1800947, rs1130864, rs2794521 and rs1205 (polymorphisms of the CRP gene) and rs361525, rs1800629, rs1799724, rs1800630, rs1799964 (of the TNF-α gene) in Mexican individuals who present coronary artery disease. Ethics and dissemination: a written informed consent will be obtained from all the participating subjects. An article detailing the results of the study will be submitted for publication in an international peer-reviewed journal, in accordance with STROBE criteria.Background: The C-reactive protein ( CRP) and the tumor necrosis factor-α (TNF-α) [CRP gene lies on chromosome 1q21–1q23, spans approximately 1.9KB and contains two exons, which are joined by a 280 base pair intron [CRP gene are associated with plasma CRP levels [CRP gene in association with CAD are the 1059G>C (rs1800947), 1444C>T (rs1130864), 717A>G (rs2794521) and 3872G>A (rs1205) variants [TNF-α) also plays a key role in cardiovascular pathophysiology. TNF-α gene is located on chromosome 6 (p21.1–p21.3) and consists of 2.76 kb distributed across its promoter region, four exons, three introns and untranslated regions 5′ and 3′ [TNF-α promoter region are reported to be associated with the TNF-α serum levels, these levels are correlated with the first-time cardiovascular disease and are also a marker for recurrent coronary events after a previous myocardial infarction [TNF-α gene have been reported, including 308G>A (rs1800629), 238G>A (rs3615525), 857C>T (rs1799724), 863C>A (rs1800630) and 1031T>C (rs1799964) [TNF-α, because several studies have found that patients with the A allele show high levels of TNF-α in serum, modifying the risk of developing CAD [Coronary artery disease (CAD) has often been considered a problem of wealthy, industrialized nations ,2. In fa (TNF-α) ,12,13. T (TNF-α) ,15. The r intron ,17. StudP levels ,19. The 72G>A rs15 variantvariants . Tumor n′ and 3′ . Geneticfarction ,26. Nume717A>G rs94521 andA rs18006, 238G>A 4C>T rs11864, 717Aping CAD  and bettCRP and TNF-α genes: rs1800947, rs1130864, rs2794521, rs1205 and rs361525, rs1800629, rs1799724, rs1800630, rs1799964, respectively. It will also investigate whether any polymorphisms of the CRP and TNF-α genes affects the serum levels of CRP and TNF-α proteins in Mexicans patients with CAD confirmed by angiography.The present study is designed as a protocol to determine the association between CAD and the polymorphisms of the The sample will be recruited from the Cardiology Service of the Mexican Social Security Institute , clinic UMF 46, Tabasco, Mexico . The stuetc.). Blood analyses will include: CRP and TNF-α serum levels, complete blood count, blood sugar, biochemistry and lipid profiles; patients without complete demographic data and/or inappropriate processing laboratory specimens will be excluded from the study. We will collect the following information from the participants: age, sex, BMI (body mass index) and risk factors for atherosclerosis , we will obtain information on CAD patients including: new onset AMI  multi-vessel disease group (two or more coronary arteries affected) and (b) single-vessel disease group (one coronary artery affected). Moreover, to reveal the genetic effect that CAD may have on acute myocardial infarction (AMI), gy −ACC− ) and hisAll subjects included in the study will sign an informed consent after explaining in detail the objectives of the study to the patients and their relatives; the participants will not receive any economical remuneration. This study has already been approved by two Ethics Committees: one from the Mexican Institute of Social Security, clinic UMF.49 (IMSS2013121) and the Research Committee of the University of Tabasco, México . Also, the study will be performed in accordance with ethical standards convened in the 1964 Declaration of Helsinki. An article detailing the results of the study will be submitted for publication in an international peer-reviewed journal, in accordance with Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) criteria .et al. [CRP and TNF-α genes chosen for the study will be: rs1800947, rs1130864, rs2794521 and rs1205, as well as rs361525, rs1800629, rs1799724, rs1800630, rs1799964, respectively . Also, genomic DNA from leukocyte blood sample will be extracted using a modified version of the protocol by Lahiri et al. . The polectively . These vt test or Mann-Whitney test. Also, the chi-squared test or Fisher’s exact test will be used to compare genotype and allele frequencies between controls and cases, using the following models: co-dominant , dominant , recessive , additive  and heterozygous advantages . For all the association analyses the level of significance will be set at p < 0.05, however the p value will be corrected according with the number of specificities tested and the comparisons made; the confidence interval will be set at 95%. Logistic regression analysis will be performed to estimate the risk of CHD between cases and controls against the CRP and TNF-α gene variants. Statistical calculations will be performed using the SPSS software, version 15. The Haploview 4.1 software will be used to construct haplotypes and linkage disequilibrium (LD) between polymorphisms. Finally, the sample size will measure of association using Quanto version 1.2 software (http://biostats.usc.edu/Quanto.html). The power of the analysis will 0.99 .First, we will evaluate the Hardy-Weinberg Equilibrium (HWE) using Pearson’s goodness-of-fit chi-squared test. We will use measures of central tendency with the mean ± SD and median with minimum and maximum values in accordance with their distribution. Moreover, continuous variables will be compared using Student’s CRP gene variants [CRP gene  and TNF-α gene  in a Mexican population in association with CAD. On the other hand, we want to observe the role of the genotype distribution in CRP and TNF-α serum levels, which are considered a marker of CAD. The outcomes of the study will provide new and unique information of the CRP and TNF-α genes participation in CAD, in Mexican individuals. This type of study is relevant for the Mexican population because the CAD has increased considerably (similar to European populations) and it cannot be explained merely by environmental factors , furthermore, the investigation of probable genetic markers of CAD in Mexico has not advance as much as in European countries [CRP and TNF-α in CAD. The purpose of the genetic research in the Mexican population will try to establish possible markers in the pathogenesis of CAD and the results of this study will be of great scientific relevance. It is well known that the first causes of mortality in adults worldwide are cardiovascular diseases; particularly, coronary artery disease . For Mexvariants . Other pvariants . Moreovevariants ,41, thervariants . Due to ountries ,44,45. ACRP and TNF-α genes variants; it will also analyze how such polymorphisms are involved in CRP and TNF-α serum levels in subjects with CAD. Therefore, the outcomes of this protocol will provide information to gain a better insight into CAD, which is considered a leading cause of death in Mexico and the world.The limitations of this study will be the lack of genetic ancestry evaluation of the participants, as well as the lack of search for a possible effect that could cause the stratification population. In conclusion this protocol study will try to unravel the role of some The cardiovascular heart disease has increased in Mexico, and its pathology can not be only explained by environmental effects. The role of genetic risk factors has been considered in several studies and this is an important point that needs to be considered by all health professionals. Due to this we considered that the development of this protocol can provide valuable data to the goal of a prevention through genetics markers.


However, we show that concurrent escape at rates 0.1–0.2 day−1 across multiple epitopes is consistent with our patient datasets.Recent studies have highlighted the ability of HIV to escape from cytotoxic T lymphocyte (CTL) responses that concurrently target multiple viral epitopes. Yet, the viral dynamics involved in such escape are incompletely understood. Previous analyses have made several strong assumptions regarding HIV escape from CTL responses such as independent or non-concurrent escape from individual CTL responses. Using experimental data from evolution of HIV half genomes in four patients we observe concurrent viral escape from multiple CTL responses during early infection (first 100 days of infection), providing confirmation of a recent result found in a study of one HIV-infected patient. We show that current methods of estimating CTL escape rates, based on the assumption of independent escapes, are biased and perform poorly when CTL escape proceeds concurrently at multiple epitopes. We propose a new method for analyzing longitudinal sequence data to estimate the rate of CTL escape across multiple epitopes; this method involves few parameters and performs well in simulation studies. By applying our novel method to experimental data, we find that concurrent multiple escapes occur at rates between 0.03 and 0.4 day Since the early 1990s, cytotoxic T lymphocytes (CTLs) have been known to play an important role in HIV infection with CTLs targeting HIV epitopes and, in turn, HIV escapes arising through mutations in the targeted epitopes. Over the past decade, studies have shown that CTL responses concurrently target multiple HIV epitopes, yet the effect of concurrent responses on HIV dynamics and evolution is not well understood. Through an analysis of patient datasets and a novel statistical method, we show that during early HIV infection concurrent CTL responses drive concurrent HIV escapes at multiple epitopes with significant pressure, suggesting a complex picture in which HIV simultaneously explores multiple mutational pathways to escape from broad and potent CTL response. During Human Immunodeficiency Virus 1 (simply HIV hereafter) infection, cytotoxic T lymphocyte (CTL) responses play a significant role in shaping viral dynamics and evolution . HoweverHIV-specific CTL responses first arise about 3 weeks into infection, several days prior to peak viral load, and initially target roughly 3–5 epitopes , 11. TypViral escape from CTL-mediated killing follows a temporal pattern similar to CTL response, although not all responses elicit an escape and escapes do not always occur in the same order as the CTL responses . The firPrevious studies have measured the rate of CTL escape as a means of quantifying the strength of CTL response , 15–20. A common approach for estimating escape rates, introduced in , 21, 23 In this work we consider the CTL response associated with the viral escapes that occur during the first 2–3 months of infection in four previously analyzed patient datasets from the Center for HIV/AIDS Vaccine Immunology (CHAVI) cohort: CH40, CH58, CH77, and CH256 , 13. We Pandit and De Boer  recentlyUsing specific examples and simulations, we show that estimates of escape rates based on the logistic model can lead to bias in the presence of multi-epitope escape, with the direction of bias depending on the structure of the escape. Furthermore, we show that application of the logistic model when CTL escape is sampled at a single time point leads to estimates that are severely downward biased . Importantly, these biases are unrelated to the presence of replicative fitness costs. Instead, bias associated with the logistic model is due to ignoring concurrent escapes and making strong assumptions regarding mutant frequency when mutants are undetectable.A, tI, and μ, that allows for unbiased estimation of escape rates given sampling of CTL escape at a single timepoint. Parameter A denotes the number of mutations that occur during the escape, tI is the time at which the first escape mutation occurs, and μ is the per epitope virus mutation rate. For a given patient, different CTL escapes will be parameterized by different values for A, tI and μ that will typically be unknown. The three parameters are the price we pay for requiring only a single sample time during each escape. Through simulation, we show that when A, tI and μ are known, our estimates are not biased by multi-epitope response and single time point sampling.To address these limitations in the logistic model, we introduce a novel method for estimating escape rates that removes bias associated with multi-epitope escape by applying the logistic model to pairs of variants, thereby generalizing the notion of wild type and mutant to the setting of multiple epitopes. This novel method still suffers from bias when CTL escape is captured at a single time point, so we introduce a further extension, involving the introduction of three parameters −1 across multiple epitopes are consistent with our dataset. Such significant escape rates across multiple epitopes would reflect a broad yet still relatively strong CTL killing. If replicative fitness costs associated with escape exist, CTL kill rates are above the range 0.1–0.2 day−1, suggesting even stronger CTL killing. Importantly, the wide range of escape rates contained within our lower and upper bound reflects a range of model assumptions consistent with our datasets. More accurate escape rate estimates require either more model assumptions, corresponding to narrower choices for A, tI and μ, or datasets with denser temporal sampling.Although we are unable to give a narrow range for the rate at which escapes proceed, our results show that escape rates in the range of 0.1–0.2 dayGoonetilleke et. al.  and Liu t1 and t2. To analyze early CTL escape, we consider only putative epitopes with mutation in at least one sequence collected at t1 and t2. Further, to avoid variation unassociated with CTL response, we only consider putative epitopes that meet at least one of two criteria: 1) the putative epitope is supported by ELISpot assays in .(2)In , 21, Fer].(2)In . Eq (2) exact freq/no mutation in the table shows, estimates are excellent when sampling is exact and no mutation occurs. The errors that do exist in assuming exact frequencies and no mutation arise from numerical error in the simulations and recombination.t1, while in n + 1) for both parent and child frequencies in sample freq columns in Tables Zero frequencies are particularly problematic in the context of t1. This difficulty exists in our simulations as well, where roughly 90% of all variants have a true frequency of less than 0.1 at t1 = 30 days, reflecting the time needed for mutant variants to arise and expand to significant frequencies. As a result, For our patient escape graphs, most vertices are expansion vertices and have zero frequency at t2:To estimate escape rates for edges pointing to expansion vertices, we replace A parameterizes the number of parent to child mutations, tI is the time of the first parent to child variant mutation, and μ is the rate at which a parent variant mutates into a child variant. Given data providing the frequencies fC(t2), fP(t2) and a choice for the parameters A, tI and μ, an estimate ϵ is determined through tI, t2] rather than . In practice, since ϵ, a numerical equation solver must be used to calculate ϵ.In A, tI, we consider different models for the ratio fC(t2)/fP(t2). Let mP(t) and mC(t) be the parent and child variant population sizes at time t, respectively. A deterministic model of parent-child dynamics assumes growth rates r − ϵ* and r for parent and child variants, respectively, and a parent to child mutation rate μ, leading to the following differential equation system:To explain ϵ* be the modeled (true) selective advantage of mutants to distinguish it from the escape rate estimate ϵ in ϵ = ϵ*. Integration of t2 under the initial condition mC(0) = 0 and mP(0) = m0 givesIn A = 1 and tI = 0, reflecting the assumptions implicit in μmP(t). While we have presented r and μ, the derivation of r and/or μ vary.t = 0 is unrealistic, since parent variants do not even exist at initial infection unless the parent is the founder variant, which is not the case for any of the expansion edges in our patient escape graphs. But we can alter tcutoff before which no mutations occur and after which mutations occur at rate μmP(t). Under such a model we would have the relation,A = 1 and tI = tcutoff.Starting mutation at μmP(t) once t > tI, but that the actual number of mutations varies around this average due to mutational stochasticity, we arrive at A parameterizing an excess or dearth of mutations relative to the average. When A = 1, the number of parent to child mutations exactly equals the average. When A > 1 and A < 1, the number of mutations is greater and less, respectively, than what we would expect.Other models of parent-child variant dynamics and mutation are possible. If we assume that parent variants mutate into child variants at a rate A, tI, μ on escape rate estimates. For the simulations presented in A, tI and μ value for each expansion edge. The columns of A = 0.75, we set A = 0.75 while tI and μ were set to their true values. The true value of μ was 10−4 for all parent-child pairs. As the table shows, changing any one of the parameters within a reasonable range can lead to estimates that are upper or lower bounds, reflected by a positive or negative relative error, respectively.A or tI for a parent-child pair within a given patient and μ is only roughly known from existing mutation studies  and tI ∈  for roughly 75% of expansion variants. Since peak viral load generally occurs 21 days post infection . Given a per nucleotide, per reverse transcription mutation rate of 3 × 10−5 [A = 10, tI five days prior to peak viral load and μ = 3 × 10−4 and with A = 1, tI two weeks post peak viral load and μ = 3 × 10−5, respectively, gives us lower and upper bounds for the escape rate. We also consider intermediate values for our parameters determined by the medians of A and tI: A = 1.25 and tI four days post peak viral load. We choose μ = 10−4 as an intermediate value of μ.In our simulations, nfection , tI falral load , making 3 × 10−5 , the ranA, tI and μ.While the parameter ranges we have chosen are relatively broad, the possibility exists that we have missed the true parameter range of HIV infection. Our method does not eliminate parameter dependence, but by choosing a broad range of parameter values, we are more likely to have captured true dynamics than if we simply specified particular values for t1 and t2  or denser temporal sampling.Overall, our analysis strongly suggests that escape in acute HIV infection occurs concurrently from multiple CTL responses and proceeds at significant rates. However, whether escape rates reach the ranges of 0.1–0.2 dayWe have presented a novel method for estimating the rate of concurrent escape of HIV from multiple CTL responses that can be applied to our dataset and potentially other datasets. The method is based on an escape graph representing the mutation pathways through which HIV evades CTL response. Our method extends the logistic model of , 21 to a−1 across multiple epitopes. The upper bound of 0.4 day−1 is in-line with upper bounds for CTL escapes found using the logistic model [Our results suggest that CTL escape can occur concurrently at multiple epitopes, with escape rates ranging between 0.03 and 0.4 dayic model , 14, 15,Pandit and De Boer  recentlyKessinger et. al.  present Ne) for HIV of 102–103 during early infection based on a census population on the order of 107. In simulations of CTL escape based on a Wright-Fisher model with Ne of 102–103, da Silva observed that escapes do not happen concurrently. Since our simulations assume census population sizes similar to da Silva’s (i.e. 107), combining our results with those of da Silva raises the possibility that a Wright-Fisher model does not accurately reflect HIV escape dynamics, as has been suggested by previous authors [Da Silva calculated an effective population size  be the CTL kill rate at time t of a variant possessing epitope i, we set ki(t) = 0 for t ≤ Tion,, where Tion, represents a time at which CTL response to epitope i initiates. For t > Tion,, ki(t) varies according toki = 0.01 giving the initial condition of the response. (See ki(t). In our simulations, the CTL response to epitope 1 occurs early and is relatively strong compared to responses to the other epitopes, which expand later. Strong and weak CTL simulations differ based on the value of kimax,. Rates of expansion and contraction for the immune response (α and β) are taken from parameters for the CD8 T cell response to a virus in mice [To model CTL response at . al. in , 45. Let in mice , 45. The in mice . In our N—not to be confused with effective population size—is expanded from 1 to 107 within the first 21 days, following estimates of the total number of infected cells in the body, and then collapses to 104.5 over the following two weeks and subsequently holds steady [7 to 104.5 variants is larger than estimated in [μ and ρ. All mutations occur independently at each epitope and to each variant according to a Poisson process run at rate μ = 10−4.At time 0, the population starts with 1 variant possessing all 6 epitopes. The population size s steady . The redmated in , but matρ/5)NfAfB with ρ = 1.4 × 10−5 and where fA, fB are the frequencies of the recombining variants A and B. When recombination does occur, we assume 5 breakpoints uniformly selected over a 10000 nucleotide genome, matching the number of breakpoints measured through in-vitro studies [ρ, matching the rate measured in [A or B variants by 1, where we choose to decrease either A or B with probability 1/2.Recombination occurs at rate Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Figt1 = 30 and t2 = 60. The census population size N was chosen to rise exponentially from 1 to 107 over the first 3 weeks of infection, collapsed to 104.5 over the next two weeks, and then hold steady. We assumed no fitness cost of the escape mutations in these simulations . In Panel D, the rate (day−1) at which 000000 variants mutates rises to roughly 1000, we plot on a more modest scale to make the other variant mutations rates visible. The sudden changes in slope seen in Panel D for variant 100000 at times 21 and 35 reflect the sudden change in the N profile at peak viral load (day 21) and the end of population collapse (day 35). This particular example assumes strong subdominant CTL responses that rise after the first CTL response.Shown are CTL kill rate profiles targeting 6 viral epitopes (panel A), the epitope mutation frequencies (panel B), the variant frequencies (panel C), and the rate at which each variant population produces mutants (panel D). The kill rate for a given variant was the sum of the kill rates across all epitopes in the variant haplotype, meaning that we assumed additive killing across epitopes for which there was a ‘0’ in the variant label shown in the legend. Epitope mutation frequencies were computed by summing up the frequencies of all variants mutated at the given epitope. The simulation was run with (TIF)Click here for additional data file.S6 Figt1 or t2, and panel B shows the escape graph generated by random sampling of 15 sequences at times t1 and t2. For example, 2 of the 15 samples at t1 were viral variant 100000, which is a frequency of 13% as shown in the panel B. Edges in the escape graph give the epitope mutated in moving from parent to child. Initial and expansion variants are colored red and yellow, respectively.We simulated HIV evolution using a stochastic model as described in the Methods and graph the pathways of viral escape from 6 CTL responses. Panel A shows the escape graph generated by considering all variants with frequencies greater than 0.01 at either (TIF)Click here for additional data file.S1 TableShown are the total number of nucleotides sites spanned by all such epitopes and in parenthesis the percentage of the viral genome covered by such epitopes (epitope sites), the total number of variable sites within all such epitopes and in parenthesis the percentage of these variable sites relative to the number of variable sites across the viral genome (epitope variable sites), and the p-value assuming all sites across the genome are equally likely to be variable .(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Tablet1 and t2, respectively.All times are in units of days since the onset of symptoms. 5’ samples and 3’ samples give the number of sequences sampled for each 1/2 genome at (PDF)Click here for additional data file.


In the subgroup analysis by tumour–node–metastasis stages, the highest TNF-α level was found in stage IV (42.7 ± 12.5 pg/mL) and was significantly elevated compared to earlier stages of CRC and controls. The survival rate of CRC patients with low TNF-α serum level, estimated as median survival, was significantly higher than that of patients with high levels of TNF-α  In conclusion, we can affirm that TNF-α affects tumour development along with disease progression which has an impact on the survival of CRC.Colorectal cancer (CRC) development is strongly associated with innate immune mechanisms and intestinal inflammation. The aim of the study was to investigate the pre-operative serum levels of TNF-α and its correlation with cancer progression and survival in CRC patients taking into account the genotype of –308G/A promoter polymorphism in TNF-α gene (rs1800629). TNF-α –308G/A genotypes of 119 CRC cases and 177 no CRC controls were determined by restriction fragment length polymorphism assay (RFLP-PCR). TNF-α serum levels were measured by enzyme-linked immunosorbent assay (ELISA). Although no significant differences in allele and genotype frequencies between CRC and controls were observed, it should be noted that the minor allele-A and its homozygous genotype were overrepresented among CRC. In addition, allele-A was more frequent in early CRC patients compared to advanced cases. TNF-α serum level was significantly higher in CRC patients than in controls (36.1 ± 8.4 pg/mL vs. 18.66 ± 11 pg/mL;  Colorectal cancer (CRC) is one of the most common malignancies and represents a significant cause of morbidity and mortality worldwide. It is well known that the inflammatory process together with the genetic background has a great impact on the carcinogenesis.Pro-inflammatory and anti-inflammatory cytokines play a key role in the regulation of inflammation. Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine, primarily released by activated monocytes/macrophages as well as tumour cells. TNF-α has several actions, such as stimulating the acute phase response, mediates the early inflammatory response and regulates the production of other cytokines, hemokines and endothelial adhesion molecules. TNF-α alRecent experimental and clinical studies on the role of TNF-α have demonstrated that the TNF-α is a key player in progression of human CRC. BalkwillIn addition, some studies have reported that a substitution G/A at –308 position of TNF-α gene promoter is correlated with cytokine production and increased cancer risks. A-allele was associated with higher circulating TNF-α level and may alter the immune response. Such genIn respect to the above, the aim of the current case-control study was to investigate the effect of –308G/A polymorphism in TNF-α gene promoter on serum level of TNF-α and its correlation with cancer progression and survival in CRC patients in ethnic Bulgarian population.2 = 2.698, df = 3, p = 0.44). The mean age at diagnosis of male vs. female among the cases was 65.07 ± 10 years vs. 66.16 ± 11 years (p = 0.57). Demographic data and disease status of 119 CRC patients are presented in A group of 119 Bulgarian patients with CRC were included in the study of distribution of the –308G/A TNF-α polymorphisms. Cases with new diagnosis of CRC attending the University hospital in Stara Zagora and an Oncology centre in Pleven, Bulgaria were selected. All patients had no previous diagnosis of inflammatory bowel disease or any of the known hereditary cancer syndromes. Patients did not receive chemotherapy or radiation therapy before surgery. The histopathological examination confirmed the diagnosis of cancer. Tumour grading and staging was performed according to the tumour–node– metastasis (TNM) classification. The patients’ group was composed of 71 (60%) male and 48 (40%) female. Different stages of CRC were found in both sex groups in approximately equal proportion  kits , following the manufacturer's protocol. Developed colour reaction was measured as OD units at 450 nm. The concentration of TNF-α was determined by using the standard curve constructed with the kit's standards and was expressed in pg/mL. The minimum detectable concentration of the TNF-α ELISA kit was less than 2 pg/mL.Genotyping for the –308G/A polymorphisms in TNF-α (rs1800629) was performed by restriction fragment length polymorphisms (RFLP)-PCR assay after amplification of 150 bp fragment with forward primer: 5′–AGG CAA TAG GTT TTG AGG GCC AT-3′ and reverse primer: 5′–TTG GGG ACA CAC AAG CAT CAA GG–3′. The cycling parameters were as follows: initial incubation step of 2 min at 95 °C; 35 cycles: 45 sec at 95 °C, 45 sec at 65 °C and 45 sec at 72 °C and a final extension step of 5 min at 72 °C completed the reaction. Amplified products were digested using 10 units of NcoI (Thermo Scientific) per reaction for 16h at 37 °C. The –308G allele yields two fragments, 128 bp and 22 bp, respectively. PCR amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems). Primers and reagents for PCR reactions were supplied by Metabion GmbH (Germany) and Thermo Scientific, respectively. PCR products and restriction fragments were visualized on an agarose gel stained with ethidium bromide (0.5 mg/mL). In each PCR run, heterozygous control template was used to ensure accuracy. For quality control, 10% of random selected samples containing both cases and controls were analysed a second time without finding any discrepancies.p-value less than 0.05 (two-tailed) was considered significant.Statistical analysis was carried out using SPSS software, version 21 . The independence of qualitative outcomes was tested using Pearson's chi-square test and Fisher's exact test, where appropriate. Differences in serum levels of TNF-α between groups and across genotypes were assayed by one-way ANOVA, and if significant, Fisher LSD test for post-hoc pairwise comparisons was applied. The survival curves were made using the Kaplan–Meier method and comparison was with the log rank test. In all cases p = 0.612 and cases p = 0.668).Genotype and allele frequencies of –308G/A polymorphism in promoter of TNF-α gene were determined in 177 healthy donors and in 119 CRC patients from Bulgaria . The genp = 0.64; df = 2). However, there was a trend of higher frequency of AA genotype (2.5%) among cases compared to controls (1.1%) and to allele-A (14.3% vs. 12.4%) but without statistical significance.The observed genotype frequencies of –308G/A TNF-α promoter polymorphism were similar among CRC patients and no-CRC controls (p = 0.519). As a result, allele-A was more frequent in early CRC patients compared to advanced cases . However, no statistically significant differences in distribution of –308G/A TNF-α polymorphism between different tumour stages were detected.Furthermore, we investigated the association of –308G/A TNF-α polymorphisms and severity of CRC. The results are presented in In respect to limited number of homozygous AA genotype, statistical analyses were performed between individuals carrying the variant allele-A (genotype AG and AA) and individuals with homozygous GG genotype. However, no significant associations were observed between investigated polymorphism in TNF-α gene and CRC.p < 0.0000001). After grading and staging the cases according to the TNM classification, the highest TNF-α level was found in stage IV of CRC (42.7 ± 12.5 pg/mL) and it was significantly higher when compared to the earlier stages of CRC and control group (p < 0.05). Serum TNF-α was approximately equal among stage I (34 ± 4 pg/mL); stage II (34.5 ± 6.3 pg/mL) and stage III (34.6 ± 6.7 pg/mL) of CRC and significantly elevated compared to the controls.TNF-α serum levels were measured in 68 CRC patients and 47 controls by the ELISA method and are presented in p = 0.042; for the stage II: 34.9 ± 6 pg/mL, p = 0.018; for stage III: 34.4 ± 7 pg/mL, p = 0.013). The same tendency was observed for AA + AG genotypes without reaching statistical significance.Moreover, we investigated the relationship of serum TNF-α and genotypes of –308G/A polymorphisms in TNF-α gene in CRC patients and healthy donors . In respp = 0.024).However, among control group, the carrying of variant allele-A was associated with increased serum level of TNF-α. Significant elevated serum TNF-α was detected in individuals with genotypes-AA + AG (23.3 ± 12.1 pg/mL) compared to GG genotype . However, it should be mentioned that a poor survival rate among CRC patients with genotype-AA (33.3%) was observed when compared to patients with AG and GG genotypes, without reaching statistical significance. Additionally, no significant difference was observed between CRC patients with GG genotype and those with genotypes carriers of variant allele-A (AA + AG) with χ2 = 0.061; df = 1, log rank test p = 0.805. Stratification by tumour stage according to TNM classification showed no significant association between investigated polymorphism and survival-rate of CRC patients.The Kaplan–Meier survival curves representing the survival rates of the patients according to their genotypes are presented in p = 0.00015; We further evaluated the correlation of serum level of TNF-α and survival in CRC patients. Taking a 51.66 pg/mL, mean value + 3* standard deviation of serum level detected among control group, as the threshold level, the total group of CRC was subdivided to patients with low TNF-α level (below 51.66 pg/mL) and patients with high level (above 51.66 pg/mL). The pre-operative serum TNF-α concentration was high in four patients. Three of patients with high TNF-α were in stage IV of CRC and one was in stage III of CRC. Interestingly, all four patients with high serum TNF-α died during the follow-up period of the present study. As a result, the survival rate of CRC patients with high TNF-α serum level was significantly lower than that of patients with low levels of TNF-α. CRC patients with high TNF-α concentration showed a poor survival of 0% as compared to 43.1% with low TNF-α concentration  compared to GG genotype. Obviously carrying AA genotype has a greater disadvantage in respect to survival among CRC patients than the carrying of variant allele-A in heterozygous genotype.As it is well known, when a selectively favourable gene substitution occurs in a population, changes in variant allele frequencies will affirm depending on the aggregate functional effect of the substitution. Bearing in mind the above simultaneously with the extremely low frequency of variant homozygotes in our current study, as well as in previous studies among other European populations, we couldp = 0.00015). Our results are in agreement with other previous studies.[The strong evidence of the TNF-α role in CRC development is the fact that an approximately twofold increase of serum TNF-α in CRC patients was found when compared to controls, regardless of TNF-α –308G/A polymorphism. In addition, the highest level of TNF-α was found in stage IV of CRC. Evaluating the relationship between the survival and serum TNF-α, it was observed that patients with high level (above 51.66 pg/mL) showed significantly inferior survival capability than patients with lower TNF-α levels (log rang,  studies.,18 Kim eWe could conclude that TNF-α has a great impact on development, progression and survival of CRC. The elevation of serum TNF-α level was associated with advanced stage of CRC and reduced survival of CRC patients.


Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear.We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo.TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α.Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies. Loss of intestinal barrier function plays an important role in the pathogenesis of IBD.Tnf-α and toll-like receptor (TLR) stimulation can induce a transient increase in epithelial cell shedding with loss of epithelial barrier function.Escape from apoptotic cell death due to caspase-8 deficiency results in necroptosis of Paneth cells at the crypt base. The role of necroptosis in cell shedding from the villus is unknown.Caspase-8 controls cell shedding and cell death on the villus induced by TLR ligands in vivo.When caspase-8-dependent apoptosis is disabled, cell death and cell shedding switch from apoptosis to Rip3-dependent necroptosis causing villous destruction which is more severe than from apoptosis.Blockade of necroptosis prevents mortality and cell shedding following TLR stimulation, and preserves mucosal integrity.Tnf-α produced by non-epithelial cells is responsible for TLR4-mediated epithelial necroptosis.TLR3-triggered necroptosis is directly mediated via TRIF-dependent pathways, and independent of Tnf-α and type III interferons.This study expands the current knowledge of regulation of apoptotic and necroptotic pathways controlling cell shedding from intestinal villi. Our findings show that therapies that inhibit apoptosis must be accompanied by manoeuvres that also inhibit necroptosis. This has important implications for the design of treatment of inflammatory and infectious intestinal diseases and, thus, for clinical practice.The gut represents the largest surface of the human body to the external environment, which is characterised by food antigens and the mucosal microbiota, including bacteria, viruses and parasites. Intestinal epithelial cells (IEC) constitute an important barrier in controlling the access of luminal antigens into the body. Owing to these essential functions, it is quite evident, that the intestinal epithelium plays a crucial role for intestinal homeostasis as well as for systemic health. Dysfunction of the intestinal epithelium, as indicated by increased permeability or deregulated epithelial defence functions, is believed to result in the excessive translocation of the commensal microbiota into the bowel wall and subsequent development of gastrointestinal diseases, including IBD.Intestinal epithelial homeostasis is maintained by a strict equilibrium between cell proliferation in the crypt and cell shedding from the villus tip.Here, we demonstrate that activation of TLR3 and TLR4 leads to a rapid and transient activation of caspase-8 and caspase-3 as well as increased cell shedding. Surprisingly, deletion of caspase-8 from the intestinal epithelium did not protect the animals from TLR-induced inflammatory cell loss, but instead, led to severe tissue destruction, excessive cell death and mortality due to Rip3-dependent necroptosis. Our analysis further revealed that TLR3-triggered necroptosis was directly mediated via TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways but independent from Tnf-α and type III interferons, whereas LPS-induced necroptosis was strictly Tnf-α dependent. Therefore, from our data, we propose a novel model of villus cell shedding in response to a range of inflammatory stimuli in which the activation of caspase-8 is essential during the first line of defence, by maintaining the gut barrier in response to mucosal pathogens through induction of inflammatory shedding and inhibition of necroptosis in infected cells.−/−mice, Tnf-R1−/− mice, TRIF−/− mice, IL-28Rα−/− mice, VillinCre mice and mice carrying loxP-flanked caspase-8 alleles were described earlier.ΔIEC mice) were generated by breeding floxed caspase-8 mice to VillinCre mice as described earlier.Rip3−/− mice, InvivoGen). Viability was monitored by visual examination and by measuring body temperature and weight loss.Mice were injected intraperitoneal with LPS  or poly(I:C)  Cy3 system, as recommended by the manufacturer (PerkinElmer), and the following antibodies were used: the primary antibodies anti-Myeloperoxidase (MPO) (Abcam), anti-Tnf-α , anti-activated caspase-3 (R&D Systems) and anti-activated caspase-8  and anti-CD324 (E-cadherin) Alexa Fluor 488 (eBioscience), and a biotinylated secondary anti-rabbit antibody (Dianova). Cell death was analysed using the in situ cell death detection kit (Roche) for TUNEL.Proteins were separated using a MiniProtean Precast gel  and transferred to a nitrocellulose membrane (Whatman). Membranes were probed with the following primary antibodies: TLR3 (Santa Cruz), Rip3 (Enzo), cleaved caspase-3, cleaved caspase-8 XP  and actin (Abcam). HRP-linked anti-rabbit  was used as a secondary antibody.HPRT).Total RNA was extracted from the gut tissue, or organoids, using the RNA isolation kit . cDNA was synthesised by reverse transcription  and analysed by real-time PCR with SYBRGreen (Roche) reagent and QuantiTect Primer assays (Qiagen). Experiments were normalised to levels of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase  or LPS, high mortality was observed in Casp8ΔIEC mice already during the first hours following TLR ligand exposure  animals . A reduced length of the small intestine was also observed in Casp8ΔIEC but not in wild-type animals  or LPS into Rip3IEC mice  and 4E. ΔIEC mice, particularly in Paneth cells at the crypt base. Therefore, we evaluated the role of Tnf-α in TLR-induced epithelial cell death. Indeed, we detected significant levels of Tnf-α mRNA in the gut of ligand-exposed wild-type mice -induced lethality. Tnf-R1−/−Casp8ΔIEC mice died with a similar kinetic as compared to Casp8ΔIEC mice  administration (see online supplementary figure S3B). However, IL-28Rα−/− animals showed massive epithelial cell shedding associated with caspase activation indistinguishable from IL-28R-sufficient animals 2 h after poly(I:C) administration (see online supplementary figure S3B).In sharp contrast with the Tnf-α-dependent effect of LPS, combined deletion of Tnf-R1 in Casp8IEC mice B. They aIEC mice C. Besidefl) and Casp8ΔIEC mice were treated in vitro with poly(I:C). By contrast with the observation made after LPS stimulation, targeting of TLR3 by administration of poly(I:C) to the organoid culture, resulted in rapid epithelial cell death of caspase-8 deficient organoids but not of organoids derived from control mice -induced epithelial gut damage (see online supplementary figure S4A). No epithelial cell shedding or signs of SIRS were observed in poly(I:C)-treated TRIF−/− animals. Moreover, poly(I:C) did not induce the disturbance of E-cadherin expression in adherence junctions or the activation of caspase-8 and caspase-3 (see online supplementary figure S4A–C) in TRIF-deficient epithelial cells. To determine if poly(I:C)-induced necroptosis was also mediated via TRIF, we generated small intestinal organoids derived from TRIF-deficient mice. Epithelial cell organoids were treated in vitro with poly(I:C) in the presence or absence of the caspase inhibitor zVAD-fmk. This allowed us to directly investigate the response of epithelial cells lacking caspase activity and/or TRIF-dependent signalling in response to poly(I:C). As shown by light microscopy and also PI staining, addition of poly(I:C) in combination with zVAD-fmk to the culture medium resulted in rapid cell death of wild-type but not TRIF-deficient organoids .It has previously been shown that TLR3 and TLR4 activation triggers the recruitment of TRIF, which interacts with Rip1 and caspase-8 via the RHIM domain of Rip1. Consistent with published results,ΔIEC mice within only a few hours, we examined whether this was a direct effect of TLR3-signalling or if bacteria are involved, since we could identify that intestinal damage in these mice was accompanied by a breakdown of the intestinal barrier, as demonstrated by staining for the adherence junction protein E-cadherin (Given that poly(I:C)-induced mortality of Casp8cadherin A.ΔIEC mice, fluorescence in situ hybridisation (FISH) with an eubacterial rRNA-specific probe revealed enhanced translocation of intestinal bacteria from the luminal side into subepithelial areas of the lamina propria . As expected, antibiotic treatment significantly prolonged the survival of Casp8ΔIEC mice after poly(I:C) treatment -challenged Casp8 propria A. Even m outcome B, C. To reatment D. Additireatment D–F. CollΔIEC mice could not inhibit Paneth cell necroptosis and inflammation, suggesting that cell death in these animals is either activated by Tnf-R1 independent pathways or that cell death in vivo is triggered by redundant death-inducing signals.Defects in intestinal barrier function due to tight junction loss or cell death-driven cell shedding and barrier loss can exacerbate or directly trigger intestinal inflammation.ΔIEC mice. Thus, our data for the first time show a crucial role for caspase-8-mediated Rip3 inhibition in controlling the epithelial response to TLR ligands.Our results clearly demonstrate that activation of caspase-8 by ligand binding to TLRs is associated with increased epithelial cell loss in wild-type animals. Challenge of mice lacking caspase-8 selectively in the intestinal epithelium via TLR3 or TLR4 ligation did not protect them from epithelial loss but, instead, caused severe gut injury due to excessive Rip3-mediated necroptotic cell death. By contrast with caspase-8-mediated inflammatory cell shedding, epithelial necroptosis resulted in a total disruption of the mucosal surface, finally causing a lethal systemic inflammatory shock in Casp8−/− mice, demonstrating that LPS-induced cell death is strictly dependent on Tnf-α. Additionally, we discovered that Tnf-R1−/−Casp8ΔIEC mice were protected from LPS-induced cell death, demonstrating that TLR4-mediated necroptosis is also mediated via this cytokine. The fact that Tnf-α is produced upon LPS administration by gut immune cells and not by IECs, indicates that bacterial toxins do not directly induce cell death via TLR4 on epithelial cells. This is supported by only low expression of TLR4 in IECs and the fact that organoids did not show cell death in response to LPS. Therefore, initial recognition of LPS associated with caspase-8 or Rip3 activation occurs via TLR4 ligation in lamina propria cells rather than epithelial cells (see online supplementary figure S5). One may speculate that in the situation of an increased intestinal permeability, an enhanced paracellular flux of intestinal pathogens may result in the activation of innate immune cells promoting their secretion of Tnf-α which subsequently induces epithelial cell death via Tnf-R1 on epithelial cells (see online supplementary figure S5). Given that the epithelium is constantly exposed to bacteria-derived TLR ligands, it seems that it would be perilous if the commensal flora could directly trigger caspase-8-dependent cell loss via TLR4 on epithelial cells. Our findings in IECs are in contrast with a recent publication in which it was shown that LPS is able to activate necroptosis in FADD-deficient dendritic cells directly via the TLR4 pathway.Surprisingly, our study identified that TLR3 and TLR4 both trigger epithelial cell death, but that the signalling pathways differ. Caspase-8 activation and gut damage, upon TLR4 stimulation, was prevented in Tnf-R1The observation that Tnf-R1 deficiency did not protect mice from TLR3-mediated cell death indicates that viral products, such as dsRNA, can directly mediate caspase-8-dependent cell death. These results are in line with a previous publication demonstrating that dsRNA rapidly induces epithelial shedding associated with caspase-8 activation independent of Tnf-α production after TLR3 ligation.−/−Casp8ΔIEC double deficient mice) and inhibition of Rip1 in vitro (nec-1) were sufficient to block cell death. Taken together, our data for the first time demonstrate in vivo that viral products, such as dsRNA induce intestinal epithelial necroptosis independent of immune cells, in a situation in which caspase-8 activity is impaired by directly targeting TLR3 on IECs (see online supplementary figure S5).Previous in vitro studies indicated that recognition of poly(I:C) by TLR3 under certain conditions can result in the formation of an intracellular platform containing the TRIF adapter that directly recruits Rip1 and caspase-8 to the TLR3, suggesting that this complex is able to mediate caspase-dependent and caspase-independent cell death pathways.In summary, our data highlight a crucial role for caspase-8-mediated Rip3 inhibition in controlling the epithelial response to bacteria and virus-associated molecular patterns. This has important clinical implications since therapies that inhibit apoptosis are likely to carry substantial toxicity in the intestinal epithelium, and might induce necroptosis and inflammation. Future therapeutic strategies might benefit from the simultaneous inhibition of apoptosis and necroptosis.


A means of in situ modulation of the DC phenotype in the host would be more accessible. Here we report a novel innate immune modulator, 1Z1, generated by conjugating a TLR7 ligand to six units of polyethylene glycol (PEG), which skews DC phenotype in vivo. 1Z1 was less potent in inducing cytokine production by DC than the parent ligand in vitro and in vivo. In addition, this drug only modestly increased DC surface expression of activation markers such as MHC class II, CD80, and CD86; however, the expression of negative regulatory molecules, such as programmed death ligand 1 (PD-L1), and interleukin-1 receptor-associated kinase M (IRAK-M) were markedly increased. In vivo transfer of 1Z1 treated DC into prediabetic NOD mice delayed pancreatic insulitis. Daily administration of 1Z1 effectively prevented the clinical onset of hyperglycemia and reduced histologic islet inflammation. Daily treatment with 1Z1 increased PD-L1 expression in the CD11c+ population in peri-pancreatic lymph nodes; however, it did not induce an increase in regulatory T cells. Pharmaceutical modulation of DC maturation and function in situ, thus represents an opportunity to treat autoimmune disease.Autoimmune diabetes mellitus (DM) results from the destruction of pancreatic islet cells by activated T lymphocytes, which have been primed by activated dendritic cells (DC). Individualized therapy with  Dendritic cells (DC) contain heterogeneous populations that contribute to initiating and perpetuating inflammatory processes as well as immunologic tolerance . Variousin vivo efficacy of 1V136, our laboratory modified the parent compound by click chemistry to link it to a six unit oligo-ethylene glycol (PEG) moiety and a carboxyl tail  / 4 × (n0 + n1+ n2 + n3 + n4), where n0, n1, n2, n3, and n4 were the number of islets scored in grade 0, 1, 2, 3, 4, respectively [th injections, and levels of IL-6 and TNFα were determined by Luminex bead assay. Single cell suspension of pancreatic lymph nodes and spleens were prepared using collagenase/DNase digestion. Single cell suspension of pooled pancreatic lymph node cells, splenocytes or buffy coats from treated NOD mice were stained for CD3+, CD4+ and CD8+, CD11c+, PD-L1+, CD69+ or CD25+ and intracellular Foxp3.Female NOD mice (n = 5 to 10 /group) were daily treated with vehicle, or 1Z1 400 nmol  from weeectively . Sera we515–524 (100 μg/mL) [BMDC were prepared from 8–9 week old female NOD mice. BMDC were incubated with 1Z1 or vehicle overnight and washed three times with medium. 1Z1- or vehicle- treated cells were intravenously transferred into 8 week old female NOD mice. Four weeks after the adoptive transfer, mice were sacrificed and histologic examination of the pancreatic tissues was performed as described above. In some experiments, NOD BMDC were incubated with the islet peptide GAD650 μg/mL) , 24 withΔΔCt method was used to assess fold changes in expression of RNA transcripts between control and drug-treated mice. ΔCt values were determined by subtracting the average GAPDH RNA gene Ct values from each test Ct value. ΔΔCt values were normalized by the control calibrator ΔCt value. Taqman Gene Expression Assays  used for the qPCR analysis were purchased from Life Technologies.Total RNA was extracted from tissues or cells. Harvested tissues were immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated using RNeasy Mini Kit (Qiagen). cDNA synthesis was performed using the iScript (Bio-Rad) and real time PCR were performed on the Bio-Rad iCycler IQ. The comparative 6) were stimulated with 10 μM 1Z1 or vehicle overnight. Cell lysates were separated on a 4 to 12% SDS-PAGE gel (Thermo Fisher Scientific). After the proteins were transferred onto a PVDF membrane, and probed with an anti-IRAK-M antibody  and developed by ECL (Thermo Fisher Scientific). The blot was then probed with an anti-actin antibody .BMDM . The Mann—Whitney U test was used to compare two groups. One-way ANOVA with Bonferroni or Dunnett’s post hoc test was used for multiple comparisons to a control group. Kaplan-Meier Survival Analysis with a log-rank test was used to monitor the incidence of diabetes.Prism 6.0  was used for statistical analyses including regression analyses. Data were plotted and fitted by nonlinear regression assuming a Gaussian distribution with uniform standard deviations between groups and ECin vitro. Splenocytes from C57BL/6 and NOD mice containing mixed cell populations were stimulated with graded doses of each compound and IL-6 production was measured. 1Z1 was 1000 to 100 times less potent than 1V136 for IL-6 stimulation (C57BL/6: EC50 (1Z1) = 75 μM vs. EC50(1V136) = 0.07 μM, NOD: EC50(1Z1) = 73 μM vs. EC50(1V136) = 0.4 μM)  underwent three times more cell divisions compared to C57BL/6 (PI = 1.52 at 1 μM). In contrast, minimal proliferation was induced by 1Z1 at 5 μM in both strains  . Using b 0.4 μM) . Since B 0.4 μM) . The par strains .in vivo, 1Z1 was subcutaneously injected to C57BL/6 or NOD mice and cytokine levels in sera were measured 2 h post administration  cells with a stable NF-κB reporter that expresses secreted embryonic alkaline phosphatase (SEAP) when stimulated through human TLR7, was therefore treated with 1V136 and 1Z1. The 1Z1 treated reporter cells produced detectable but low SEAP release compared to 1V136 treated cells, indicating that this compound retained the ability to stimulate human TLR7, albeit at a lower potency . The effTo evaluate the immunologic characteristics of 1Z1 treated BMDC, the surface expression of co-stimulatory molecules was evaluated by flow cytometric assay. Compared to the lipopolysaccharide (LPS) treated or reference TLR7 ligand, 1V136, treated cells, 1Z1 induced only minimal increases in expression of CD80, CD86, CD40 and MHC class II at 0.4 and 2 μM . Howeverin vitro. WT C57BL/6 or TLR7 null BMDC were incubated with ovalbumin (OVA)-primed CD4+ T cells and OVA in the presence of 1Z1 or vehicle. The vehicle treated cultures showed a robust T cell proliferation as monitored by CFSE  or vehicle for one week. Flow cytometric assays revealed an increase in the expression of PD-L1 on the gated CD11cc+ cells . The aveted mice . Expressted mice . The uprted mice . Howeverted mice  and 5I.+, or CD8+ T cells, nor increase the Treg population (CD25+/Foxp3+) in the gated CD4+ T cells in the pancreatic lymph nodes (+) CD4+ T cells in the pancreatic lymph nodes , while similar proportions of activated T cells were detected in the spleens   .+ T cells and CD4+CD25+ Tregs [+CD25+ Treg proliferation, and treatment of pre-diabetic mice with a synthetic TLR2 agonist diminished the onset of T1D, and increased the number and function of CD4+CD25+ Treg [Recent data suggest that TLRs are also expressed on CD45+ Tregs , 37. DC 25+ Treg , 38, 39.+ T cells were incubated with 1Z1 in the presence or absence of anti-CD3 antibody. The stimulatory activity was measured by CFSE dilution   .ex-vivo generated BMDC treated with 1Z1 are able to induce Treg, the number of Treg in the spleens or pancreatic lymph nodes of recipient NOD mice was assessed by flow cytometry seven days after transfer of 1Z1(2 μM)-treated BMDC (+/CD25+/CD127lo Treg population between the splenocytes of the 1Z1-DC recipient NOD mice compared to controls (53.1 ± 2.5% vs. 42.1 ± 1.4%)[Next, to study whether ted BMDC . There w1 ± 1.4%), 41. Thein vivo.Lastly, the suppressive function of Treg isolated from 1Z1 or vehicle treated NOD mice were compared . NOD micType 1 diabetes (T1D) is induced by auto-reactive T cells that attack beta cells in the pancreatic islets. Although the pathogenesis of this disorder depends on adaptive T cell immune responses, DC activation plays an important role in the onset and progression of the disease –45. DC aIn vitro studies indicated that 1Z1 was a potent inducer of negative immune regulators, such as PD-L1, IRAK-M, or IL-10, compared to the immunostimulatory cytokine, IL-6. However, there was no detectable expansion of Treg cells in vivo in 1Z1 treated mice.Prior reports have indicated that treatment with TLR ligands or TNFα could induce a suppressor phenotype in DC , 46–50. in vivo after transfer has not been easily achieved. Exposure of DC to 1Z1 ex vivo induced robust PD-L1 expression, and 1Z1 treated cells were then able to reduce antigen specific T cell proliferation. However, a single transfer of 1Z1 treated DC was not sufficient to abrogate the onset of diabetes.Adoptive transfer of tolerogenic DC has been successful in reducing the incidence of diabetes in NOD mice –54; howein vivo by treating NOD mice with a DC modulating drug. In NOD mice, infiltration of lymphocytes and macrophages into the pancreatic islets begins at 4 to 5 weeks of age followed by overt diabetes beginning at approximately 12 weeks of age [An alternative strategy to DC therapy is to induce the same PD-L1 expressing DC phenotype s of age . The thes of age , suggest+CD25+ Treg that inhibit the onset or progression of autoimmune diseases [in vitro in the presence of anti-CD3, but not independently. However, neither repeated injection of 1Z1 nor adoptive transfer with 1Z1 treated DC increased the frequency of Treg cells in the pancreatic lymph nodes or spleens. Furthermore, Treg isolated from NOD mice after repeated administration of 1Z1 showed similar suppressive effects as the Treg isolated from vehicle treated mice. This result suggests that the mechanisms of treatment with a TLR7 ligand is different than from those previously reported for a TLR2 ligand [Activation of innate immune pathways by TLR ligands has been reported to inhibit progression of type 1 diabetes , 43, 50.diseases , 37, 38.1Z1 induced inhibitory molecules such as IL-10, IRAK-M, and PD-L1. In 1Z1 treated NOD mice, increased PD-L1 expression on DC was detected in local pancreatic lymph nodes . Furtherin vivo administration, and did not induce any weight loss [TLR agonists have been successfully used in NOD mice to delay T1D by inducing tolerogenic responses , 39, 50.ght loss , in contght loss . Furtherght loss . Thus thin vivo in the local pancreatic lymph nodes, characterized by effective induction of negative feedback molecules, including PD-L1. 1Z1 has bioactivity in the draining lymph nodes of inflamed organs, but induces minimal adverse proinflammatory cytokines and B cell proliferation. 1Z1 may be a new class of drug that selectively induces suppressive DC in local lymphoid organs with resulting benefit in treatment of autoimmune diabetes and possibly other autoimmune diseases.In summary, 1Z1 induced a suppressive DC phenotype S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.


To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.in vitro. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint. Biomechanical factors play important roles in several forms of osteoarthritis (OA), including temporomandibular joint disorder (TMD) . Based oOne of the main pathological changes in cartilage in OA is enhanced chondrocyte apoptosis , 10 or aThe initial morphological characteristics of the mitochondria-dependent or intrinsic apoptotic pathway include condensation of chromatin and the swelling of mitochondria, followed by the release of cytochrome c . In the Pro-inflammatory cytokines, most notably TNF play a pivotal role in apoptosis, inflammation and tissue damage , 22, 23.In the present study, we investigated if apoptosis occurs in UAC-induced TMJ cartilage and if TNF is involved.Sixty-two 6-week-old, female, Sprague-Dawley (SD) rats (weight 140–160 g) were provided by the animal center of the Fourth Military Medical University in Xi’an, China. The animal care and all procedures were performed according to institutional guidelines and were approved by the Ethics Committee of the Fourth Military Medical University. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Fifty-six rats were equally divided into seven groups and time-points. In the 2-week Control and 4-week Control groups, a mock operation was conducted on the rat incisors, and the animals were sacrificed either 2 or 4 weeks later, respectively. In the 2-week UAC and 4-week UAC groups, unilateral anterior crossbite (UAC) was induced in the left incisors as described below, and the animals were sacrificed either 2 or 4 weeks later. In the UAC+Inhibitor, Control+Inhibitor, and UAC+PBS groups, a TNF monoclonal antibody (see below) or PBS was injected locally into the TMJ area after 2 weeks. The TMJs were sampled after 4 weeks. The mandibular condylar chondrocytes of the other six rats were isolated and treated with TNF in vitro.The unilateral anterior crossbite (UAC) model was created following our previous reports , 8. BrieTwo weeks after UAC stimulation, rats were laid sidelong after induction of deep anesthesia with intraperitoneal sodium pentobarbital. Fifty μl (10 μg/ml) of an anti-TNF monoclonal antibody, TN3-19.12  without sodium azide was diluted in PBS and injected locally into right and left TMJ areas of the UAC+Inhibitor and Control+Inhibitor groups. The same volume of PBS was similarly injected bilaterally into the TMJ areas of rats in the UAC+PBS group. The injections were performed once every two days for 2 weeks  until thAll rats were sacrificed by pentobarbital overdose, and the TMJs were sampled after 2 or 4 weeks of UAC stimulation n = 8). Similar to our previous study [. SimilarFor morphologic analysis, a tissue block containing the right TMJ was dissected out. The histological samples were fixed in 4% paraformaldehyde for 1 day and decalcified in 10% EDTA for 35 days. After dehydration, the tissue blocks were embedded in paraffin. A rotary microtome  was used to make serial 4-μm-thick sections through the TMJ in the sagittal plane. The sections were mounted onto poly-L-lysine-coated glass slides. Hematoxylin&eosin and Safranin O staining was performed to determine changes in morphology and proteoglycans, respectively. TUNEL staining was performed to detect apoptosis in samples from every group. To ensure a reliable comparison between the specimens from different groups, the central sagittal sections of each joint were selected.For Western blot and real-time PCR analysis, the cartilage was carefully isolated to exclude bone tissue and preserved at -80°C for protein or mRNA preparation. To ensure that the technical parameters of each stain were identical and that the results were comparable between groups, the sections from each group for one type of staining were stained at the same time.Hematoxylin&eosin (H&E) and immunohistochemical (IHC) staining was performed, as previously reported . Immunohin situ Cell Death Fluorescein Detection Kit (Roche) following the manufacturer’s protocol. Briefly, after deparaffinization and rehydration, sections were incubated with proteinase K  for 50 min at 37°C. After rinsing with PBS, the sections were incubated with the reaction mixture solution for 120 min at 37°C. The slides were mounted and analyzed under a fluorescence microscope  with 450–500 nm excitation filters and an emission of 515–565 nm. Positive and negative controls were incubated with 0.01 mg/ml bovine pancreatic DNase I (Boehringer Mannheim GmbH) or labeling solution, respectively. Immunofluorescent positive cells in cartilage were counted and compared between groups.Apoptotic chondrocytes in the condyles were assessed by TUNEL analysis with an 7 cells/cm2. The medium was replaced every 2 days, and the cells reached confluence after approximately 5 days in culture. The isolated chondrocytes were treated with 10, 50 or 100 ng/ml of TNF for 24 h. Chondrocytes in another group were treated with TNF 100 ng/ml and TNF inhibitor 10 ug/ml together for 24 h and were then isolated for further examination.Primary chondrocytes were isolated from the 6-week-old rat mandibular condylar cartilage through enzymatic digestion for 20 min with 0.25% parenzyme, followed by 2 h with 0.2% type II collagenase in RPMI 1640. The isolated cells were collected by brief centrifugation and were then resuspended in RPMI 1640 supplemented with 10% (v/v) FBS, 50 mg/ml streptomycin and 50 units/ml penicillin (Gibco). The cells were plated in culture dishes at a density of 5×107 cells) or condyle cartilage (100 g) samples were collected by trypsinization and centrifuged at 600 g for 5 min at 4°C. Then, the samples were incubated with 800 μL of mitochondria isolation reagent and homogenized on ice in a Dounce homogenizer. Unlysed cells and large debris were pelleted by centrifugation at 700 g for 10 minutes at 4°C. The supernatant was further centrifuged at 12,000 g for 15 minutes at 4°C. The supernatant (cytosolic fraction) was collected, and the pellet  was lysed in 100 μL of mitochondria splitting reagent to obtain mitochondrial fractions.Mitochondria were isolated from chondrocytes from the TMJ condylar cartilage, which was carefully separated from bone tissue according to previously described procedures . MitochoWestern blot analysis was used to detect the cytochrome c content in the mitochondria and cytosol of chondrocytes in TMJ condylar cartilage, which was carefully removed to exclude bone tissue, and of cultured chondrocytes. The protein concentrations were quantified using the bicinchoninic acid (BCA) method with a protein assay kit (Pierce). The protein aliquots (30 μg) were separated using 12% SDS/polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore). After blocking in 5% non-fat dried milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST), pH 7.4, for 60 min at room temperature, the membranes were incubated overnight with purified rabbit anti-cytochrome c monoclonal antibodies  at 4°C. After washing with TBST, the membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated IgG at room temperature for 1 h. The blots were then visualized by enhanced chemiluminescence with ECL reagents (Pierce).The activity of caspase-8 and caspase-9 were determined using fluorogenic assay kits . The chondrocytes and TMJ condylar cartilage, which were carefully removed to exclude bone tissue, were lysed, and the extracts (30 mg protein) were incubated for 2 h with 2 mM of the specific fluorogenic peptide substrates Ac-IETD-pNA (acetyl-Ile-Glu-Thr-Asp p-nitroanilide) for active caspase-8 and Ac-LEHD-pNA (acetyl-Leu-Glu-His-Asp p-nitroanilide) for active caspase-9. The samples were read in a microplate spectrophotometer  with a 405-nm emission filter.-ΔΔCt. The results were calculated as the relative fold increase/decrease of the target gene compared to the 2-week Control group.The condylar cartilage samples from every group were homogenized and the total RNA was extracted using the Tripure Isolation Reagent (Roche). Primers for the targeted genes were designed as shown in 6 cells were harvested and placed in EP tubes. The cells were rinsed with 0.1 mmol/L PBS (pH = 7.4) and collected by brief centrifugation. After that, the cells were resuspended in 190 μL of binding buffer, mixed with 5 μL of Annexin V-FITC, and then kept in darkness for 10 min at room temperature. Then, 10 μL of propidium iodide (PI) was added and the samples were kept in darkness for 10 min at room temperature. Approximately 300 μL of binding buffer was added, and the samples were analyzed using flow cytometry within 1 h. The blank control was the cells of the control group without Annexin V-FITC and PI. The Annexin V-FITC single-labeled control consisted of cells labeled with Annexin V-FITC. The PI single-labeled control consisted of cells labeled with PI.After 24 h of treatment, 1×10t-test was used to compare two groups. For multiple comparisons of three or more groups, one-way analysis of variance with Tukey’s post hoc test was used. The results were expressed as the mean values with 95% confidence intervals (95% CI). p values less than 0.05 were considered to be statistically significant in all cases, and each experiment was performed in triplicate.Statistical analysis was accomplished using the SPSS 21.0 software. All data acquisition and analysis were completed blindly. Student’s p = 0.026 for the 2-week group, p = 0.002 for the 4-week group; posterior third: p = 0.128 for the 2-week group, p = 0.014 for the 4-week group)  , but not at 2 weeks  (p = 0.002) and IL-6 (p = 0.002) in the UAC group were down-regulated at 2 weeks and were identical to the control group at 4 weeks  . The prok group) . TNF-posk group) . The per= 0.063) . The mRN 4 weeks . There wp = 0.02 and p = 0.008, respectively). The TUNEL-positive cells were located mainly in the proliferative and hypertrophic layers of the condylar cartilage  (p = 0.002) (p = 0.003) . The level of cytochrome c release from the mitochondria into the cytosol was also increased at both 2 and 4 weeks   . However= 0.002) . Further= 0.003) , whereasctively) .p = 0.024; TNF mRNA: p = 0.041) (p>0.05) (p = 0.021) (p = 0.023), and the levels of active caspase-8 units (p = 0.006), but had no effect on the mRNA expression levels of caspase-9, the levels of active capsase-9, or the cytosolic levels of cytochrome c   in the c= 0.041) . The mRN(p>0.05) . The TNF= 0.021) , the mRNchrome c . The TNFp = 0.007; 50 ng/ml: p = 0.029; 100 ng/ml: p = 0.034), though the mRNA expression levels of caspase-9 was not up-regulated (p>0.05) (p = 0.029 and p = 0.005) (p = 0.009) and of active caspase-8 (p = 0.017)  . Fluorog= 0.005) . However= 0.005) . Additio= 0.017) , but had= 0.017) .in vitro data agree with this finding, demonstrating that caspase-8 is involved in the TNF-stimulated apoptosis of chondrocytes without involvement of either caspase-9 or cytochrome c at all three dosages. At the highest dose of TNF (100 ng/ml) used to treat chondrocytes, the TNF inhibitor (10ug/ml) prevented the caspase-8 increase without affecting either caspase-9 or cytochrome c. This compliments the in vivo data showing that injection of the TNF inhibitor down-regulated the levels of caspase-8, but not caspase-9 or the release of cytochrome c. Moreover, this treatment effect of the TNF inhibitor in the presence of TNF confirmed that this anti-TNF monoclonal antibody works as a neutralizing antibody.Temporomandibular joint disorder (TMD) is common and is induced by various pathologic conditions, including aberrant occlusion . TMJ-OA Some studies have suggested that the mandibular condylar cartilage plays a significant role during mandibular development and has been referred to as secondary cartilage , which iIntra-articular injection of a TNF monoclonal antibody model has been reported to control local inflammation and inhibit the degradation of the cartilage matrix in an animal model of TMJ OA , and alsTNF plays a pivotal role in induction of chondrocyte apoptosis and cartilage degeneration in OA  and has In summary, we report that UAC stimulation induced apoptosis of chondrocytes in osteoarthritic cartilage, which was initiated by the mitochondrial pro-apoptotic signaling pathway and was secondarily promoted by increased expression of TNF and the extrinsic apoptotic pathway. Our results demonstrate limited efficacy of injecting a TNF inhibitor to prevent chondrocyte apoptosis in OA and the progression of OA lesions and our recently published findings indicate that removal of the aberrant biomechanical stimulation is pivotal for rescue of the OA lesions.S1 Appendix(PDF)Click here for additional data file.