Parecoxib (5.0 mg/kg once daily) or vehicle was administered intraperitoneally for 22 consecutive days. Skin lesions were classified histologically. Toxicological endpoints included genotoxic parameters, hepatic oxidative stress, transaminases and histology. Parecoxib completely prevented the onset of epidermal dysplasia in HPV16+/− treated animals . Parecoxib decreases lipid peroxidation (LPO) and superoxide dismutase (SOD) activity and increases the GSH:GSSG ratio in HPV16+/− treated animals meaning that oxidative stress is lower. Parecoxib increased genotoxic stress parameters in wild-type and HPV16-transgenic mice, but didn’t modify histological or biochemical hepatic parameters. These results indicate that parecoxib has chemopreventive effects against HPV16-induced lesions while maintaining an acceptable toxicological profile in this model.Carcinogenesis induced by high-risk human papillomavirus (HPV) involves inflammatory phenomena, partially mediated by cyclooxigenase-2. In pre-clinical models of HPV-induced cancer, cyclooxygenase-2 inhibitors have shown significant efficacy, but also considerable toxicity. This study addresses the chemopreventive effect and hepatic toxicity of a specific cyclooxigensase-2 inhibitor, parecoxib, in HPV16-transgenic mice. Forty-three 20 weeks-old female mice were divided into four groups: I (HPV16 Persistent high-risk human papillomavirus (HPV) infections are among the leading causes of cancer in the world. This infection is mainly associated with cervical cancer as well as other anogenital malignancies and a subgroup of oropharyngeal cancer . Followi−+/ mice showed higher relative masses of the lungs, spleen, liver, and kidneys  showed normal histology . HPV16-n+/− mice (group III) showed a 100% incidence of dysplasia, but dysplastic lesions were absent in all mice . HPV-transgenic mice showed increased numbers of tumor-associated leukocytes compared with wild-type animals  showed normal histology . HPV16-n animals . Parecox animals .+/− mice compared with matched untreated controls. However, it slightly reduced the oxidative damage revealed by Fpg (+/− mouse control (group IV) showed mild hepatitis, characterized by Kupffer cell hyperplasia and multifocal microabscesses, while mice in all other groups showed normal liver histology. In line with these findings, the plasma concentrations of albumin (Alb), total proteins (TP), glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gama glutamyl transerase (GGT) were not statistically different between groups  in HPVd by Fpg . Only onn groups .p = 0.0072), glutathione S-transferase (GST) activity had no changes and glutathione reductase (GR) had an increase in its activity, but differed only between wild-type groups (p = 0.0028). Lipidic peroxidation showed a statistical decrease for wild-type groups  and the ratio of GSH:GSSG increased in treated groups, but is only statistically different to the wild-type ones (p = 0.0042).The results presented in Chronic inflammation is associated with factors like reactive species of oxygen and nitrogen, cytokines, chemokines, growth factors, and specific microRNAs, which often contribute to cancer development . In most+/−), and wild type-mice (HPV16−/−) treated or not with parecoxib. K14-HPV16 mice in an FVB/n background express all the early genes of HPV16 under the control of the human cytokeratin 14 promoter. These animals develop multi-step lesions, associated with progressive inflammation, in squamous epithelia such as the skin, similar to HPV-induced lesions in patients ,26,27. TIn the present study, we made two major sets of observations. The first, concerning the efficacy of parecoxib against HPV16-induced lesions, showed that this drug completely abrogated the onset of epidermal dysplasia in our animal model. Secondly, we observed that parecoxib displayed a favorable toxicological profile concerning several general and liver-specific parameters and the proper functioning of antioxidant defenses.+/− mice treated with parecoxib showed only epidermal hyperplasia that did not progress to dysplasia, while 63.6% of untreated HPV+/− mice showed epidermal dysplasia. This is of great importance, given that the dysplastic stage precedes the development of squamous cell carcinoma in this model, suggesting that parecoxib may able to block the development of HPV16-induced cancer. A previous study using celecoxib showed similar results [We observed that HPV results . Another results . We foun results . FurtherO’Donoghue et al.  employedDuring the experimental protocol, the animals were monitored for signs of disease and distress that could reflect toxicity. Importantly, no changes in the behavior and physiology of the animals were observed. There were also no significant differences concerning body weight or food consumption, supporting the hypothesis that parecoxib was safe in the present experimental conditions. Transgenic animals had a higher water consumption, as previously reported for this animal model . We also observed significant differences concerning the relative mass of internal organs between wild-type and transgenic mice, which had also been reported for these animals. Transgenic animals treated with parecoxib showed near-normal values for the relative masses of internal organs, suggesting that this compound may act systemically to re-establish homeostasis in this setting. Fpg. This is likely reflecting the hepatic biotransformation of parecoxib into valdecoxib, and suggests that DNA-repair mechanisms could cope with parecoxib-induced genetic damage, so that it didn’t translate into to biochemical or morphological changes. Parecoxib did not induce detectable hepatotoxicity at the biochemical or histological levels. In wild-type animals, parecoxib did not alter the baseline levels of genetic damage. However, in transgenic animals, parecoxib increased the total genetic damage index (GDI) while slightly reducing the GDIOxidative stress is caused by an imbalance between oxidants–antioxidants that favors the oxidants, leading to the excessive generation of free radicals, particularly reactive oxygen species (ROS), and subsequently to biological damages . Our res2•−), and GR, GST, and CAT didn’t show any differences. GST activity does not show differences since parecoxib is rapidly converted to valdecoxib, the pharmacologically active substance, by enzymatic hydrolysis in the liver, after that cytochrome P450 system metabolizes it to hydroxyvaldecoxib [Regarding the results of transgenic mice HPV16) treated with parecoxib (group III) and its control (group IV), it seems that parecoxib leads to a decrease in oxidative stress since lipid peroxidation decreases and the GSH:GSSG ratio increases, although the latter is not statistically significant. The reduction of ROS production, and therefore the lower oxidative stress observed in groups treated with parecoxib will reduce COX-2 expression, leading to the inhibition of prostaglandin synthesis and, subsequently, proinflammatory cytokines which are also responsible for increased free radical production . For oxi treated ldecoxib . The GSH−/−) and 22 transgenic (hemizygotic HPV16+/−) animals. The animals were genotyped using previously described methods [The K14HPV16 strain was generously donated by Drs. Jeffrey Arbeit and Douglas Hanahan, from the University of California, through the USA National Cancer Institute Mouse Repository. Forty-three female, 20 weeks-old, FVB/n mice were used, including 21 wild-type ; group II ; group III ; group IV . Parecoxib (5.0 mg/kg once daily) [The animals were kept under controlled conditions such as temperature (23 ± 2 °C), relative humidity (50 ± 10%) and light-dark cycle (12h light/12h dark). Food and water were provided ad libitum and the health of animals was checked daily. The 43 animals were divided into four groups: group I (HPV16e daily) ,32 was ae daily) . Skin saThe chest skin and ear samples were fixed in 10% neutral-buffered formalin, included in paraffin and stained with hematoxylin and eosin for histological analysis. Each sample was classified as normal skin, epidermal hyperplasia and epidermal dysplasia, as previously described . Total iThe skin samples were tested immunohistochemically for expression of the proliferation marker ki67. Briefly, endogenous peroxidase was blocked using Refine Peroxide Block (Leica) and the slides were incubated with the primary antibody  for 60 min. The refine (rabbit) polymer HRP (Leica) was used as secondary antibody and the antibody reaction was detected with 3.30-diaminobenzidine tetrachloride (DAB) for 10 min and counterstained with Mayer’s hematoxylin (4 min). Mouse spleen samples were used as positive controls and mouse IgG was used as an isotype control. Samples were classified as normal when ki67-positive cells were restricted to the basal layer of the epidermis or as pathological when suprabasal layers showed positive cells as well.Humane endpoints were assessed weekly by the same researcher, using a previously published scoring sheet . Animalsg for 15 min, and the plasma was separated and frozen at −80 °C for further analysis. Plasmatic concentrations of Alb, TP, glucose, ALT, AST and GGT were determined by spectrophotometric methods using an autoanalyzer . Histologically, liver samples were classified as normal liver or mild hepatitis, characterized by Kupfer cell hyperplasia with or without microabscesses.Blood collected by cardiac puncture during euthanasia was stored in heparinized tubes, centrifuged at 1400× The alkaline comet assay was performed based on the method described by Collins , as adapn = 7) were used. The liver samples (≈ 0.25 g) were washed and added to 10% (p/v) phosphate buffer . After homogenization and sonication , the samples were subjected to three centrifugation cycles, at 4 °C: 800× g for 10 min, 10,000× g for 20 min and 12,000× g for 20 min. The supernatant obtained in the last centrifugation was used to measure antioxidant enzymes activities and GSH:GSSG ratio. The lipid peroxidation was determined using the pellets of second and third cycles. The total protein content of the supernatant and pellets obtained was evaluated by the Biuret method, using BSA as standard [−• generated by xanthine/xanthine oxidase system, according to the method originally described by Payá [−1·mg protein−1. CAT activity was assayed polarographically using a Clark-type oxygen electrode as described before [2O2·min−1·mg protein−1. GR activity was assayed as described previously [−1·mg protein−1. GST activity was evaluated, as we described previously [−1 cm−1) in 100 mM potassium phosphate buffer, 10% Triton X-100, 100 mM CDNB and 100 mM GSH. Lipid peroxidation was assayed by a modified TBA-based method for measuring the aldehydic lipid peroxidation decomposition derivatives (such as MDA), which form fluorescent products after reacting with TBA [g, 3 min), the fluorescence of the upper butanol layer was measured at excitation 535 nm and emission 550 nm. The results are expressed as µmol MDA·mg protein−1. The GSH:GSSG ratio was calculated based on individual concentrations of glutathione reduced and oxidized that are determined spectrofluorimetrically using ortho-phthalaldehyde (OPA 1 mg/mL methanol) at excitation and emission wavelengths of 350 nm and 420 nm, respectively [N-ethylmaleimide (NEM) at room temperature and then incubated with 100 mM NaOH (pH 12.0) and 200 μL OPA to determinate GSSG content. Standard curves were prepared for calculation of GSH and GSSG concentrations.Twenty-eight mice livers from the four experimental groups , version 25. A Chi-squared test was performed to study the distribution of histological lesions between groups. Student’s The present results indicate that parecoxib is highly effective against HPV16-induced lesions, altogether abolishing the development of epidermal dysplasia. The mechanisms underlying its mechanism of action should be the focus of additional studies on this subject. Furthermore, parecoxib seems to attenuate oxidative stress induced by HPV-16 infection and showed a favorable toxicological profile, suggesting it is safe at the current dose in this model.


Left bundle branch area pacing (LBBaP) is a new physiological pacing strategy that produces comparable clinical effects to His bundle pacing (HBP).The purpose of this study was to investigate the immediate clinical outcomes of LBBaP vs RVP.P < .001) and X‐ray exposure times . The lead threshold of the LBBaP group was increased , while the R‐wave amplitude and ventricular impedance did not significantly differ between the two groups. The conventional RVP procedure significantly widened the QRS complex , whereas the LBBaP had no effect on QRS complex . Furthermore, the LBBaP procedure significantly narrowed the QRS complex in patients with left bundle branch block (LBBB) .From April 2018 to September 2018, we included 44 patients under continuous pacemaker implantation. Patients were randomly divided into the LBBaP group and conventional RVP group. Compared to the RVP group, the LBBaP group displayed significantly increased operative (90.10 ± 19.68 minutes vs 61.57 ± 6.62 minutes, LBBaP is a new physiological, safe and effective pacing procedure with a high overall success rate. Compared to conventional RVP, LBBaP can correct LBBB, thereby improving cardiac electrical dyssynchrony. AFatrial fibrillationCRTcardiac resynchronization therapyECGelectrocardiogramHBPHis bundle pacingHFheart failureHFHheart failure hospitalizationHYHANew York Heart AssociationLAOleft anterior obliqueLBBleft bundle branchLBBaPleft bundle branch area pacingLVleft ventricularLVDDleft ventricular end‐diastolic dimensionLVEFleft ventricular ejection fractionRAOright anterior obliqueRBBright bundle branchRVright ventricularRVPright ventricular pacing1Right ventricular pacing (RVP) is gradually being recognized as a conventional pacing method which may lead to cardiac electro‐mechanical dyssynchronize.Although clinical experts have attempted pacing multiple positions  in the right ventricle, these do not lead to physiological pumping of the heart and so the clinical effect does not make a difference.22.1All consecutive patients from Beijing Anzhen Hospital received implantable pacemakers between July and September 2018, according to the established guidelines.33.1. Subsequently, the 3830 lead and C315 sheath were pushed together clockwise in the ventricular apex direction (1‐3 cm)Figure B.max) was similar for selective and non‐selective pacing; (e) the paced morphology was the left anterior branch block; and (f) under the X‐ray, the final position of the leads in the patient performed HBP and LBBaP at the same time (Appendices S1 and S2).The basic criteria of LBBaP Figure A‐D were 3.2RV leads were implanted in a standard fashion at the RV apex or septum.3.32 test or Fisher's exact test  and used to examine the aforementioned differences. All the tests were two‐sided. A P‐value < .05 was considered statistically significant.SPSS version 19.0  was used for all statistical analyses. Normally distributed continuous data were expressed as the mean ± SD. Categorical data were described as the number (%) and χ44.1P = .012). In the LBBaP group, a higher incidence of heart failure was observed ; left ventricular ejection fraction (LVEF) was lower , left ventricular end‐diastolic dimension (LVDD) was larger , and the electrocardiogram(ECG) QRS interval was wider . Other clinical baseline data did not statistically differ between the two groups.A total of 44 consecutive patients were enrolled and divided into two groups by the methods of RV lead implantation, that is, RVP and LBBaP groups. In the LBBaP group, 23 patients underwent pacing in the LBBa region, among which the surgery was successful in 20 patients (87.0%). A total of three patients 13.0%) failed LBBaP and underwent RV septum pacing instead. In the RVP group, 21 patients underwent conventional RVP and all surgeries were success (100%)  and X‐ray exposure time  were significantly increased for the LBBaP group. Intraoperative testing of the ventricular lead parameters showed that the LBBaP group lead captured the threshold increase , while the R wave amplitude did not significantly differ between the two groups. In addition, no surgery‐related complications occurred in either group  were implanted with dual‐chamber pacemaker and 1 (5%) was implanted with single‐chamber pacemaker. On the other hand, 21 patients of the RVP group were implanted with a dual‐chamber pacemaker. Compared to the RVP group, the surgery time . Furthermore, we analyzed patients with LBBB in the LBBaP group (n = 7) and observed significantly narrowed QRS waves . The traditional RV pacing procedure (n = 21) significantly widened the QRS complex . (Table We compared the electrocardiogram (ECG) parameters before and after surgery. In the LBBaP group (n = 20), the QRS wave narrowed  LBBaP is a safe and effective physiological pacing procedure with a high success rate (87.0%), and (b) LBBaP does not increase the QRS complex width, particularly in patients with LBBB, and significantly narrows the QRS interval.The conventional RVP method results in artificial LBBB‐like ventricular activation and lost ventricular electromechanical synchronization. This significantly increases the incidence of AF, hospitalization rates, and mortality due to HF.P = .001), the average threshold of 0.68 V and the pulse width of 0.5 ms were lower than the output threshold of HBP. The R wave amplitude of the lead in 20 patients with LBBaP was 9.28 ± 5.00 mV which did not statistically differ from the RVP group. The R wave amplitude of HBP was significantly increased compared to previous reports. Abdelrahman et alHuang et alP = .094). Surprisingly, LBBaP could significantly reduce the QRS interval  in patients with LBBB. The LBBaP procedure rotated the 3830 lead into the interventricular septum and allowed it to reach the left ventricular surface LBB area, directly pacing the conduction system and rapidly agitating the entire ventricle to achieve electro‐mechanical synchronization. A total of seven patients with HF combined with LBBB through the LBBaP had achieved electrical synchronization. Hence, LBBaP could significantly improve the clinical prognosis of patients with cardiac asynchrony, and achieve CRT efficacy, which may represent an alternative strategy to traditional CRT. The LBBaP procedure was similar to HBP since the heart was electrically synchronized and would be effective for patients with distant sites at His. At the same time, the advantages of a low threshold, high sensing, and firm lead were achieved. In addition, LBBaP can theoretically achieve cardiac resynchronization in patients blocked in His bundle.In this study, the LBBaP procedure reduced the QRS interval  The 3830 lead better should be placed under the X‐ray RAO and the lead under LAO should be screwed to ensure that the pre‐shaped sheath is perpendicular to the interventricular septum surface; (b) during the screw‐in process, the intermittent lead parameter should be measured and when the impedance drops by approximately 40% of the initial value, which highly suspected that the 3830 lead entered the LV through the interventricular septum; (c) upon entering the LV through the interventricular septum, calm should be retained for favorable results when the lead is retracted; (d) for patients with an enlarged heart, to quickly identify the ideal screw‐in point, the C315 sheath should be properly shaped. After the inner sheath and outer sheath are assembled, the large bending angle of the sheath will be gently reduced; (e) patients with LBBB must have temporary pacemaker protection. Upon searching for the ideal screw point of the C315 sheath, it would be easy to damage the vulnerable RBB, so the patient will have a third‐degree atrioventricular block; and (f) for patients with myocardial infarction, particularly in patients with anterior myocardial infarction, a clear magnetic resonance imaging should be performed prior to surgery to clarify whether the interventricular septum is fibrotic. If fibrosis is evident in the interventricular septum, then the patients are not suitable for LBBaP.6There were several limitations associated with our study. First, it was not a random, controlled, multicenter prospective study, therefore, the LBBaP surgery was not representative. Second, the small sample size may produce bias and not all patients received a follow‐up. As such, the clinical effect of LBBaP remains unexplained.7In summary, LBBaP is a new type of safe and effective physiological pacing, with an overall high success rate. With increased surgical proficiency and continuous improvement, success rates might further improve. Similar to HBP, LBBaP can achieve cardiac electrical synchronization. Compared to RVP, LBBaP does not increase patient electrical disconnection. In addition, LBBaP can correct LBBB, thereby improving electrical synchronization.The authors declare that there are no conflicts of interest regarding the publication of this paper.APPENDIX S1The morphology and duration of QRS in different pacing modes. A, Normal QRS morphology in the patient without conduction disease;B, LBBB pattern and wide QRS interval in the RVP patient with a complete atrioventricular block.C and D, LBBB pattern and the wide QRS interval before LBBaP, while left anterior branch block pattern and narrow QRS interval after LBBaP in the same patient with LBBB.LBBB, left bundle branch block; LBBaP, left bundle branch area pacingClick here for additional data file.APPENDIX S2 The final position of leads in one patient with HBP and LBBaP at the same time. HBP, His bundle pacing; LBBaP, left bundle branch area pacingClick here for additional data file.


The intestinal epithelial monolayer, at the boundary between microbes and the host immune system, plays an important role in the development of inflammatory bowel disease (IBD), particularly as a target and producer of pro-inflammatory TNF. Chronic overexpression of TNF leads to IBD-like pathology over time, but the mechanisms driving early pathogenesis events are not clear. We studied the epithelial response to inflammation by combining mathematical models with in vivo experimental models resembling acute and chronic TNF-mediated injury. We found significant villus atrophy with increased epithelial cell death along the crypt-villus axis, most dramatically at the villus tips, in both acute and chronic inflammation. In the acute model, we observed overexpression of TNF receptor I in the villus tip rapidly after TNF injection and concurrent with elevated levels of intracellular TNF and rapid shedding at the tip. In the chronic model, sustained villus atrophy was accompanied by a reduction in absolute epithelial cell turnover. Mathematical modelling demonstrated that increased cell apoptosis on the villus body explains the reduction in epithelial cell turnover along the crypt-villus axis observed in chronic inflammation. Cell destruction in the villus was not accompanied by changes in proliferative cell number or division rate within the crypt. Epithelial morphology and immunological changes in the chronic setting suggest a repair response to cell damage although the villus length is not recovered. A better understanding of how this state is further destabilised and results in clinical pathology resembling IBD will help identify suitable pathways for therapeutic intervention. Recent findings suggest a primary role for focal injury of the epithelial lining and selection for aggressive microbial communities preceding the establishment of Crohn’s-like ileitis4. Likewise, the murine dextran sodium sulfate (DSS) colitis model highlights the importance of the severity of epithelial injury in the establishment of IBD. Depending on the DSS dose, animals present either severe intestinal injury with impaired “mucosal healing” and fatality, or mild injury with rapid restoration of mucosal integrity6. Ultimately, re-establishment of the epithelial barrier leads to sustained clinical remission and resection-free survival in IBD patients7.Inflammatory bowel disease (IBD) is associated with excessive epithelial death in the ileum and colon10. A single exogenous high dose of TNF induces transient intestinal damage with rapid epithelial cell apoptosis, predominantly at villus tips, villus shortening, fluid exudation into the gut lumen, and diarrhoea13. Animal models with persistent elevated TNF exhibit IBD-like inflammatory changes in the mucosa and are widely used to study intestinal chronic inflammatory processes15. Such models reveal the role of epithelial cells as targets and producers of TNF in apoptotic death, leading to barrier breach and ultimately to IBD-like pathology18.TNF is a cytokine produced by immune, mesenchymal and epithelial cells, and regulates the epithelial barrier in multiple ways, including mucus secretion, barrier permeability, proliferation/differentiation and wound healing21. although heterogeneous responses are detected upon differences in signal transduction downstream of the receptor binding24. TNFRII can play an additive role in enterocyte death13 or diverse roles in epithelial cell survival, proliferation and migration, and immune regulation28.Numerous studies using TNFRI and TNFRII knockout mouse models suggest TNF-induced cell apoptosis in the small intestine is triggered primarily through TNFRI signalling29: the duodenum which, is usually not compromised by IBD, and the ileum, which exhibits typical lesions during IBD episodes. We combined cell labelling and tracking techniques with mathematical modelling to quantify cell dynamics along the crypt-villus epithelial unit (CVEU), a one-dimensional column of cells running from the base of a crypt to the tip of an adjoining villus31. We used Bromodeoxyuridine (BrdU) to quantify the progression of labelled cells along the CVEU, from which we inferred the absolute cell production rate, henceforth referred to as epithelial turnover. This rate quantifies the cell yield resulting from proliferation and death along the CVEU and differs from the number of cells generated per proliferative cell per unit time, which we referred to as division rate. Epithelial turnover depends on the number of proliferative cells, the division rate, and the rate at which cells die along the crypt-villus axis. To study each of these parameters in our experimental models, we combined mathematical models with BrdU S-phase cell labelling, Vincristine mitosis arrest, and TUNEL staining. Concurrently, we measured the intracellular concentration of TNF and the spatial distribution of TNF receptors along the CVEU. Applying this methodology, we aimed to gain insight into the loss of epithelial homoeostasis preceding IBD development.We here investigated epithelial cell dynamics in the small intestine of experimental mouse models of acute and chronic intestinal inflammation. Acute inflammation was induced by a single intraperitoneal delivery of recombinant TNF, while chronic inflammation was induced by delivery of a TNF-expressing plasmid, resulting in lower, but persistent, levels of circulating TNF Fig. . We studFollowing a single intraperitoneal delivery of rTNF (0.5 mg/kg) Fig. , intense15. These relatively low TNF levels result in no effect if delivered as a single dose, but lead to pathology if maintained for longer periods15. Core retraction, tip constriction and shedding were not observed in the samples collected post-induction of chronic injury  to induce low-level TNF production in our chronic model of inflammation. We quantified circulating TNF at 48 h, 1 week and 2 weeks post-plasmid delivery and observed constant average values of 216 ± 82 pg/ml plasma, similar to those reported in genetically altered mouse models of TNF-driven intestinal damage, 90–430 pg/mlury Fig. . Howeverury Fig. . Chronicury Fig. . Thus, aWe studied the intestinal immune response by analysing expression of inflammation-associated proteins and assessing the mucosal immune cell composition with flow cytometry. Levels of CXCL5, MCP-1 and M-CSF, which are all involved in recruitment of immune cells to sites of inflammation, were increased in chronic inflammation . Flow cytometry analysis showed a shift from T-effector to T-regulatory profile in chronic inflammation, compared to acute TNF-injured and healthy mucosa .31. We generated a BrdU labelling dataset by injecting a single dose of BrdU and sampling repeatedly in the following 48 h for both the acute and chronic models . To calculate the number of proliferative cells in the crypt, we, therefore, administrated a combination of vincristine sulphate to halt cell division during metaphase, leaving visible mitotic figures, and BrdU to label cells in S phase. Vincristine arrested the division cycle of all cells entering metaphase for a period of 8 h post-injection (data not shown). BrdU was administered together with the initial vincristine injection and approximately every 2 h thereafter to label cells already in, or entering, S-phase over the following 8 h Fig. . The totOur results showed no differences in the size of the proliferative compartment between duodenum and ileum in control animals Fig. . During 11  positive cells mainly on the villus tips of both inflammation models indicating cell death by apoptosis in agreement with previous reports11 Fig. . The num11 Fig. . While t11 Fig. . An incr11 Fig. .Fig. 3CeWe then quantified the temporal dynamics of cell death along the crypt-villus axis. The estimation of death rates enabled the further assessment, as detailed below, of whether the decreased epithelial turnover observed in chronic inflammation is associated with increased cell death. We developed a mathematical model which assumes that death commences when cells become TUNEL-positive and culminates in cells detaching from the epithelium (equations (6–7) in supplementary material). The model considers two cell ‘compartments’, (i) healthy cells and (ii) TUNEL-positive cells, and describes how the number of cells in each compartment changes over time, as healthy cells die, detach, and are lost from the system. The parameters governing these dynamics are the rate of death of healthy cells and the rate of detachment of apoptotic cells (equations (6–7)). Identification of parameter values is described in the supplementary material. Model fits, estimates and errors are given in Fig. Cell loss in duodenal and ileal CVEUs  during the first hour of acute inflammation Fig.  is not c34. Two compartments comprise crypt cells: one with proliferative cells and the other with non-proliferative cells; the third compartment, or villus compartment, contains all remaining non-proliferative cells of the CVEU  and II (TNFRII) along different regions of the CVEU. Following a single acute TNF injection, we detected variation in intraepithelial levels of TNF, with an increasing concentration from villus base to tip. Levels of intracellular TNF in villus tips were ~100-fold higher than in healthy mice Fig. . The detImmunofluorescence staining for TNFRI Fig.  in healtAltogether, these results indicate that cells on the villus tip respond rapidly to TNF by upregulating TNFRI and TNFRII. TNFRI spatial pattern of upregulation is associated with elevated intracellular levels of TNF and cell apoptosis, which results in rapid loss of the tip in acute inflammation. We hypothesise that the maintenance of elevated circulating TNF impairs the recovery of the TNF-sensitive villus tips in our chronic injury model, in which we observe recovered epithelial continuity but reduced villus length.35, while DSS injury is reported to increase apoptosis, but decrease proliferation in BALB/c mice36. Increased cell migration but unaffected proliferation is reported in DSS-treated colon37. In a surgical biopsy injury model, increased proliferation was reported exclusively in the colonic crypts adjacent to the site of biopsy38.The integration of labelling techniques with mathematical models here demonstrated a lack of proliferative compensatory response to chronic inflammation. In other reports, proliferative responses to sustained epithelial injury are reported in the colon, but vary with species and injury model. Increased proliferation and apoptosis is reported in DSS colitis in rats39 and the detection of pro-apoptotic cleaved villin fragments specifically in tip cells40, have also been implicated in TNF-regulated cell death. Upregulation of these factors may be promoted by signalling from tip-localised subsets of sub-epithelial myofibroblasts, which are morphologically and phenotypically distinct from fibroblasts located lower in the villus body44.We found increasing TNF intracellular concentration toward the villus tip in acute inflammation Fig. . We also18.In chronic inflammation, we did not observe tip-localised expression of TNFRI Fig. , yet cas45 and ileal L-cells46, but does not reproduce conflicting descriptions of more widespread apical29 or basolateral24 staining of duodenal epithelial cells. Our ileal TNFRI staining following TNF challenge is similar to previous reported patterns in healthy ileum29. Disparities likely reflect differences in animal housing and specific antibodies used. We detected widespread TNFRII expression in epithelial cells in our inflammation models in agreement with previous reports47. The differences we observed in TNFRI and TNFRII staining patterns suggest distinctive location-specific regulation which results in co-localisation of TNFRI, but not of TNFRII, with elevated intracellular TNF and TNF-induced apoptosis.The TNFRI staining pattern we observe in homoeostasis is in agreement with previous reports describing staining of lamina propria fibroblasts14 remains to be specifically determined. Strong evidence suggests that the microbiota is an essential driver of IBD development14, which may explain why we detect epithelial alterations in both duodenum and ileum in our chronic setting, whereas subsequent IBD lesions are reported to affect the ileum and ileocolonic region14. Of note is that subclinical lesions detected by confocal endomicroscopy are reported in the duodenum of IBD patients48.In chronic TNF injury, we quantified that increased cell death in the villus body and tip was responsible for the slower epithelial turnover and blunted villi, respectively. Together with inflammatory protein expression analyses and mucosal immune cell profiles, these results suggest a repair response to cell damage, which leads to villus re-epithelialisation but fails to recover the original villus length. What causes this state to further destabilise and lead to overt clinical pathology resembling IBD in later weeksIn summary, we have developed a strategic combination of in vivo models, labelling techniques and mathematical models to assess cell dynamics during acute and chronic injury aiming to understand the response of the epithelium to inflammation. Future detangling of the epithelial response, and the immune and microbial changes during the onset of IBD, is essential to discover new strategies for therapeutic intervention.2 and cervical dislocation, at the time points described in the text, prior to subsequent tissue collection. All animals were regularly monitored for clinical signs; any displaying signs beyond those expected within the moderate limits of the procedures would be immediately sacrificed by the above methods and were not included in experimental data. Osmotic minipumps  were inserted subcutaneously to anaesthetised animals.All animal experiments were conducted in strict accordance with the Home Office Animals (Scientific Procedures) Act 1986. Female C57BL/6 mice, aged 10–12 weeks and weighing at least 25 g prior to use in experiments, were housed and maintained in SPF conditions at the University of East Anglia, Norwich, UK in accordance with HO regulations, and all procedures were performed by fully-trained and licenced researchers. Experimental animals were closely monitored and were killed by rising CO30. BrdU was delivered simultaneously with TNF in the acute injury setting. In the chronic inflammation experiments, BrdU time courses were performed once elevated blood TNF levels had been established  at 0.5 mg/kg. Chronic inflammation was initiated by hydrodynamic tail vein delivery of 10 µg TNF-expressing plasmid  in a volume of 10% of body weight in Ringer’s Solution . Alternatively, low-level TNF  was delivered continuously for 2 weeks at 35 ng/h by subcutaneously implanted osmotic minipump. The minipump delivery setting was used to validate the epithelial turnover results observed in the plasmid mediated setting with similar TNF circulting concentrations. Plasma concentration of TNF was confirmed by specific ELISA  for elevated levels in blood plasma over a minimum of 14 days, and in liver and intestinal tissue lysates post-mortem. The thymine analogue 5-bromo-2-deoxyuridine, BrdU  was administered at 50 mg/kg body weight by single intraperitoneal injection. In the case of acute inflammation, the experimental ‘time zero’ of the BrdU labelling process was set to 2 h after the single-dose BrdU injection, which we previously demonstrated to be adequate for all mouse models and tissue sampleshed Fig. .To halt cells in mitosis, 0.5 mg/kg vincristine together with 50 mg/kg BrdU was delivered by single intraperitoneal injection. In the acute model, these two compounds were delivered simultaneously with the injection of TNF, while in the chronic model vincristine and BrdU were administered after ~2 weeks of TNF induction, once elevated plasma levels had been confirmed by ELISA. BrdU was then delivered at 2.33, 4.66 and 7 h post-vincristine injection both in the acute and chronic model Fig. . Tissues30. The numbers of labelled and unlabelled cells were recorded by position, from crypt base to villus tip, for 30–50 individual hemi crypt-villus units per tissue section per mouse, with counts recorded as binary values.At sampling time points post BrdU administration, mice were killed and intestinal tracts were removed, dissected, formalin-fixed and paraffin embedded. Transverse sections of duodenum and ileum were prepared at 5 μm and were immunostained for BrdU using biotinylated anti-BrdU antibody , Neutravidin-HRP , and diaminobenzidine reaction . Ki-67-positive cells were detected with Rabbit anti-Ki-67 and goat-anti-rabbit Alexa-568 (AbCam). TNFRI and TNFRII labelling was performed on FFPE sections with rabbit polyclonal antibodies against TNFRI  or against TNFRII  followed by secondary labelling with goat-anti-rabbit-Alexa-568 (Life Technologies). Villus cell apoptosis was confirmed histologically by Caspase-3 , and general cell death by TUNEL assay  in FFPE duodenal and ileal sections counterstained with H&E or DAPI. Labelled cell counts for BrdU, Ki-67, caspase 3 and TUNEL were obtained following the format described in ref. Small intestinal epithelial cells were isolated by removal of whole small intestine which was flushed, opened longitudinally, washed and cut into small (<1 cm) pieces which were then sequentially digested by three rounds of incubation at 37 °C, shaking for 8 min in DPBS/1 mM HEPES/2 mM EDTA. Detached epithelial cells in the resulting supernatants were pelleted and resuspended in IMDM, spun over 40% Percoll layer, aspirated, washed and re-pelleted, prior to preparation of cell lysates for protein analysis, as described below.49.Cell lysates of isolated epithelial cells or whole small intestinal tissue pieces (50 mg) were lysed using Lysing Matrix D ceramic beads  and Cell Lytic solution (Sigma-Aldrich) containing protease inhibitors. Samples were centrifuged at 13,000 rpm for 10 min at 4 °C. Supernatants were collected and total protein concentration determined using the BCA method . Samples were adjusted to normalise total protein concentration before inflammatory protein analysis using Mouse Inflammation Antibody Array . Array blots were imaged using chemiluminescence detection and imager . Densitometry analysis of images was performed in Fiji50. Briefly, small intestinal pieces (obtained as above) were incubated at 37 °C for 15 min in HBSS/1 mM HEPES/2 mM EDTA, washed four times  before collagenase/dispase digestion for 1 h at 37 °C. Supernatants were filtered, centrifuged, and pellets resuspended in chilled FACS buffer (DPBS/2% FBS 1 mM EDTA) on ice before antibody staining and flow cytometric analysis.Lamina propria lymphocytes and myeloid cells were isolated from small intestine using a method adapted from Scott et al.51 and are shown in Fig. S1B. 100,000 cells were analysed per sample. Live/dead discrimination was determined using Zombie UV dye . Cells were stained with a panel of immunophenotyping antibodies  for 40 min protected from light, at room temperature. Data were acquired with a BD LSRFortessa flow cytometer using BD FACSDiva software .For flow cytometry identification, gating strategies were based on those previously describedO} > 0.95, where O represents the dataset used to fit the model.The mathematical and statistical models developed to describe temporal cell dynamics in the CVEU are described in supplementary material. Model parameters were estimated using Bayesian inference. Statistical comparison of our experimental groups was carried out by simulating the posterior probability distribution of the difference between model parameters using Markov Chain Monte Carlo methods (MCMC). Differences between groups were considered significant when P{parameter (Group i) > parameter (Group j) | p-value <0.05) of differences between means of observed quantities in our experimental mouse models was assessed by ANOVA followed by a Tukey multiple comparison test.The significance (Parameter inference and statistical analysis was performed using SAS 9.4Supplemental Material.


Crassostrea gigas, while the gills and mantle lacked any HBGA structures. By using Virus-like particles (VLPs), which are antigenically and morphologically similar to native virions, we were able to demonstrate that VLPs of GI.1 West Chester norovirus reacted with the digestive tissues and palps. Despite of the lack of HBGA expression in mantle, dominant GII.4 Sydney strain readily bound to all the oyster tissues, including the digestive tissues, gills, palps, and mantle. In contrast, no binding of the epidemic GII.17 Kawasaki308 VLPs to any of the investigated oyster tissues was observed. In synthetic HBGA and saliva-binding assays, GI.1 reacted with A type, H type, and Leb (Lewis b) HBGAs. GII.4 Sydney VLPs showed a broad binding pattern and interacted with various HBGA types. Compared to GI.1 and GII.4 VLPs, the GII.17 Kawasaki308 VLPs only weakly associated with long-chain saccharides containing A type, B type, H type, and Leb blood group epitopes. Our findings indicate that GI.1 and GII.4 noroviruses are likely to be concentrated in oysters, by binding to HBGA-like glycans, and therefore potentially leading to increased long term transmission. In regards to the GII.17 Kawasaki308 strain, we suggest that oysters can only function as short term transmission vector in periods of high environmental virus concentrations.Noroviruses are the major cause of foodborne outbreaks of acute gastroenteritis, which are often linked to raw oyster consumption. Previous studies have suggested histo-blood group antigens (HBGA)-like structures in the oyster tissues as ligands for norovirus binding and persistence. To better understand how oysters function as vectors for the most common human noroviruses, we first tested the ability of the norovirus strains GI.1 West Chester, the pandemic GII.4 Sydney, and the epidemic GII.17 Kawasaki308 strains to interact with oyster tissues. Secondly, we explored how the HBGA preferences of these strains can affect their persistence in oyster tissues. We found limited HBGA expression in oyster tissues. HBGAs of A and H type 1 were present in the digestive tissues and palps of the Pacific oyster  Caliciviridae family. According to the most recent classification, there are seven genogroups (GI- GVII) in the norovirus genus  and human-like histo-blood group antigens (HBGA). Human HBGAs are carbohydrate epitopes at the terminal end of O-glycans on glycoproteins and of glycolipids on the surface of red blood cells, the mucosal epithelium of the gastrointestinal, and the respiratory and the genitourinary tracts. Specific HBGAs, similar to blood group A type and H type, have also been identified in the digestive tracts of different oyster species .Crassostrea gigas , Leb-PAA-biotin (01-042), H type1(tri)-PAA-biotin (01-037), Lex-PAA-biotin (01-036), Lea-PAA-biotin (01-035), H type 2 (tri)-PAA-biotin (01-034), Blood type A (tri)-PAA-biotin (01-032), Blood type B (tri)-PAA-biotin (01-033). The following monoclonal antibodies (MAbs) were purchased for the HBGA phenotyping: A type antibodies , B type antibodies , H type 1 antibodies , Lewis a antibody , Lewis b antibody , Lewis x antibody , Lewis y antibody . Porcine gastric mucin type III (PGM) was purchased from Sigma-Aldrich (M2378). Goat α-mice IgG HRP conjugated antibodies were purchased from ThermoFisher (62-6520).We examined VLP binding to multivalent-HBGAs conjugates purchased from GlycoTech . They included LeSpodoptera frugiperda (Sf9) cells using baculovirus expression system as described previously , GII.4 Sydney (X459908), GII.17 Kawasaki308  VLPs were produced in eviously . In shorCrassostrea gigas. The oysters were collected from an uncontaminated area on Sylt, North Sea, Germany, 54.937962° N, 8.313825° E, and shipped the same day (within 24 h) on ice to the laboratory in Mannheim. Upon arrival, the oysters were immediately dissected and the digestive tissues, mantles, gills, and pulps were collected from five to six different oysters. The tissues were homogenized in phosphate-buffered saline (PBS) pH 7.4, boiled for 10 min at 95°C and centrifuged at 8000 rpm for 5 min . The supernatants were collected and used for ELISA experiments after the protein concentration was measured with the Bio-Rad protein assay kit. The tissue homogenate was tested for the presence of preexisting GI or GII norovirus contamination by a GeneXpert commercial assay according to the manufacturer’s instructions  (data not shown). The oysters which were artificially exposed to norovirus-containing stool samples were used as a positive control.All experiments were performed with the Pacific oysters Saliva samples collected from 17 healthy adult persons were boiled for 10 min at 95°C and centrifuged at 8000 rpm for 5 min . Approval was obtained from the ethics committee of the Medical Faculty of Mannheim, Heidelberg University, #2017-528N-MA.6 nanobodies (Nb85) for GI.1 VLPs, α-rabbit-polyclonal antibodies for GII.4 VLPs, or α-VLP-biotinylated nanobodies (Nb26) for GII.17 VLPs  were added. Finally, the washed plates were developed for 30 min at room temperature with o-phenylenediamine (OPD) and H2O2. The reaction was stopped after 30 min and absorbance was measured at 490 nm on a Tecan infinite M200 Multiwell reader . Negative controls included a VLP-negative control and primary antibody-negative controls. PGM and secretor saliva samples, described below in the results, were used as positive controls.The binding of the GI.1 West Chester, the pandemic GII.4 Sydney, and the epidemic GII.17 Kawasaki308 norovirus VLPs to oyster tissues was measured in triplicate for each oyster . Briefly.17 VLPs . After wx-PAA-biotin) conjugates were diluted to a final concentration of 20 μg/ml in PBS and applied on streptavidin-coated plates overnight at 4°C. The plates were blocked and a serial dilution of VLPs  was applied. The concentration of bound VLPs was detected as described above.For the synthetic HBGA assays, carbohydrate-polyacrylamide - biotin  was added to carbohydrate buffer pH 9.4, and were incubated overnight at 4°C, then washed and blocked. The plates were incubated with 10 μg/ml VLPs in PBS and the concentration of bound VLPs was detected as described above.2O2 mixture for 30 min and the intesity was measured on the Tecan reader.The HBGAs phenotypes expressed in the oyster tissues and saliva samples were determined using MAbs as previously described . Each int-test, and a P-value of below 0.05 was considered as statistically significant .Means were compared by using the Student GI.1 West Chester VLPs bound to the digestive tissues were easily detected even at the lowest concentration of 1.25 μg/ml, while no binding to mantle and gills was observed Figure . In contSimilar to the less prevalent GII.4 variant , the domSurprisingly, no binding of the epidemic GII.17 Kawasaki308 VLPs to any of the oyster tissues was observed Figure . Althouga,b,x,y HBGA epitopes. The binding cut-off was set at OD490 = 0.18, which was three times the value of the PBS control. Only A type and H type 1 carbohydrates were found to be present in the tissues  and palps (OD490 = 0.27). Likewise, H type 1 HBGAs were present in the digestive tissues (OD490 = 0.31), gills (OD490 = 0.26), and palps (OD490 = 0.27). None of the other tested HBGAs  were detected in the oyster tissue extracts.The HBGA phenotypes present in oyster tissues were examined by eight MAbs which recognize A type, B type, H type 1 and Les Figure . The A tb and Ley antigens were defined as secretor positive probes, while the absence of the A, B, H, Ley, Leb antigens suggested a non-secretor status. Overall, thirteen samples were secretor positive and four samples were from non-secretors  . Prior to the VLP binding assay, the saliva samples were tested for the ABO, Lewis, and secretor phenotypes using the MAbs. Saliva containing Le) Figure .490 > 1.0), containing A/H1/Lea,b,x,y and A/Lea,b,x,y type glycans, respectively , C , D , G , I , O . Weak and no GI.1 VLP binding was observed for saliva samples B, P, E, F, K, which lack A type and H type 1 carbohydrates. Overall, the GI.1 West Chester VLP binding profile matched well-established preferences of GI.1 Norwalk VLPs toward H1, Leb and A type antigens , J , L, N   and Q . In contrast, subjects B, K, P, E containing predominantly the Lea and Leb antigens displayed absorbance readings that were below or just above the cut-off limit (OD490 < 0.2).The GII.17 VLPs demonstrated the highest signal, with an OD) Figure . Moderatb, A type, but also B type antigens.With some exceptions, the saliva binding pattern of GII.17 VLPs closely resembled the binding patterns of GI.1 VLPs Figure . Howevery and Leb types  domain of the major capsid VP1 protein . Distincantigens . As was  oysters .b type tetrasaccharide-PAA, Ley type tetrasaccharide-PAA conjugates. However, the latter results have to be treated with caution, due to that fact that the short carbohydrate chains are attached to a polymer linker, and are therefore less accessible for the bulky VLP structure. Our parallel work, using carbohydrate mixtures from breast milk , revealed the specificity of GII.4 Sydney VLPs toward longer saccharides containing 4–15 sugar units and the terminal blood group H type 1 or Lewis-b epitopes , and,thus, just weakly attach to the HBGA epitopes expressed in the digestive tissues of pacific oysters.Enhanced HBGA binding capabilities have been put forward as a factor to explain the rapid emergence of the GII.17 strains . HoweverConsidering these binding assay results it is surprising that oysters can be contaminated with GII.17 noroviruses . One expFrom the combination of binding assays applied in this study, we conclude that the pandemic GII.4 Sydney norovirus strains are likely to employ H type 1-mediated interactions to accumulate within oysters, which could potentially lead to long-term transmission. The epidemic GII.17 noroviruses, on the other hand are likely to have low persistence in oysters and transmission is suggested to be short-term, when high environmental concentration of these viruses is found. Understanding these strain specific characteristics of persistence will allow the prediction of norovirus epidemiology, which is vital for the development of control measures.This study was carried out in accordance with the recommendations of the ethics committee of the Medical Faculty of Mannheim, Heidelberg University, 2017-528N-MA, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the Medical Faculty of Mannheim, Heidelberg University, 2017-528N-MA.VM, KMW, and HS conceptualized the idea. VM, F-GH, KMW, and HS analyzed the data and wrote the original draft. VM performed the experiments. All authors had read and approved the final version of the manuscript.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.


Considering serum levels of TNF-alpha, there was a statistically significant difference, comparing the resistance training group at Pre (66.27 ± 10.31 pg/mL) and Post (37.85 ± 9.05 pg/mL) moments (p=0.01). In molecular analysis of TNF-alpha gene expression, there was a statistically significant decrease (p=0.007) between Pre (0.010 ± 0.01 ng/ml) and Post (0.0002 ± 0.0001 ng/ml) moments. Among CRP data, in the resistance training group, there was a statistically significant reduction, between Pre (2.04 ± 0.32 mg/L) and Post (0.90 ± 0.22 mg/L) moments (p=0.001). In the Control group, there was no statistical significance between these two moments. Therefore, the resistance training demonstrated changes in the TNF-alpha gene expression in elderly women, as well as decreased serum levels of interleukin-6, TNF-alpha, and CRP. Such conditions may be related to immune modulation and anti-inflammatory effects, since resistance training releases cytokines, especially interleukin-6, which acts as a TNF-alpha antagonist during exercise.The aim of this study was to determine the effects of resistance training on the immunologic response, body composition, tumor necrosis factor-alpha  gene expression obtained from blood leukocytes, and the cytokines interleukin-6, TNF-alpha, and C-reactive protein (CRP), in the elderly women (mean age 63 ± 2 y). A randomized controlled trial was performed using a bi-set training method for eight weeks in nineteen elderly women. Peripheral blood samples were collected by puncture in pretraining (Pre) and posttraining (Post) moments. In the resistance training group, there was a statistically significant decrease from 38.43 ± 9.48 pg/mL to 11.76 ± 5.19 pg/mL ( In recent years, life expectancy has grown considerably. Nowadays, there are 14.3 million Brazilians over 60 years. Until the year 2025, Brazil will become the sixth country in the number of elderly people [α), and C-reactive protein (CRP) [Human aging is a process characterized by biological and physiological changes with repercussion on body composition and onset of chronic systemic inflammation, pointed by the increase of inflammatory markers such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-in (CRP) , 4. Accoin (CRP) , severalα is the main mediator of acute inflammatory response whose physiological function is stimulation of leukocytes, signaling to sites of inflammation and activating them in order to eradicate microorganisms and reduce inflammation [Cytokines are proteins that respond to pathogens with cells of innate and adaptive immunity and also act on inflammatory processes . Among cammation , 7.α. Thus, the body returns to its resting state as the inflammatory process is eradicated [Another important cytokine in the inflammatory process is IL-6. It is synthesized from mononuclear phagocytes, vascular endothelial cells, fibroblasts, and adipose and muscle tissues. Its function is stimulation of neutrophil production by bone marrow progenitors, promoting synthesis of proinflammatory cytokines and inhibiting TNF-C-reactive protein is a risk marker for cardiovascular disease, which also features an important function in the inflammatory process, especially in the acute phase, when CRP can increase up to 1000 times its normal serum level. This protein is produced and released into the bloodstream by hepatocytes, but it can also be produced by arterial walls. Its synthesis is stimulated by IL-1 and IL-6, which are released by macrophages . Among sα, IL-1β, and IL-8), being also recommended for health promotion in the elderly [Several systemic, tissue, cellular, and molecular changes happen in the aging process, featured by progressive dysfunction of the immune system, with an imbalance in the concentrations of pro- and anti-inflammatory cytokines and other inflammatory markers associated with the development of cardiovascular diseases, and the increase in morbidity and mortality, in the elderly , 12. Res elderly –16.α, IL-6, and CRP in the face of chronic resistance training in the elderly can be a good tool for several health professionals and, especially, for the general population, because of the beneficial effects of exercise in this senile age. Thus, this study is mainly aimed at analyzing the immunological response, gene expression, inflammatory markers through cytokines such as TNF-α, IL-6, and CRP, and body composition, in a group of elderly women, so as to better understand these events.Therefore, the role of immunological and inflammatory markers such as TNF-α, IL-6, and CRP in relation to resistance training in the elderly can be a good appliance for several health professionals, and especially for the general population, as these effects will be beneficial in this age by reducing proinflammatory cytokines and thereby reducing various types of chronic degenerative diseases due to aging. Thus, this study is aimed at analyzing the behavior of the immune system, through gene expression, inflammatory markers of TNF-α, IL-6, and CRP, and body composition in a group of elderly women to better understand these events.Therefore, the role of immunological and inflammatory markers such as TNF-It is an experimental and controlled study. The Ethics and Research Committee of the CEUMA University, through the Brazil Platform Ministry of Health, National Health Council, National Commission of Ethics in Research-Division of Experimental Research Involving Human Beings, approved this study under CAEE permit number 10863313.2.1001.5084 (protocol no. 372453/2013). All participants signed an individual consent form, so their identification is included in this article. All procedures performed in this study were in accordance with the Ethical Standards of the Institutional and/or National Research Committee and with the Helsinki Declaration of 1975 and its later amendments or comparable ethical standards.2 and WHR ≤ 1.00); and voluntarily sign the consent term. For exclusion criteria, participants should not have untreated hypertension or diabetes mellitus . The total initial sample consisted of 23 elderly women. After eight weeks of RT, the final sample was 19 participants. Two people from the initial sample presented leukocyte values above the reference for inclusion criteria, and the other two attended less than 85% of the training sessions.This is a nonprobabilistic sample. A list with names and telephone contacts of women aged between 60 and 70 was initially requested from the Integrated Seniors University (UNITI/UFMA). From the telephone contacts available, subjects were invited to take part in the study, and those who had not entered any other structured RT program in the last six months were selected. A meeting was held for explanation about project logistics, for delivering the schedule of activities to all participants, and for the consent form to be signed by them. Also, there were scheduled days, times, and locations for anthropometric evaluation and blood collection. For inclusion criteria, participants should be between 60 and 70 years; not be enrolled in structured RT programs in the last six months; be nonsmokers; have no degree of obesity according to the World Health Organization , using bhttp://www.randomization.com, Protocol no. 2538 08/2014, available on 08/18/2014) divided into the control group (Control) and experimental group (RT). The Control received no intervention, and the RT performed resistive training. It happened this way: first, the entire sample (n=19) was submitted to anthropometric assessment and blood collection; then, the entire sample was randomly assigned. One group (n=9) performed RT, while the other (n=10) received no intervention. After 24 hours, corresponding to eight weeks, all participants returned for a new anthropometric evaluation and blood collection. Then, the Control group started the intervention (n=10), performing RT for eight weeks as well. Afterwards, they were again submitted to anthropometric evaluation and blood collection. Then, the groups that performed RT were paired, forming the RT group (n=19) and the Control group (n=10) (A pretest/posttest with a control group design was adopted for the study. Both groups were pretested and posttested at eight weeks. The participants were randomly (p (n=10) .The RT protocol was based on a critical analysis of the databases in , 18–20. For the protocol procedure, before and after all training sessions, participants rested for 5 minutes and then blood pressure was measured. The RT was supervised by one physiotherapist and two physical education professionals. The model of resistance training was by means of 8–12 maximal repetitions (MRs), aiming at muscular hypertrophy. In relation to the weekly frequency, the training protocol was performed three times per week, where eight exercises were performed in LL and UL: sitting leg press, sitting supine, knee extension, pulley (back), lying down knee flexion, low pulley elbow flexion, seated leg press, and pulley elbow extension. As for the increase in intensity (load (kg)), two criteria were adopted: (1) exercise intensity and load increase were obtained using the BORG scale; in other words, participants were instructed to mention any number from this scale, provided that the number actually corresponded to their perceived effort after each exercise series was performed. (2) After that, all participants performed their RT program within a maximum repetition (MR) zone of 8 to 12 repetitions; thus, whenever the participants exceeded the limits of this zone, a new increase of load occurred which would remain within the established zone, corresponding to 15% of the initial load. The time interval between the repetitions was one minute for each series, and the duration of the training protocol sessions lasted on average 50 minutes. In relation to the speed of execution of the exercise preestablished in four seconds for each movement, the repetition rate was performed with a concentric action of 2 seconds and an eccentric action of 2 seconds, controlled by visual and verbal commands to standardize angles of movement. The intervention of resistance training was very intense, and the participants were elderly and had not trained for at least six months, so in order to avoid possible side effects and possible withdrawal of the participants in the research, we chose to do a one-week adaptation training in the participants performed two sets of 15 submaximal repetitions with low loads, aiming at neuromuscular adaptation avoiding late-onset muscular pain.All participants were submitted to anthropometric assessment in pretest and posttest (eight weeks later). Body mass was measured using a digital scale  with a maximum capacity of 300 kg. Height was measured using an anthropometric scale between 1.00 and 2.00 m. These measurements were used to calculate the body mass index (BMI). Also, waist and hip circumference was measured using the metric tape  to calculate the waist-to-hip ratio. Prior to the measurements of body composition, participants were instructed to not eat 2-3 hours before, not drink alcohol, not perform any physical exercise for 24 hours, monitor fluid intake, and urinate 30 minutes prior to assessment. To begin the assessment, the participants were asked to lie down on the hammock, and then electrodes were put at predetermined points which were sanitized using 70% alcohol.The emitter electrodes were placed at the dorsal surface of the right hand, near the metacarpophalangeal joint, and at the transverse arch of the upper surface of the right foot. The detector electrodes were placed between the distal prominences of the radius and ulna of the right wrist, and another one was placed between the medial malleolus and lateral malleolus of the right ankle . This prα gene expression and (b) the tubes containing the separating gel were used to perform the biochemical analysis of the high-sensitivity CRP and the ELISA immunoassays for cytokines IL-6 and TNF-α.Blood samples were collected by a trained pharmaceutical and in accordance with the biosafety standards recommended by NR32. The blood collection was performed at Pre (fasting) and Post (24 hours after the last day of RT) moments. At the moment Pre, all participants were instructed to go to the Laboratory of Physiology and Exercise Prescription (LAFIPEMA-UFMA), at 6 am, fasted for 8–12 hours. At the time Post, participants in the Control group made blood collections after a time period of 8 weeks. Already, the participants who performed RT made blood collections 24 hours after the last day of training. A total volume of 14 ml, approximately, was collected at vacuum and then distributed into a 4 ml EDTA tube (Vacuette) and two dry tubes containing 5 ml separating gel (Vacuette). The samples collected proceeded with the following usage: (a) the tube containing the EDTA anticoagulant was used to perform the complete blood count and the analysis of TNF-This was analyzed to measure the CRP serum levels and feature the degree of risk for cardiovascular diseases. Therefore, biochemical analysis was performed by immunoturbidimetry.α was performed through ELISA tests at the Laboratory of Immunology and Microbiology of Respiratory Infections (LAMIR), CEUMA University. To process the immunoassay, serum aliquots and the IL-6 ELISA kits  and TNF-α ELISA kits  were used, according to the manufacturer's instructions. This technique was performed using IL-6 and TNF-α monoclonal antibodies (MAbs), marked with enzymes, for chromogenic reactions. The quantification of IL-6 and TNF-α concentrations was performed by calorimetry, measuring the absorbance proportional to the concentration of each cytokine studied. A calibration curve was drawn, and the concentrations of both cytokines (IL-6 and TNF-α) were determined by interpolation from the curve.The analysis of the blood concentrations of IL-6 and TNF-α were determined by an enzyme immunoassay, using a microplate reader device with an 8-channel optical photometric system to measure the optical density of vertical light .All concentrations of IL-6 and TNF-μL was used for RNA extraction, according to the protocol using the GeneJET RNA Purification Kit , in line with the manufacturer's recommendations. The total RNA was resuspended in 20 μL of RNase-free water and quantified using a GeneQuant 100 spectrophotometer ; after that, cDNA was synthesized from 200 μg of total RNA, using the RevertAid First Strand cDNA Synthesis Kit , according to the manufacturer's recommendations. In short, approximately 200 μg of total RNA was reverse transcribed in a final volume of 20 μL of reaction. The resulting cDNA was stored at −30°C until real-time PCR analysis.The RNA isolation was performed from blood leukocytes collected in a vacuum tube containing the EDTA anticoagulant. A sample volume of 20 α gene analysis from blood leukocytes. A pair of primers presenting the following sequences were synthesized: TNF-α-5′-GTAGGACCCTGGAGGCTGAAC-3′ (sense) and 5′-CCCAAAAGAAATGGAGGCAAT-3′ (antisense), and GAPDH 5′-GGAAGGTGAAGGTCGGAGTCA-3′ (sense) and 5′-CTGGAAGATGGTGATGGGATTTC-3′ (antisense). Their construction was based on the gene sequences described by Schattner et al. [μL of the cDNA sample, 1.0 μL of forward and reverse primers (200 nM), 10 μL of Supermix, and 7.0 μL of nuclease-free water. The oligonucleotides used in PCR were selected using the Primer Premier 6.1 Program . The amplicons were generated under the following thermocycling conditions: 1 cycle at 50°C for 2 min (UNG activation), 1 cycle at 95°C for 10 min (UNG inactivation), and 40 cycles at 95°C for 15 s and 60°C for 1 min.Real-time quantitative PCR assays were performed for TNF-r et al. . GAPDH mp > 0.05). The descriptive analysis of the variables, between the groups, was presented as mean and standard deviation, followed by Student's T-test for independent samples. To compare the anthropometric variables, body composition and lipid profile, at Pre and Post moments, the paired Student's T-test was performed. A one-way ANOVA test—paired for repeated measures and followed by Tukey's post hoc test—was performed to compare dependent samples of the load increase in the RT group. A two-way ANOVA test was performed to compare outcome variables between intra- and intergroups. The significance level was set at p < 0.05.The statistical analysis was performed using the software GraphPad Prism 6.0. First, the Shapiro–Wilk normality test was performed, considering parametric tests (p=0.01) and body weight (p=0.04)  .p=0.02), body fat percentage (p=0.01), and WHR and increased lean mass (p=0.02), lean mass percentage (p=0.01), and body weight   .p=0.05) between the groups (Control vs RT) and their respective moments (Pre vs Post) was noted, as well as a significant reduction (p=0.01) in the RT group between their respective moments (Pre vs Post)  .α, there was a statistically significant interaction (p=0.05) between the groups (Control vs RT) and their respective moments (Pre vs Post), as well as a significant reduction (p=0.01) in the RT group between their respective moments (Pre vs Post) (With respect to serum concentration of TNF-vs Post) .α gene expression between groups (Control vs RT), showed that although Pre values in the RT group were higher than the Pre values in the Control group, these values did not interfere in the statistically significant decrease observed in the RT group (p=0.007)  .p=0.01) between the groups (Control vs TR) and their respective moments (Pre vs Post), as well as a significant reduction (p=0.001) in the RT group between their respective moments (Pre vs Post)  .α, the inflammatory marker of high-sensitivity CRP, and the TNF-α gene expression, as well as to analyze body composition in elderly women. Thereby, the initial hypothesis was confirmed, and there were observed a decrease in the serum concentrations of IL-6, TNF-α, and CRP after eight weeks of TR and a decrease in cellular activation, translated into the TNF-α gene expression. In addition, we observed a decrease in fat mass and an increase in lean mass and training load.This study aimed to evaluate the effects of eight weeks of RT on the immune response of IL-6 and TNF-α and inflammatory marker of high-sensitivity PCR, as well as the gene expression of TNF-α in elderly women after eight weeks of intervention through the resistance training protocol. Thus, the initial hypothesis was confirmed, and a decrease in the serum concentrations of IL-6, TNF-α, and CRP after eight weeks of RT and a decrease in the cellular activation, translated into expression of the TNF-α gene, were observed. In addition, we observed a decrease in fat mass and an increase in lean mass, along with an increase in strength represented by the evolution of load along the resistance training.The objective of this study was to evaluate the behavior of the immune system of IL-6 and TNF-α, and CRP in the elderly women. TNF-α and IL-6 are cytokines produced by immune, endothelial, adipose, and smooth and skeletal muscle cells [α) have receptors on several cell types, including hepatocytes, and signal the synthesis of acute-phase proteins, such as CRP [α also stimulates the process of phagocytosis to repair and regenerate tissue [α and IL-1Ra). The IL-6 has an important function in this muscle repair, since it increases soon after the beginning of the exercise and promotes anti-inflammatory actions and also inhibitory effects on the production and secretion of TNF-α (mainly), which cause a chronic adaptation to exercise [In this study, eight weeks of RT significantly decreased the serum concentrations of IL-6, TNF-le cells , 25. Thee tissue , 24, 27.e tissue , Costa ae tissue , Petersee tissue , physicaexercise .α, IL-1a, IL-1b, IL-12, IL-6, and IL-10) in women with metabolic syndrome. No statistical difference was observed; however, there was a trend to decrease TNF-α concentration and to increase IL-1b and IL-6 concentrations 60 minutes after the training. This result indicates that a single RT session can modulate and reduce proinflammatory cytokines and shows that periodized and systematized training programs have a positive effect on cytokine concentrations. Rossetti et al. [α). This process occurs in acute and consecutive training sessions and supports the systemic decrease in serum concentrations of total cytokines and, hence, in the acute inflammatory marker (CRP) [Therefore, to analyze the chronic effects of physical exercise on inflammatory markers, it is essential to understand the acute effects of exercise on cytokine concentrations. Pereira et al.  investigi et al.  explain er (CRP) , 33.α, and IFN-γ in elderly women. The study demonstrated that RT in elderly women is associated with low serum concentrations of proinflammatory cytokines, evidenced the impact of RT on these markers, and showed that sedentary lifestyle is a risk factor for diseases related to the aging process. Ho et al. [α, IL-6, and CRP in overweight and obese individuals and reinforced that idea because all studied variables decreased in the training group compared with the control.This was observed by Córdova et al. , who invo et al.  observedα gene expression, influencing this proinflammatory mediator production. The results of the present study showed a significant decrease in TNF-α gene expression after 8 weeks of training, and this may be related to immunomodulation mediated by IL-6, since this cytokine acts as a natural TNF-α antagonist during long-term physical exercise and also promotes stimulation of anti-inflammatory cytokines production, like IL-1 antagonist receptor (IL-1Ra) and interleukin-10 (IL-10) [Searching evidence about the molecular mechanisms behind these findings, it was verified that RT produces modulatory effects on TNF- (IL-10) , 6, 25.α gene expression in terms of molecular mechanisms, Keller et al. [knockout mice for the two main signaling pathways: TNF-α receptor 1 (p55) and TNF-α receptor 2 (p75). After training, TNF-α gene expression levels decreased in both knockout models, thus indicating that the activation of TNF-α receptors is one of the main mechanisms by which physical exercise regulates the TNF-α gene expression, although it is believed that other pathways may also be involved in this regulation.In order to understand the control of TNF-r et al.  used an α gene expression, since, in obesity, adipose tissue produces a low-grade systemic inflammatory state, characterized by an increase in transcription and elevated levels of circulating TNF-α mRNA [As well, it is important to point out that the RT group presented significant gains in lean mass and loss of fat mass. These findings may also have contributed to the suppression of TNF-F-α mRNA , 35.α and CRP concentrations, which reverberates in their increased serum concentrations [α, and CRP). This is beneficial because it has a protective effect against cardiovascular diseases [α, caused by two training modalities (aerobic and concurrent training), for eight weeks, in elderly women, and they observed a reduction in fat mass and an increase in lean mass, especially in aerobic training. Considering inflammatory markers, concurrent training decreased serum concentrations of CRP, IL-6, and TNF-α, compared to aerobic training. Based on the previously presented studies, it is possible to speculate that the decrease in fat mass and the increase in lean mass, verified in this study, were determinant for the decrease in serum concentrations of TNF-α, IL-6, and CRP, in the elderly women.Based on these data, adipose tissue contributes to the increase in TNF-trations , 36. Phydiseases , 37. Thediseases  corrobordiseases , in whicdiseases  followedα, and CRP), as these variables have a strong relationship with each other [α, via TNF-α receptor, TNF-R.In this research, an increase in muscle strength was also observed, evidenced by the progressive increase in training load and in muscle volume, over the eight weeks of training. According to Prestes et al. , an adjuch other . For Rosch other  and Petech other , this rep=0.01). Thereby, they corroborate the findings of the present study. Moreover, Lera et al. [Mavros et al.  and Santa et al.  observedα) and CRP and also induces molecular changes in TNF-α gene expression, in elderly women.The systematized and progressive RT program is developed through appropriate and specific training purposes. Among several protocols available, the bi-set method shows an efficient proposal for a well-structured and individualized RT program, which follows the RT prescription recommendations for the elderly. In view of this, it was decided to determine the training intensity by means of an RM range between 8 and 12 –20, 43. A possible limitation of this study may be related to the representativeness of the sample, since it is a limited group of women who are available to participate in institutional care research. Another limitation of the study was in relation to the elderly women who have passed th0065 menopause period, and it is known that the hormonal changes that occur in the postmenopausal period can influence the responses to the cytokines through body composition, mainly in the fat weight. In this way, we controlled this limitation with the decrease of the fat weight with the resistance training in relation to the basal values.α gene expression, in the elderly women, as well as a decrease in serum levels of IL-6, TNF-α, and CRP. This may be related to an immunomodulation and anti-inflammatory effect, since RT releases myokines (cytokines produced in active muscles), particularly IL-6, thus acting as a TNF-α antagonist during exercise. Therefore, the performance and adaptation to chronic RT controlled inflammatory conditions, which resulted in a balanced action between pro- and anti-inflammatory effectors in different tissues, especially in the striated skeletal muscle.In summary, the results of the present study showed that eight weeks of RT caused molecular changes in TNF-


Petersburg (http://redox.vub.ac.be/events/embo-redox-biology-conference.html) and in September of the same year in San Feliu de Guixols (Spain) (http://meetings.embo.org/event/17-thiol-ox). We could then submit the idea to long time collaborators and redox friends but also to other colleagues with whom we had the chance to get in touch with at these meetings. In general, although Antioxidants is a rather recent creation and its credentials were at the time not so well known, the idea of participating in a special issue was very well received and many of the contacted colleagues have answered positively. Of course, as our background is in plant sciences, this special issue mostly contains papers dealing with oxygenic phototrophs but other experimental model organisms are also addressed . Overall the special issue contains 16 papers, 12 of those reporting experimental research data, and 4 others being more review-like although some of them also contain original bioinformatics data. The special issue on Thioredoxin and Glutaredoxin systems (The two volume editors (J.P.J. and M.Z.) wish to testify that the reviewing process has been done in a very professional way by Antioxidants. We have been phased out of the few papers that presented a conflict of interest, but asked to give a final approval for those. All papers were reviewed by at least two international experts, very often three and more rarely four. Occasionally the participants were asked to cross review papers of the special issue but on average this happened quite rarely. This very thorough evaluation system has helped improve the quality of several of the papers by pointing out some weaknesses that were fixed in a second or third round of evaluation.Thus overall, we are very pleased with the outcome of this editorial effort and we wish here to give a brief summary of its content and most exciting features. 2+- and phosphorylation-dependent cascades, thus transmitting ROS-related information in transduction pathways. Lourenço dos Santos, Petropoulos and Friguet  type likely ligated by the classical catalytic cysteines present in the conserved WCGPC signature. This situation is somewhat similar to those of various GRXs which also bind ISCs in a homodimer although the nature of the center is different. Remarkably, their results suggest that a novel regulation mechanism may prevail for mitochondrial o-type TRXs, possibly existing as a redox-inactive Fe–S cluster-bound form that could be rapidly converted in a redox-active form upon cluster degradation under specific physiological conditions. NFU proteins could be target of the catalytically active form. It remains to be seen whether the ISC-replete form could be involved in ISC transfer/assembly as suggested for GRXs in many species and sub-compartments. The paper by Stéphane Lemaire et al.  reports io group  providesd et al.  provide er group  describef-type TRX are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and RuBisCO activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the TRX family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Among the GRX targets is cytosolic isocitrate dehydrogenase (cICDH), the activity of which is controlled by S-nitrosylation upon nitrosoglutathione (GSNO) treatment as shown by Reichheld and colleagues [Arabidopsis thaliana MDHAR isoform 6 was specifically and strongly increased by reduced TRX y, and not by other plastidial TRXs. In addition, TRX y mutant plants showed reduced stress tolerance in comparison with wild-type (WT) plants that correlated with an increase in their global protein oxidation levels. The last of the papers dealing with redox regulated enzymes provides results concerning chlorophyll biosynthesis. It is well known that chlorophyll synthesis requires light and one key regulatory enzyme is aminolevulinate dehydratase (ALAD). Berhanrd Grimm and colleagues [f, TRX m and NTRC in chloroplasts. The reduced and oxidized forms of ALAD differed in their catalytic activity and they conclude that (i) deficiency of the reducing power mainly affected the in planta stability of ALAD; and (ii) the reduced form of ALAD displayed increased enzymatic activity. In the next four papers, we now turn our attention to target enzymes of the TRX systems in plants. Yoshida and Hisabori  investiglleagues . In partlleagues . They folleagues  show thaThe last paper of this special issue is by Nicolas Navrot et al. . This coIn conclusion, we believe that this special issue had indeed provided new information about a number of proteins involved in the redox regulatory networks, either oxidants, reductants, TRXs, or targets. Of course, we would have liked to cover additional topics, in particular the field of peroxiredoxins and glutathione peroxidases was not addressed at all. Likewise, two recent papers reported on the interesting diversity of protein disulfide isomerases in plants  and this