We compared patterns of visual field loss detected by standard automated perimetry (SAP) to saccadic vector optokinetic perimetry (SVOP) and examined patient perceptions of each test.A cross-sectional study was done of 58 healthy subjects and 103 with glaucoma who were tested using SAP and two versions of SVOP (v1 and v2). Visual fields from both devices were categorized by masked graders as: 0, normal; 1, paracentral defect; 2, nasal step; 3, arcuate defect; 4, altitudinal; 5, biarcuate; and 6, end-stage field loss. SVOP and SAP classifications were cross-tabulated. Subjects completed a questionnaire on their opinions of each test.We analyzed 142 (v1) and 111 (v2) SVOP and SAP test pairs. SVOP v2 had a sensitivity of 97.7% and specificity of 77.9% for identifying normal versus abnormal visual fields. SAP and SVOP v2 classifications showed complete agreement in 54% of glaucoma patients, with a further 23% disagreeing by one category. On repeat testing, 86% of SVOP v2 classifications agreed with the previous test, compared to 91% of SAP classifications; 71% of subjects preferred SVOP compared to 20% who preferred SAP.Eye-tracking perimetry can be used to obtain threshold visual field sensitivity values in patients with glaucoma and produce maps of visual field defects, with patterns exhibiting close agreement to SAP. Patients preferred eye-tracking perimetry compared to SAP.This first report of threshold eye tracking perimetry shows good agreement with conventional automated perimetry and provides a benchmark for future iterations. Perimetry is central to the management of patients with glaucoma, and is used to aid diagnosis, gauge disease severity, and detect and measure rates of progression. Automated threshold perimetry using a white stimulus on a white background  has become the accepted gold standard. SAP involves measuring the differential light sensitivity (DLS) at several test locations, with algorithms used to vary stimulus brightness and determine threshold values. Defining thresholds using a full threshold test is time-consuming and onerous for the patient, which can lead to reduction in test reliability and increased visit-to-visit variability. Although testing times have been reduced significantly using Swedish Interactive Threshold Algorithm (SITA) strategies, the common perception of visual field testing from patients is that it is difficult to perform.1 Similar findings have been reported recently using patient focus groups, with patients expressing the opinion that SAP was in need of modernization.2 In addition, patients undergoing SAP have been found to experience higher levels of anxiety than those undergoing other tests, such as retinal imaging, and those who were anxious before undergoing SAP produced poorer reliability test results.3 There is a need for a more acceptable visual field test for patients.An investigation of patients' opinions of tests used in the management of glaucoma, found SAP to be the least popular commonly performed procedure.4 but only recently have several groups explored the possiblity of performing perimetry using computerized eye tracking.58 Studies using eye tracking also have shown that assessing the speed and accuracy of saccadic eye movements also may be useful for detecting disease, with differences in saccades noted between healthy individuals and those with glaucoma.912 Eye tracking can be used to monitor patient gaze responses to peripheral stimuli, with the advantage that it does not require the patient to press a response button. Additionally, by continually monitoring the patient's eye location and adjusting the position and size of the stimuli accordingly, perimetry can be performed without the need for a chin rest. We previously described a method of eye tracking perimetry termed saccadic vector optokinetic perimetry (SVOP).1316 SVOP initially was developed as a suprathreshold test for children who struggle to maintain fixation and produce the timely and reliable responses needed for SAP. We recently developed a threshold SVOP test and have shown that threshold visual field sensitivities obtained using this device are correlated closely with SAP threshold values.17 The ability to perform threshold visual field testing using an eye tracker has the potential to offer an alternative method of perimetry to those who struggle with conventional testing. In the present study we compared patterns of visual field loss detected by SAP and threshold SVOP and examined patient perceptions of each technique.Pioneering work by Damato has shown that the visual field could be evaluated by moving the eye rather than the test stimulus (oculokinetic perimetry),17This cross-sectional study included patients with glaucoma and healthy subjects. Patients with glaucoma were recruited from a nonconsecutive series of patients attending the glaucoma clinic at the Princess Alexandra Eye Pavilion, Edinburgh. Healthy subjects were required to have no previous history of glaucoma or visual field defect and no neurologic conditions that might affect the visual field. All subjects provided written informed consent. The study adhered to the tenets of the Declaration of Helsinki and was approved by the South-East Scotland Research Ethics Committee, NHS Lothian. Details of the inclusion and exclusion criteria have been described previously.All patients attending the glaucoma clinic underwent best-corrected visual acuity, slit lamp biomicroscopy, intraocular pressure measurement, pachymetry, gonioscopy and dilated funduscopy. The diagnosis of glaucoma was made by a glaucoma specialist, based on the presence of typical glaucomatous changes in optic disc morphology and a glaucomatous visual field defect on SAP using the Humphrey visual field analyzer (HFA) SITA Fast 24-2 test . All subjects performed SAP and threshold SVOP in both eyes and a random sample of subjects underwent repeat SAP and SVOP testing in one eye during the same session with suitable breaks if required. Patients with strabismus or a history of eye movement disorders were excluded. All patients had a best corrected vision of better than 0.3 logMAR. In the healthy group, eyes with a visual acuity worse than 0.15 logMAR were excluded.17 Threshold SVOP consists of a personal computer with an examiner screen, a 24″ high-resolution LCD patient screen (Eizo ColorEdge CG243W) and an eye tracker  if required, while also occluding the nontest eye with an infrared bandpass filter that enabled the eye tracker to detect the position of the occluded eye. The test commenced with the patient fixating on a central fixation stimulus. A test stimulus then was presented peripherally. During testing, the patient was instructed to follow their natural reaction to fixate towards any peripheral stimulus perceived. The eye tracker evaluated responses to the stimuli presented on the display screen, and custom software determined whether stimuli had been perceived based on the direction and amplitude of the subject's gaze response. The subject then was required to fixate on the peripheral stimulus until another was perceived. Stimuli were equivalent in size to Goldmann III and each stimulus was presented for 200 ms using coordinates equivalent to the SAP 24-2 test pattern. The size and position of the stimuli were automatically adjusted to compensate for changes in the patient's position during testing to ensure the perceived stimulus size remained constant.2) and stimulus luminance levels. Stimuli luminance levels replicated the luminance values corresponding to 14 to 40 dB on the HFA. Thresholds were obtained using a 4-2 bracketing strategy and began by testing four “seed” locations (one in each quadrant), which then were used to set the starting stimulus luminance levels for neighboring locations, which in turn were used to calculate the remaining starting luminance levels. The SVOP threshold sensitivity values were matched in luminance to those of the HFA to allow direct comparison.The screen was calibrated using a Look-Up Table (LUT) pairing the grey-levels of each pixel to the corresponding required background .During the study, improvements were made to version 1 (v1) of the SVOP software and version 2 (v2) was developed. Modifications included: (1) an indicator of correct patient position, (2) an improved interactive demonstration test, (3) an increased time for determining fixation and a new fixation target with central pulsating crosshairs to improve fixation, and (4) modification of the starting luminance levels, calculated from “seed” location and neighboring location threshold levels to ensure they were never below 18 dB. This ensured that threshold point results would never be determined from a single stimulus decision. Software v2 also included two bug fixes, one relating to incorrect screen stimuli positions calculated under rare circumstances, and a second relating to incorrect calculation of the starting luminance level for one field point location. Subjects completing testing using v1 and v2 were asked to complete the questionnaire, however only results from software v2 were used for analysis of visual field defect patterns.18 The purpose of this classification was not to represent the natural progression of glaucomatous field loss, but rather to allow broad stratification of different patterns of glaucomatous field loss. Graders were permitted to assign visual field results to multiple categories if more than one type of defect was felt to be present. For example, a plot with paracentral and arcuate loss could be classified as categories 1 and 3.To compare the patterns of visual field loss detected by threshold SVOP and SAP, visual field threshold sensitivity plots  and sensitivity values were reviewed by a panel of three graders  who reached a consensus on each visual field result. Grading was performed in two batches, each batch corresponding to a device, so that SVOP and SAP results from a single patient were not performed consecutively. Graders assigned visual fields into seven groups: 0, normal; 2, paracentral; 3, nasal step; 4, arcuate; 5, altitudinal; 6, biarcuate; and 7, end-stage field loss .18 The pThe agreement between SVOP and SAP classifications, using the highest number category if more than one was assigned, was compared qualitatively by cross-tabulation. Sensitivity and specificity of SVOP (v1 and v2) were calculated by comparison of normal (category 0) and abnormal  results using SAP as the reference standard. The agreement between SVOP v1 and v2 and SAP also was assessed qualitatively by cross-tabulation. A random subset of patients underwent repeat SAP and SVOP testing to determine whether the patterns of visual field loss detected using each device were repeatable. Repeated results were cross-tabulated and performance assessed using descriptive statistics.t-tests were used for normally distributed variables and the Wilcoxon rank-sum test for continuous nonnormal variables. For comparison of more than two groups, 1-way analysis of variance (ANOVA) was used. All tests were 2-sided and a P value less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS version 21.For group comparison tests, normality assumption was assessed by inspection of histograms and using Shapiro-Wilk tests. For comparison of two groups, Student's P < 0.001). The study included 59 healthy subjects and 103 with glaucoma. Healthy subjects were younger than those with glaucoma  or abnormal (any other category), the sensitivity and specificity of SVOP were determined using SAP as the reference standard. The 2 × 2 contingency table for SVOP v2 is shown in The detailed cross tabulation of the visual field defect categories between SVOP v2 and SAP is shown in Repeat field tests were compared to the initial tests for each patient and the agreement in classification of pattern of field defect was compared . SVOP v2The results of the patient questionnaire examining test preferences are shown in Our study showed that SVOP can be used to obtain threshold visual field sensitivity values in patients with glaucoma and produce maps of visual field defects with patterns exhibiting good agreement with SAP. This is an exciting finding as SVOP has the major advtange over SAP of being a totally objective test of visual function sindce it does not rely on a subjective patient response. Additionally, it negates the requirement of the patient to press a response button. Therefore, SVOP represents a potentially important new test for the evaluation of the visual field. An additional benefit is the finding that 71% of participants preferred SVOP to conventional SAP testing.We used two versions of the SVOP software, with v2 found to be superior to v1, which is now retired. Compared to the gold standard of SAP, SVOP v2 correctly identified 42 of 43 abnormal visual fields, which equates to a sensitivity of 97.7%. Of 68 normal visual fields on SAP, 15 were identified as abnormal on SVOP v2. This indicated a specificity of 77.9% for SVOP compared to SAP SITA-fast; however, some of the normal SITA-fast tests might have been false-negatives. The study population for SVOP v2 testing included 70 eyes with glaucoma, of which 67 (95.7%) were identified as abnormal by SVOP and only 43 (61.4%) by SITA-fast . Patient17 However, it is possible that some cases of apparantly worse visual fields on SVOP compared to SAP were due to earlier detection of damage using SVOP.We also found good agreement between patterns of field loss detected by SAP and SVOP. SVOP v2 produced visual field maps that were assigned to the same category as SAP for 76 of 111 (68%) eyes, with a further 20 (18%) differing by only one category. Only 15 of 111 tests (14%) differed by more than one category. For those cases where SAP and SVOP did not agree, the SVOP visual field tended to be graded with a higher number, normally indicating more severe visual field loss with 31 of 35 (89%) higher and 4 of 35 (11%) lower than SAP. The most frequent disagreement was normal (category 0) on SAP being classified as 1 or 3 by SVOP, and SAP category 3 or 4 being classified as 6 by SVOP. The tendancy for SVOP to produce results suggestive of a worse visual field than SAP is likely due to the difficulty of detecting small eye movements using the eye tracker, which may cause some inaccuracies in precise mapping of the central visual field in particular. Although the patient may perceieve and move fixation to a central stimulus, there may be instances where the eye tracker fails to detect this movement, resulting in an apparent scotoma that is not present on SAP. This finding also is supported by our separate analysis of agreement between SAP and SVOP threshold values, which found overall good agreement between these values, but poorer correlation between sensitivities in the central test locations.We found SVOP v2 to be superior than v1 and it is likely that further improvements are possible, particularly through software refinements and improvements in eye tracking technology. Towards the end of the study, an improved eye tracker became available , with a higher sampling frequency (60 Hz compared to a variable 25–40 Hz used for the eye tracker used in v1 and v2). We subsequently evaluated the improved eye tracker (using software version 2) in 15 subjects , 11 of whom had experienced problems with the eye tracker and had incomplete tests during original testing. All 15 patients produced a complete test with 100% agreeing with the visual field defect classification from SAP. The results of testing in these 15 patients  compared to the two-dimensional response used by SAP; that is, visual recognition and a following motor component (button press) and it is possible that this different methodology itself could have an influence in the measurement. Additionally, subjects were tested using the HFA “SITA Fast” thresholding algorithm, which is known to have larger test–retest variability than the “SITA Standard” algorithm.17 However, despite the longer test time, there was little difference in how the participants perceived the testing time, with most subjects scoring the time “just right” for both tests . Further work is required to evaluate SVOP v3 on a larger cohort of subjects while also collecting useful normative threshold data and saccadic latency data in glaucoma patients and healthy subjects.Supplement 1Click here for additional data file.Supplement 2Click here for additional data file.Supplement 3Click here for additional data file.Supplement 4Click here for additional data file.


Approximately 30 members of the tumor necrosis factor receptor superfamily (TNFRSF) have been identified. They are transmembrane proteins with cysteine‐rich motifs in their extracellular domains that bind to their cognate ligands Bone cells , fibroblast‐like synoviocytes, chondrocytes, and immune cells that infiltrate the arthritic joint will at different times express a wide range of TNFRSF members and TNFSF ligands. An overview of the current status of our knowledge in this regard is provided in Table DR‐3 and its TNFSF ligand TNF‐like molecule 1A (TL1A) contribute to the pathogenesis of autoimmune and rheumatic diseases PGRN, also known as granulin‐epithelin precursor, proepithelin, acrogranin, and GP88/PC cell–derived growth factor, is a 593–amino acid autocrine growth factor. PGRN contains 7.5 repeats of a cysteine‐rich motif (CX5–6CX5CCX8CCX6CCXDX2HCCPX4CX5–6C) and forms a unique “beads‐on‐a‐string” structure Accumulating evidence indicates that TNF orchestrates OA pathology The direct impact of PGRN on bone remodeling has yet to be determined, with current knowledge derived from a model of bone healing. In mice at least, PGRN deficiency delayed bone healing, while recombinant PGRN enhanced bone regeneration The inflammatory and catabolic actions of TNF are largely mediated through its interaction with TNFRI. However, we continue to have limited understanding of the impact of TNFRII‐mediated signaling. Recent studies indicate that TNFRII signaling is beneficial and protects against joint destruction Unlike etanercept, mouse monoclonal antibody to TNF (infliximab) and humanized monoclonal antibody to TNF  are specific for TNF and have been shown to be protective against OA in animal models DR‐3  was discovered simultaneously by multiple groups in the middle‐to‐late 1990s, when a combination of BLAST homology searches to Fas and TNFRI The identification of ligand(s) for DR‐3 has been complicated by the number of potential candidates and their altering nomenclature −/− mice −/− mice The generation of transgenic mice genetically deficient for DR‐3 or TL1A or overexpressing TL1A or dominant‐negative forms of DR‐3 has given rise to many in vivo studies describing the essential requirement for the DR‐3/TL1A pathway in models of multiple autoimmune and inflammatory diseases. These have supported an ever‐growing list of in vitro human functional and genetic studies that have associated DR‐3 and TL1A with human diseases ranging from inflammatory bowel disease (IBD) and primary biliary cirrhosis to leprosy  −/− mice were resistant to exacerbation of disease induced by exogenous addition of TL1A to DR‐3+/+ animals, while antagonism of the pathway with anti‐TL1A monoclonal antibody ameliorated disease in CIA The development of the main effectors of bone resorption, osteoclasts, is also regulated by the DR‐3/TL1A axis, at least in a setting of inflammation. While osteoclastogenesis driven by macrophage colony‐stimulating factor and RANKL was unaffected in DR‐3−/− mouse genotype on a DBA/1 background, which results in a mouse that spontaneously develops ankylosing enthesopathy −/− mice showed significantly less thoracic spine–specific bone formation in vivo, while DR‐3−/− mouse osteoblast cultures exhibited reduced levels of alkaline phosphatase, osteopontin, and mineral apposition Finally, DR‐3 also modulates osteoblast function. Human osteoblast cell lines were first reported to express DR‐3 in 2003 Screening the associations of Atsttrin with all members of the TNFR subfamily led to the discovery that in addition to TNFR, PGRN/Atsttrin directly binds to DR‐3 and inhibits TL1A activity The association of PGRN with TNFR and DR‐3 also led to investigations of the immunologic mechanisms underlying PGRN‐mediated antiinflammatory and protective activities in autoimmune diseases high Treg cells high T cell transfer model, lack of PGRN signaling in CD4+ T cells also exacerbated experimental colitis. In addition, PGRN‐mediated protective effect was compromised in the absence of interleukin‐10 (IL‐10) or TNFRII signaling PGRN protects Treg cells from a negative regulation by TNF, and these protective effects are primarily mediated by TNFRII −/−The DR‐3 pathway may also contribute to PGRN‐mediated protective effect in inflammatory diseases, since a recent study showed that agonistic antibody to DR‐3 expanded CD4+FoxP3+ Treg cells in vivo, which in turn suppressed immune responses In contrast to Treg cells, the frequency of Th17 cells was significantly decreased in spleens of mice treated with recombinant PGRN in a CIA model Because TNF is one of the key main mediators of inflammation, it is no surprise that alterations of its physiologic antagonist PGRN have a direct impact on the initiation and progression of arthritis. The effect of TNF antagonism by PGRN should be at least comparable to that of conventional TNF blockers Mycobacterium tuberculosis or viral hepatitis, or of developing opportunistic infections A potential disadvantage of PGRN or Atsttrin compared to anti‐TNF antibodies might be that anti‐TNF antibodies can trigger apoptosis of proinflammatory T lymphocytes by binding to membranous TNF. This effect, which is also missing for TNFR‐Fc fusion proteins, appears to play a particular role in inflammatory bowel diseases and less of a role in arthritis Another question arises from the discovery of autoantibodies to PGRN. Can recombinant PGRN or Atsttrin be administered to patients with preexisting antibodies to PGRN? Frequently occurring anti‐PGRN antibodies have been identified in a wide spectrum of autoimmune diseases including RA and, surprisingly, psoriatic arthritis, which had been regarded as a seronegative disease Epitope mapping identified a binding region within the N‐terminal 112 amino acids of PGRN as a target of antibodies to PGRN in all patients. This means that autoantibodies to PGRN target the antiinflammatory PGRN and possibly cotarget only mature granulin G, the most N‐terminal granulin motif. Despite the structural similarity of granulin G and the other 6 granulins, no binding was detected against granulin motifs other than granulin G 81, was identified exclusively in an anti‐PGRN antibody–positive patient 81 prevents interaction with TNFRI, TNFRII, and DR‐3, so hyperphosphorylated PGRN has lost its antiinflammatory function. Considering these facts, it seems that a reasonable therapeutic strategy would be to compensate for the imbalance of pro‐ and antiinflammatory molecules due to lack of functional PGRN (caused by anti‐PGRN antibodies or hyperphosphorylation of PGRN at Ser81) and/or excessive secretion of TNF and TL1A by administering a recombinant PGRN derivate that cannot be neutralized by preexisting autoantibodies to PGRN (Figure As a reason for the breakdown of self‐tolerance against PGRN, a second immunogenic PGRN isoform, hyperphosphorylated at SerIn conclusion, PGRN and its interaction with TNF/TNFRI/TNFRII and TL1A/DR‐3 represent attractive new therapeutic targets Table . When weAll authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published.


Background and Aims: Endothelial dysfunction is a hallmark of cardiovascular diseases. The straight region of an artery is protected from atherosclerosis via its laminar blood flow and high shear stress. This study investigated the cytoprotective effects of a new laminar shear medium (LSM) derived from a modified cone-and-plate shear device and identified basic fibroblast growth factor (bFGF) secreted by human aortic endothelial cells (HAECs) as the dominant protective factor in the LSM.Methods: Based on a modified cone-and-plate shear device system, HAECs were exposed to laminar shear (15 dynes/cm2) and static control for 24 h to produce a new supernatant LSM and static medium (SM). Evaluation of the protective effects of LSM and SM on endothelial dysfunction induced by tumor necrosis factor (TNF)-α (10 ng/mL), which leads to production of reactive oxygen species (ROS), inflammatory monocyte adhesion, and tissue factor activity. ROS induction-, inflammation-, and thrombosis-related genes and protein expression were evaluated by quantitative-PCR and western blotting. To identify the cytokines that played a key role in the cytoprotective action of the LSM, we used cytokine antibody arrays, selected an abundant marker cytokine, bFGF, and validated the different cytoprotective effects of recombinant bFGF (rbFGF) and neutralization by monoclonal antibody (rbFGF+Ab) co-treatment. Aortic and lung tissues from different groups of C57BL/6J mice were examined by immunohistochemistry. SB203580 (specific inhibitor of p38) and BIX02189 (specific inhibitor of MEK5) were used to identify bFGF as the main cytoprotective factor acting via p38/MAPK and MEK5-KLF2 pathways.Results: Compared with traditional LSM, the new LSM not only significantly decreased TNF-α-induced intracellular adhesion molecule 1 and plasminogen activator inhibitor type 1 gene expression, but also significantly increased heme oxygenase 1 gene expression. The new LSM and bFGF attenuated TNF-α-induced ROS induction, inflammation, and tissue factor activity and inhibited the inflammatory- and thrombosis-related gene/protein overexpression both in vitro and in vivo. Mechanistically, the cytoprotective action of bFGF was mediated via the p38/MAPK and MEK5-KLF2 pathways.Conclusion: bFGF was identified as the critical factor mediating the cytoprotective effects of LSM derived from the modified laminar shear system. In contrast, laminar flow with high shear stresses (10–70 dynes/cm2) are atheroprotective, and they are associated with the following phenotypes: low expression of adhesion molecules/inflammatory genes/chemokine genes, high expression of antioxidant genes, and inhibition of leukocyte adhesion/platelet aggregation/thrombosis  on underlying endothelial cells , we observed that human aortic endothelial cells (HAECs) in the center of the culture disc, compared with those at the periphery, were easily detached on the culture disc and did not show the typical high shear stress-induced alignment of endothelial cell shape, likely because of the non-uniform shear stress levels of this device -induction as the major indicator of atherogenesis and inflammation in endothelial cells  was applied to HAECs (between passages 2 and 5) for 24 h. A HAEC monolayer fully covering a 10-cm tissue culture dish represents the traditional shear system, whereas a monolayer grown in a 10-cm tissue culture dish excluding a central circle (diameter of 5.41 cm) represents our new shear system. In this study, both systems were exposed to an arterial level of unidirectional laminar shear for 24 h by rotating a Teflon cone using the magnetic stirrer (Supplementary Data 2) and then traditional and new LSM were collected. The static medium (SM) was collected from the same cells exposed to static conditions for 24 h. The collected new LSM and SM were filtered through 0.45-μm filters and stored at −80°C until analysis.HAECs were purchased from Cell Applications, Inc.  and cultured in endothelial cell growth medium  according to the manufacturer's recommendations. The human monocytic cell line THP-1 was obtained from the American Type Culture Collection  and maintained as previously described -5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay, CellTiter 96® AQueous One Solution reagent , and absorbance measurement at 490 nm. The viabilities of the control cell groups incubated with fresh medium for 24 h were set to 100%. Cell viabilities of different percentages of LSM-treated cells were compared with those of the individual control group.in vitro studies, co-treatment of the cells with recombinant human TNF-α (TNF-α)  and 20% LSM (TNF-α+LSM group), TNF-α and 20% SM (TNF-α+SM group), and TNF-α and recombinant human bFGF  (TNF-α+rbFGF group) were performed. Additionally, for the TNF-α+LSM+Ab group, the LSM was pre-neutralized with anti-human bFGF antibody  at 37°C for 1 h before co-treatment with TNF-α. LSM pre-neutralized with anti-IgG secondary antibody  at 37°C for 1 h before co-treatment with TNF-α, and this group was named as the TNF-α+LSM+IgG group. After 6 h incubation, genetic analysis, protein expression level measurement, and multiple biochemical experiments were carried out to explore the cytoprotective efficacies of the new LSM.For GAPDH), Kruppel-like factors (KLF2), NAD(P)H quinone dehydrogenase 1 (NQO-1), heme oxygenase-1 (HO-1), thrombomodulin (TM), Tissue factor (TF), plasminogen activator inhibitor type 1(PAI-1), Kelch-like ECH-associated protein 1 (Keap-1), vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), monocyte chemo-attractant protein-1 (MCP-1), hepatocyte growth factor (HGF), granulocyte-colony stimulating factor (G-CSF), IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor receptor (EGFR), monokine induced by gamma interferon (MIG), and basic fibroblast growth factor (bFGF) genes are provided in Supplementary Data All mRNA transcript levels of HAECs were analyzed by qPCR. The individual Universal ProbeLibrary probe and primer sequences for glyceraldehyde-3-phosphate dehydrogenase HProtein expression levels in the HAECs were analyzed by western blotting as previously described . After different treatments, all groups were incubated with H2DCFDA (10 μM) at 37°C for 30 min in the dark. Unbound H2DCFDA was removed by washing with 1× phosphate-buffered saline, and H2DCFDA fluorescence was imaged using a fluorescent microscope equipped with a digital camera . The fluorescence intensity (ROS activity) was measured using ImageJ software  and expressed as fold-changes of the corresponding control.ROS accumulation was detected using 2′,7′-dichlorofluorescin diacetate (H5 cells) were grown in six-well plates. After different treatments, cells were washed twice with 1× phosphate-buffered saline, followed by incubation with human factor VIIa (FVIIa) and factor X (FX) at 37°C, which allowed the formation of a TF/FVIIa complex at the cell surface. The TF/FVIIa complex converted human FX to factor Xa, which was measured by its ability to metabolize a chromogenic substrate. A standard curve with lapidated human TF was obtained to ensure that measurements were acquired in the linear range of detection.Cellular TF-mediated procoagulant activity was measured using commercial Tissue Factor Human Chromogenic Activity Assay Kit  according to the manufacturer's instructions. Serum-starved HAECs  as previously described  according to the manufacturer's instructions as previously described  were prepared for immunohistochemistry staining.Male C57BL/6J mice (8 weeks of age) were used. All animal protocols were approved by the Institutional Animal Care and Use Committee of Hungkuang University (HK-105-29). Mice were randomized into four groups: TNF-α, TNF-α+LSM, TNF-α+LSM+Ab, and TNF-α+rbFGF groups. Intraperitoneal injection of recombinant mouse TNF-α  and 100 μL of LSM, LSM+Ab, or rbFGF (3 μg/mouse) were administered. After 24 h, mice were sacrificed by COAll aortic tissue cross-sections from the above experiments were prepared with a Bond-Max autostainer . Slides were stained with primary antibody on a fully automated Bond-Max system and VBS Refine polymer detection system (Leica Microsystems) as previously described  and 10 μM BIX02189 (specific inhibitor of MEK5)  for 1 h, followed by addition of TNF-α (10 ng/mL) and rbFGF (10 ng/mL) for 6 h. Gene expression was determined by q-PCR as described above.t-test and defined as p < 0.05.Independent experiments were conducted to assess the significance of differences between the control group and other groups or TNF-α group and other groups. Significant differences were determined using Student's 6 cells/dish) were seeded overnight into a 10-cm dish keeping a central empty area (diameter of 51.4 mm) using a 6-cm dish and 2% agarose  for 24 h, the media showed different phenotypes: LSM of traditional LSS was less clear (right), while the LSM of the new LSS system was relatively clear (left)  Figure . HAECs e) Figure . Previou) Figure . In our y Figure . Other Hy Figure .The cytokine TNF-α is an important mediator of acute inflammatory processes that occur during the progression of atherosclerosis. Examples include transcriptional regulation of various inflammation- and thrombosis-related genes  compared to the control group. The TNF-α+LSM group showed significant attenuation with an average intensity of 1.59-fold relative to the control group. The TNF-α+SM group showed an increased oxidative intracellular milieu, similar to that observed in the TNF-α group.A previous study showed that TNF-α induces a pro-oxidant environment in a cell that can be measured by HThe adhesion of circulating monocytes to endothelial cells is an important event causing vascular inflammation Hopkins, . As showTF initiates the coagulation cascade upon vascular injury. TF-specific procoagulant activity is induced in endothelial cells to initiate coagulation and thrombosis  and 1.09-fold (1.3/1.19) relative to the TNF-α group  compared to the TNF-α group. The TNF-α+rbFGF group also showed significantly attenuated levels of H2DCFDA-dependent fluorescence (average 0.87-fold) compared to the TNF-α group. The TNF-α+LSM+Ab group displayed ROS activity similar to that of the TNF-α group (average 0.98-fold). As shown in Figure To validate the HAEC-secreted autocrine rbFGF as a critical protector in the LSM, we evaluated the protective efficacy of rbFGF and LSM+Ab using the HICAM-1, VCAM-1, MCP-1, HO-1, NQO-1, Keap-1, TF, TM, and PAI-1 in the TNF-α+LSM, TNF-α+LSM+IgG, and TNF-α+rbFGF groups differed significantly  enhanced the TNF-α-induced expression of ICAM-1 at the mRNA and surface-protein levels in underlying HUVECs, but suppressed TNF-α-induced expression of VCAM-1 and E-selectin proteins  enhanced the migration of vascular smooth muscle cells  decreased podocyte monolayer resistance  is the prototype member of a family of heparin-binding growth factors, and lacks a signal peptide that directs secretion through the classical secretory pathway  that signal to receptor tyrosine kinases and intracellular non-signaling proteins (intracellular FGFs)  LSM collected from the new LSS system had more protective effects against TNF-α stimulated endothelial dysfunction, than traditional LSM. (2) LSS-exposed HAEC-secreted autocrine bFGF is the critical factor in the LSM that provides diverse cytoprotective effects against TNF-α-stimulated endothelial dysfunction. (3) The protective mechanism of bFGF via activation of the p38-MAPK and MEK5/ERK5-KLF2 signal pathway inhibits nuclear factor-κB downstream target genes Figure . Based oH-JW and W-YL designed and performed the experiments; analyzed the data and wrote the manuscript; provided the funding. W-YL supervised the study.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.


Recent work suggests that the dynorphin (DYN)/kappa opioid receptor (KOR) system may be a key mediator in the behavioral effects of alcohol. The objective of the present study was to examine the ability of the KOR antagonist norbinaltorphimine (nor-BNI) to attenuate relapse to ethanol seeking due to priming injections of the KOR agonist U50,488 at time points consistent with KOR selectivity. Male Wistar rats were trained to self-administer a 10% ethanol solution, and then responding was extinguished. Following extinction, rats were injected with U50,488  or saline and were tested for the reinstatement of ethanol seeking. Next, the ability of the nonselective opioid receptor antagonist naltrexone  and nor-BNI  to block U50,488-induced reinstatement was examined. Priming injections U50,488 reinstated responding on the previously ethanol-associated lever. Pretreatment with naltrexone reduced the reinstatement of ethanol-seeking behavior. nor-BNI also attenuated KOR agonist-induced reinstatement, but to a lesser extent than naltrexone, when injected 24 hours prior to injections of U50,488, a time point that is consistent with KOR selectivity. While these results suggest that activation of KORs is a key mechanism in the regulation of ethanol-seeking behavior, U50,488-induced reinstatement may not be fully selective for KORs. High rates of relapse following periods of abstinence are typically experienced by many alcoholics, contributing to the challenge of the long-term management of alcoholism. The underlying neuropharmacological mechanisms associated with relapse, however, are not yet fully understood. Recent work suggests that the dynorphin (DYN)/kappa opioid receptor (KOR) system may be a key mediator in the behavioral effects of alcohol and other drugs of abuse  includinPrevious work has shown that KOR antagonists block stress induced reinstatement of cocaine seeking and nicotine seeking in rats , 3. The KORs may also regulate the reinstatement of ethanol-seeking behavior. For example, the KOR antagonist JDTic reduces cue-induced ethanol reinstatement when administered 2 hours prior to testing , 7. nor-n = 26) were used in this experiment. Body weights were 200–250 g and age was approximately 60 days at the start of the experiments. Rats were group housed (2-3 per cage) with food and water available ad libitum except during the initial 3 days of ethanol self-administration training and were weighed daily. Rats were maintained on a 12-hour light/dark cycle (lights on at 22:00 h). Procedures met guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and were approved by the Institutional Animal Care and Use Committee of Grand Valley State University.Male Wistar rats -3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclo-hexyl] benzeneacetamide; Tocris Biosciences, Ellisville, MO) and norbinaltorphimine  were dissolved in 0.9% saline solution for intraperitoneal (i.p.) injections. Naltrexone  was dissolved in 0.9% saline solution for subcutaneous (s.c.) injections.U50,488  were injected with priming doses of U50,488  or saline 10 min prior to testing for the reinstatement of ethanol seeking. Rats were then placed in the operant chambers and reinstatement testing occurred under extinction conditions. Tests were conducted every 3-4 days, with rats experiencing extinction sessions daily between test days. Doses were administered in a counterbalanced order using a Latin Square design  were trained to self-administer ethanol and then underwent extinction as described. Following extinction, rats were pretreated with naltrexone  or saline 30 min prior to reinstatement testing. Rats were then injected with priming doses of U50,488  or saline 10 min prior to testing. Test sessions were conducted every 3-4 days, with rats experiencing extinction sessions daily between test days. The dose range of U50,488 was adjusted based on the results of the previous experiment, and doses were administered in a counterbalanced order using a Latin Square design  or saline 10 min prior to testing. Over the next 6–8 days, test sessions were repeated every 3-4 days with rats experiencing extinction sessions daily between test days. Doses of U50,488 were administered in a counterbalanced order using a Latin Square design. No additional saline injections were given over this testing period. After all doses of U50,488 were tested, rats were given a single injection of nor-BNI . 24 h later, rats were injected with a priming dose of U50,488  or saline 10 min prior to testing. A pretreatment time of 24 h was chosen because previous research has shown that nor-BNI is most selective for KORs at 24 h after administration as opposed to earlier times . Over thp < 0.05.Data for the experiment examining the reinstatement of ethanol seeking by U50,488 was analyzed using a one-way repeated measures analysis of variance (ANOVA) with U50,488 dose as the within subjects factor. Post hoc analysis was performed using Tukey's test to determine any significant differences between treatment conditions. Data for the antagonism experiments were analyzed using a two-way repeated measures ANOVA with naltrexone or nor-BNI dose and U50,488 dose as the within subjects factors. Sidak's test for multiple comparisons was used for post hoc analysis to determine if there were any significant differences between saline and naltrexone or saline and nor-BNI when rats were injected with U50,488. The alpha level for all statistical analysis was F = 7.94, p < 0.05, p < 0.05, Tukey's test).Injections of U50,488 significantly increased responding on the previously ethanol-associated lever  = 5.38, p < 0.05) and naltrexone dose  = 39.21, p < 0.0001) on responses on the previously ethanol-associated lever, as well as a significant interaction between U50,488 and naltrexone on ethanol-associated lever responding  = 7.56, p < 0.01, p < 0.05, Sidak's test for multiple comparisons). There were no effects on responding on the inactive lever  = 7.75, p < 0.01) and nor-BNI dose  = 13.73, p < 0.01) on ethanol-associated lever responding. Although not statistically significant, there was a trend towards an interaction between nor-BNI and U50,488 on lever responses for the previously ethanol-associated lever  = 2.44, p = 0.10, p < 0.05, Sidak's test for multiple comparisons). Inactive lever responses were not affected by any treatment  receptor antagonist CP 154,526 and the α2 adrenoceptor agonist clonidine significantly attenuate KOR agonist priming-induced reinstatement of drug seeking [1 receptor antagonist antalarmin, and nor-BNI reduces reinstatement via priming injection of yohimbine, an α2 adrenoceptor antagonist [One hypothesis is that the KOR/DYN system may work in conjunction with other neurotransmitter systems to regulate relapse. For example, type 1 corticotropin-releasing factor (CRFtagonist . DYN andtagonist –27 and stagonist  and humatagonist , which itagonist . With retagonist , increastagonist , and stitagonist . Taken tAlthough U50,488 led to a significant increase in responding on the previously ethanol-associated lever, response rates during reinstatement testing were less pronounced when compared to those observed during ethanol self-administration training. However, this lower rate of responding is consistent with previous work examining KOR agonist-induced reinstatement of drug seeking , in ratsThe current study demonstrated that the KOR agonist, U50,488, increased responding on the previously ethanol-associated lever following extinction, an effect attenuated by nor-BNI at time points consistent with KOR selectivity. These results suggest that KORs are a key mechanism in the reinstatement of ethanol seeking. However, this effect occurred to a lesser degree when compared to naltrexone, indicating that U50,488 may also interact with other neurochemical systems to reinstate ethanol-seeking behavior. When considered with previous findings demonstrating that KOR agonists interact with stress-related systems to reinstate drug seeking [


SP110 gene and TNF-α gene among pulmonary TB (PTB) and spinal TB (STB) patients.  To investigate the association of single-nucleotide polymorphisms (SNPs) in SP110 SNPs (rs722555 and rs1135791) and the promoter -308G>A (rs1800629) and -238G>A (rs361525) polymorphisms in TNF-α were genotyped. Results. TNF-α -238G>A polymorphism was involved in susceptibility to STB, but not to PTB. The TNF-α -238 A allele was a protective factor against STB . Furthermore, the presence of the -238 A allele was considered a trend to decrease the risk of STB . However, SP110 SNPs (rs722555 and rs1135791) and TNF-α -308G>A (rs1800629) showed no association with PTB and STB in all genetic models.  In a total of 190 PTB patients, 183 STB patients were enrolled as the case group and 362 healthy individuals at the same geographical region as the control group. The TNF-α -238 A allele appeared a protective effect against STB, whereas the SP110 SNPs (rs722555 and rs1135791) and TNF-α -308G>A (rs1800629) showed no association with susceptibility to PTB and STB patients in southern China. The  Mycobacterium tuberculosis (MTB) infection, remains one of the leading causes of death in the world. The World Health Organization estimated that the TB incidence was 10.4 million, and there were approximately 1.7 million TB-related deaths that occurred among HIV-negative people (1.3 million) and HIV-positive individuals (374000) in 2016  = 0.61 [0.45–0.82]; CC versus TT: OR [95% CI] = 0.84 [0.36–2.00]) and the 1135791 C allele was protective against TB . Moreover, Cai et al.  = 0.0532 [0.57–0.89]), and further haplotype analysis showed that haplotypes of CGACCG  and TGATTG  in SP110 rs1135791-rs3948464-rs1365776-rs9061-rs11556887-rs11679983 were related to TB risk. On the contrary, Cong et al.  = 1.9795 [1.1141–3.5169]) and rs722555 G allele  increased the risk of developing PTB in Chinese Han population. However, distinct races or populations might have different susceptible genetic factors. Furthermore, a wide-range geographical meta-analysis also suggested a negative association between SP110 polymorphisms and TB risk among African, European descendant, and Asian mixed populations  = 0.405 [0.147–0.657]). While GA genotype and A allele were risk factors of OA-TB in a Hebei population , this was against the view of Merza et al. ) .TNF-α -308G>A polymorphism (rs1800629) which was reported to be associated with the regulation of TNF-α levels [TNF-α SNP was -238G>A (rs361525) which was TB-associated [TNF-α -238G>A was associated with STB, but not with PTB. The -238 A allele had shown a protective effect on STB  after adjusting for age and gender. Furthermore, we observed a marginal statistically different distributions of AG genotype  and the dominant model  and TNF-α -308G>A (rs1800629) show no association with susceptibility to PTB and STB in southern China. Our study may serve to assess the susceptible genetic factors and the possible outcomes of TB infection.In conclusion, our study provides further evidence supporting the host genetic variability in TB susceptibility, especially in different types of TB infection. The


Mycobacterium tuberculosis and Listeria monocytogenes, among others. Our goal was to model infection and arthritis in mice and to compare etanercept, a currently used anti-TNF-α inhibitor, to an anti-TNF-α vaccine. We developed a murine surrogate of the TNF-α kinoid and produced an anti-murine TNF-α vaccine (TNFKi) composed of keyhole limpet hemocyanin conjugated to TNF-α, which resulted in anti-TNF-α antibody production in mice. We also used etanercept (a soluble receptor of TNF commonly used to treat RA) as a control of TNF neutralization. In a mouse model of collagen-induced arthritis, TNFKi protected against inflammation similar to etanercept. In a mouse model of acute L. monocytogenes infection, all TNFKi-treated mice showed cleared bacterial infection and survived, whereas etanercept-treated mice showed large liver granulomas and quickly died. Moreover, TNFKi mice infected with the virulent H37Rv M. tuberculosis showed resistance to infection, in contrast with etanercept-treated mice or controls. Depending on the TNF-α blockade strategy, treating arthritis with a TNF-α inhibitor could result in a different profile of infection suceptibility. Our TNFKi vaccine allowed for a better remaining host defense than did etanercept.Tumor necrosis factor-alpha (TNF-α) blockade is an effective treatment for rheumatoid arthritis (RA) and other inflammatory diseases, but in patients, it is associated with reduced resistance to the infectious agents  Tumor necrosis factor-α (TNF-α) is a central mediator of inflammation, tumor growth control, autoimmunity, and immune response to infection . In rheuTwo types of TNF-α antagonists are used for treating rheumatic diseases, including RA and spondyloarthritides: the soluble TNF-α receptor 2 etanercept and monoclonal antibodies such as infliximab, adalimumab, golimumab, or pegylated-IgG-Fc fragments such as certolizumab. Anti-TNF-α drugs have greatly changed the treatment of RA in terms of clinical control and articular damage prevention. However, these treatments have limitations, such as high cost, frequent therapeutic failures (only 20% of RA patients achieve sustained remission) , and incRecently, we developed an alternative strategy of TNF neutralization by a vaccination approach. A human TNF-α vaccine (hTNF-K) was obtained by chemically coupling the human cytokine to the carrier protein keyhole limpet hemocyanin (KLH) . PreclinIn previous studies, we demonstrated that anti-human TNF-α vaccination activates resting autoreactive B cells to produce anti-TNF-α antibodies. The carrier protein (KLH) possesses the T epitopes able to activate T cells, thereby providing the necessary help for anti-TNF-α antibody production. No T cell being directly sensitized to TNF-α has two major consequences. First the antibody production is self-limited, with serum antibody titers showing a bell-shaped curve, with peak concentration around day 50 in mice and between day 112 and 140 in humans. Second, any increase in self-produced TNF-α  does not elicit any anti-TNF-α antibody response in immunized subjects. However, the risk of infection with the hTNF-K treatment could not be easily studied in the TTG mice because antihuman TNF-α antibodies are usually species-specific and do not cross-react with murine TNF-α.Listeria monocytogenes and Mycobacterium tuberculosis infection, as reflected by a differential local immune response to the pathogens. Hence, depending on the anti-TNF-α inhibition strategy, TNF-α inhibition could control chronic inflammation without modifying the pathogen-induced immune response and, therefore, have no deleterious effect.To evaluate the risk of infection using anti-TNF-α strategies, we compared a vaccine directed against murine TNF-α we developed by coupling murine TNF-α to KLH (TNFKi) and etanercept, the anti-TNF-α treatment associated with the lowest occurrence of infections, namely iatrogenic tuberculosis , 13. OurN-succinimidyl S-acetylthioacetate (26102 Pierce)] were from Thermo Scientific Pierce, France. TNFKi resulted from the chemical coupling of murine TNF-α to the carrier protein KLH, by using heterobifunctional cross-linkers that react with primary amines. TNFKi was prepared as follows: 4 mg murine TNF-α was derivatized with 1.2 mg SATA for 2 h at room temperature. In parallel, 20 mg KLH was derivatized with 1.76 mg GMBS for 30 min. Cross-linker quantities were calculated to modify half of the lysine residues. For coupling, 1 mol KLH-GMBS was incubated with 20 mol TNF-SATA for 2 h at room temperature in the presence of 5 mM hydroxylamide (23225 Thermo Scientific Pierce). The heterocomplex (TNF-KLH) was dialyzed overnight against phosphate-buffered saline (PBS) by use of a Microcon tube (YM100 Millipore). TNFKi was sterilized by centrifugation in an Ultrafree-CL 0.22-µm tube (FC40GV 0S Millipore), then stored at 4°C. Three batches were prepared successively for different experiments. They were validated biochemically by ELISA and western blot analysis. Batches were systematically tested in vivo in the collagen-induced arthritis (CIA) model.Murine TNF-α was from PeproTech . KLH , GMBS , succinimide ester (22309 Pierce), and SATA [L. monocytogenes (LM) infection experiments involved 40 C57Bl/6 female mice  and 10 TNF-α−/− mice (TNF−/−) (M. tuberculosis (Mt) infection experiments involved 60 C57Bl/6 female mice  and 7 TNF−/− mice. Mice were kept in isolators in a biohazard animal unit. Infected mice were monitored every day for clinical status and weighed twice weekly. Mice were randomly assigned to treatment groups and randomly distributed to cages (except for naïve uninfected mice).For CIA, 36 DBA/1 male mice (6 weeks old) were used, and for collagen antibody-induced arthritis (CAIA), 24 C57Bl/6 male mice (6 weeks old) were used, all purchased from Janvier Laboratory (France). Mice were randomly assigned to treatment groups and randomly distributed to cages. Body weight was monitored weekly. (TNF−/−)  backcrosDBA/1 mice used for CIA were injected with a 1:1 emulsion of bovine type II collagen  and complete Freund’s adjuvant  on day 0 . A boost was given on day 21 with 100 µl of a 1:1 emulsion of CIIb (50 μg/mice) and incomplete Freund’s adjuvant . Arthritis onset occurs at about 30–35 days after the first collagen injection .E. coli 0111:B4 strain) on day 3. Arthritis onset occurs at about 3–4 days after anti-collagen antibody injection and peaks at about day 7–10  and lipopolysaccharide  and two joints of the two forelimbs . The maximum score for each of the 10 joints was 4, so the maximum individual arthritis clinical score on a given day was 40 . The meaFor histology, both left hind limbs of each mouse were collected, fixed, decalcified, dehydrated, and transferred to paraffin blocks. Slides of 7 µm thickness were stained with hematoxylin and eosin before optical microscopy observation. At least 10 fields per section were evaluated with blinding to treatment. In each joint, two variables were separately assessed on a 4-point scale . The first variable was inflammation, as reflected by synovial membrane thickness  and inflammatory cell infiltration. Inflammation was scored as 0, no synovial proliferation and no inflammatory cell infiltration; 1, limited or absent synovial proliferation with inflammatory cells infiltrating ≤5% of synovial membrane; 2, synovial proliferation with inflammatory cells infiltrating between 5 and 50% of synovial membrane; and 3, massive synovial proliferation with inflammatory cells infiltrating >50% of synovial membrane. The second variable was joint destruction . Joint destruction was quantified as 0, no bone erosion, smooth cartilage surface with conserved thickness; 1, presence of cartilage erosion or unevenness or thinning involving ≤50% of cartilage surface with absent or single bone erosion; 2, multiple cartilage erosions or cartilage thinning involving >50% of cartilage surface and/or >1 bone erosions involving ≤50% of articular surface; and 3, complete derangement of articular structure. For determining prevalence, histological inflammation or destruction was defined as inflammation or destruction score ≥0.5 .For clinical and histological evaluation of arthritis, blinding was ensured by a random distribution of treatments inside cages, limited by the need to have each treatment group represented, and by the use of tables including only cages and mouse numbers.n = 6) were vaccinated intramuscularly with different doses of TNFKi  on days −21, −7, and 7. In the same experiment, one group of mice (n = 6) were intraperitoneally injected three times/week with 30 mg/kg etanercept  from day 22. Negative controls were mice receiving intraperitoneal injections of PBS or intramuscular injections of KLH (10 µg) emulsified in IFA. KLH and PBS were administered on the same time schedule as TNFKi or etanercept treatment, respectively.Immunization involved the anti-mouse TNF-α vaccine (TNFKi) emulsified in IFA. DBA/1 mice (Listeria monocytogenes (L028) was grown in BHI medium . Aliquots of M. tuberculosis H37Rv kept frozen at −80°C were thawed, diluted in sterile saline containing 0.05% Tween 20, and clumping was disrupted by 30 repeated aspirations through a 26-G needle . Pulmonary infection involved delivering 2250 colony formation units (CFU) of M. tuberculosis H37Rv/lung into nasal cavities of mice under xylazine–ketamine anesthesia, as verified by determining bacterial load in the lungs on day 1 postinfection.n = 10) received intraperitoneal injections of etanercept (30 mg/kg) twice a week from days −4 to day 52 before and during infection. Controls were untreated (naïve) mice, vaccinated with KLH, or received intraperitoneal injections of PBS. All groups comprised 10 mice per group except for TNF−/− mice (n = 7). On day 25 postinfection for all TNF−/− mice or on day 28 for other groups, 5 animals were killed; lung, livers, and spleens were harvested; and the number of viable bacteria in organ homogenates was determined by plating serial dilutions in duplicate onto Middlebrook 7H11 (Difco) agar plates containing 10% OADC and incubating at 37°C. Colonies were counted at 3 weeks and results are expressed as log10 CFU per organ. Similar analyses were performed on day 56 (established infection) with the remaining mice.Mice were vaccinated before infection with four TNFKi intramuscular injections (10 µg) on days −44, −31, −17, and −4. One group of mice  received intraperitoneal injection of etanercept (30 mg/kg) on days −4, −2, 0, and 3 before and after infection. Controls were treated with KLH (n = 9 mice) or PBS (n = 10) on the same time schedule as TNFKi vaccination or etanercept treatment, respectively. All groups and TNF−/− mice (n = 10) underwent intraperitoneal injection on day 0 with 104 CFU L. monocytogenes. On day 4 postinfection, four mice/group were killed, then livers and spleens were harvested and the number of viable bacteria in organ homogenates was determined by plating serial dilutions on trypticase soy broth agar plates  and incubating overnight at 37°C, followed by counting CFU. Survival during infection was evaluated until day 11 (n = 5–6 mice/group of treatment).A similar vaccination schedule was used before Listeria study; 7 days after the third and 32 days after the fourth TNFKi vaccination in the Mycobacterium study. Serum was obtained and tested for anti-KLH and anti-TNF-α antibody content. Specific anti-mTNF-α and anti-KLH antibody content was determined by direct ELISA. Precoated ELISA plates with 50 ng per well mTNF-α (PeproTech) or KLH (Sigma) were incubated with serial dilutions of serum from immunized and control mice. Specific antibodies were detected by using phosphatase alkaline-conjugated rabbit anti-mouse IgG . Substrate PNP (Sigma-Aldrich) was added for 40 min. The optical density was measured at 405 nm.Blood samples were collected at different times in accordance with the duration of experiments: 19 and 41 days after the third TNFKi vaccination in the CIA study; 7 days after the third and fourth TNFKi vaccination in the M. tuberculosis-infected mice collected on days 25–28 and 56 were fixed in 4% buffered formalin and paraffin-embedded, and 4 µm sections were stained with hematoxylin and eosin (H&E) or underwent the Ziehl–Neelsen coloration. Sections were observed by light microscopy at 400× magnification with a Zeiss Observer D1 microscope equipped with a motorized stage connected to Histolab and Archimed software . The number and size of granulomas were quantified by using Archimed.Lungs and livers from 2O2. Endogenous biotin in the liver was blocked by using an avidin-biotin kit . Tissue sections were incubated for 2 h at room temperature or overnight at 4°C with the primary antibody CD45R  or inducible nitric oxide synthase . Sections were then incubated for 30 min at 37°C with the appropriate biotinylated secondary antibody. Avidine biotin peroxidase complexes were added to the sections for 30 min , then incubated with diaminobenzidine substrate . After a rinsing in water, sections were counterstained with hematoxylin before mounting and examined by use of a Zeiss Observer D1 microscope as described previously.For immunohistochemistry, lung and liver sections were fixed in acetone–ethanol (5 min), and endogenous peroxidase activity was blocked with PBS and 3% HListeria infection, four mice per group were euthanized on day 4 postinfection. Livers were treated as described previously; H&E-stained sections were observed by light microscopy and images were obtained by using a nanozoomer. The number and size of microabcesses were assessed by using NDPview . Immunohistology of livers, macrophages , and neutrophils  infiltration was performed as described for CD45R and iNOS staining.For Listeria infection studies, evaluation of survival involved the Kaplan–Meier method (log-rank test), and bacterial burden data were analyzed by one-way ANOVA with Newman–Keuls posttest. p < 0.05 was considered statistically significant.For arthritis studies, serial measurements of clinical variables were analyzed by the area under the receiver operating characteristic curve for each mouse as a summary measure, then as raw data for inter-group comparisons (ANOVA). Differences in arthritis onset were analyzed by ANOVA. Posttest data were compared by data distribution (Student–Newman–Keuls). For All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. For arthritis models, all procedures were approved by the Animal Care and Use Committee of the University of Paris 13 . Infectious protocols were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans (CCO) (no. CLE CCO 2015-1071). We were particularly concerned by a strict application of the 3R rules .in vivo, we used the CIA model with DBA/1 mice. Mice were vaccinated three times with 5, 10, or 20 μg/mice TNFKi . Control groups were vaccinated with KLH or PBS. Etanercept was a positive control. The first signs of arthritis appeared in all control groups at 33 ± 2 days, as expected, but in TNFKi groups at 38 ± 2 days and in the etanercept group at 41 ± 3 days. All anti-TNF treatments resulted in significantly lower clinical arthritis scores as compared with controls  was significantly lower for mice with 10 µg TNFKi or etanercept treatment than other mice . Despite this, the incidence and onset of arthritis were not significantly delayed with TNFKi or etanercept treatment as compared with KLH and PBS controls . The weights of TNFKi-vaccinated mice were comparable to that of PBS and KLH controls during the experiment (data not shown).Histology of paws of PBS and KLH control mice showed significant inflammation, with cell infiltration associated with cartilage destruction Figures B–E. By cThus, TNFKi was effective in protecting against TNF-mediated arthritis in mice.L. monocytogenes. Controls received PBS or KLH, and TNF−/− mice were a genetic control. All mice were injected intraperitoneally with L. monocytogenes (104 CFU) on day 0. Blood was collected before (day −10) and during infection (day 3) to evaluate anti-TNF-α antibody levels. ELISA revealed the production of anti-TNF-α and anti-KLH antibodies in serum from mice with TNFKi vaccination, with an increase in level in responder mice between day −10 and day 3 .Mice were vaccinated on days −44, −31, −17, and −4 with TNFKi or intraperitoneally injected with etanercept on days −4, −2, 0, and 3 before (or after) infection with −/− mice died within 5 days, and 80% of mice with etanercept treatment died by day 6, as expected; by contrast, mice vaccinated with TNFKi did not die due to infection during this period  in TNFe Figure A. Macropt Figure B.M. infection. Mice were vaccinated on days −44, −31, −17, and −4 with TNFKi (10 μg/mice) or treated with etanercept (30 mg/kg twice a week from day −4 to day 52 before or after infection). Control groups were mice with KLH vaccination or PBS treatment. TNF−/− mice, which are very sensitive to virulent M. tuberculosis H37Rv infection, were used as a positive control of total infection in the absence of functional TNF. Except for five naïve mice, all mice were infected on day 0 by intranasal instillation of M. tuberculosis H37Rv . TNF−/− mice lost weight rapidly and had to be euthanized on day 25 postinfection, but the other mice did not experience body weight loss (data not shown) and were euthanized during early infection (day 28) or established infection (day 56).We assessed the consequences of TNFKi vaccination during early and established Blood was collected before (day −10) and during infection (day 28) to evaluate levels of anti-TNF-α antibodies. As expected, ELISA revealed a sustained serum production of anti-TNF-α antibodies in mice vaccinated with TNFKi .−/− mouse organs showed a marked increase in weight as compared with the other groups, with the exception of lung and spleen of etanercept-treated mice    Figure J. Granul) Figure K,L, whic) Figure L.−/− and etanercept-treated mice than other mice .Histology of the liver at the early phase of infection Figures A–F reveae Figure G . At day 56, liver granuloma surface area was larger for etanercept-treated mice than other groups .To confirm mycobacterial infiltration in granulomas, we used Ziehl–Neelsen coloration and semiquantitative evaluation of bacterial staining in mouse lungs Figure . Microsc−/− and etanercept-treated lungs, iNOS expression in macrophages co-localized with microabscesses containing altered neutrophil number. Etanercept-treated lungs showed more macrophage infiltration, higher number of foamy histiocytes expressing iNOS, and fewer neutrophils during established than early infection . Lesions of PBS-treated lungs showed few neutrophils, and iNOS was expressed in macrophages and foamy histiocytes. Lesions of mouse lungs with KLH or TNFKi vaccination showed similar cellular infiltration as for PBS-treated lungs but a lower number of foamy histiocytes. Thus, macrophages within the granuloma of TNFKi-vaccinated lungs retained the bactericidal function controlling infection.We used immunohistochemistry of mouse lung sections to investigate the relationship between cellular infiltration or activation status and TNF-α neutralization. During early infection, iNOS was expressed in granulomas of all mouse groups . Histology revealed lesions in TNFKi, KLH, and PBS lungs characterized by macrophage infiltration and a very low number of neutrophils. Conversely, in TNFM. tuberculosis infection . The TNFKi vaccine used in the present study consisted of mouse TNF-α linked to the KLH carrier protein. TNFKi induced the production of anti-TNF-α polyclonal antibodies and alleviated arthritis in two experimental models of arthritis, CIA and CAIA, which confirms the effective neutralization of TNF-α.A major concern of anti-TNF-α treatments is an increased risk of serious bacterial, viral, and fungal infections. Tuberculosis is the most common opportunistic infection and, to a lesser extent, bacterial infections, such as listeriosis –21. AntiL monocytogenes and M. tuberculosis. In both cases, we compared the effect of TNFKi to that of complete lack of TNF-α (TNF−/− mice) and a current anti-TNF-α treatment (etanercept). Etanercept is considered the safest anti-TNF inhibitor in terms of the possible risk of serious infection (notably tuberculosis reactivation). It is a pharmacological reference for anti-TNF-α blockade in animal models of infection , whereas etanercept-treated mice showed increased bacterial load. In agreement, the size and number of liver microabcesses were similar between TNFKi and control mice at day 4 postinfection. In contrast, TNF−/− mice showed increased number of lesions and large lesions characterized by necrotic areas. Immunization with TNFKi did not alter immune cell migration into the liver during Listeria infection, whereas etanercept-treated and TNF−/− mice exhibited neutrophil accumulation and reduced macrophage recruitment in lesions.To assess to what extent this novel anti-TNF strategy would affect immunity against infectious agents, we used two experimental models of infection, with nfection . With L.M. tuberculosis infection, TNF neutralization by TNFKi vaccination did not increase the sensitivity to the pathogen, but TNF−/− mice died during the early phase of infection. Previously, mice treated with a murine homolog of etanercept died due to M. tuberculosis infection  infection, the antimycobacterial immune response was restored by infection with a TNF-α-secreting recombinant M. bovis BCG, effectively protecting these highly susceptible mice  and infection, these models only partially mimic human disease. Moreover, differences between the human and murine immune system both in response to vaccination and infection may lead to deep discrepancies that warrant caution. No safety issues or increased risk of infection were reported when anti-TNF vaccination was administered in humans . Nevertheless, the samples of those trials were limited and no specific studies on response to infection were performed.M. tuberculosis and L. monocytogenes infection in mice. This is a major point for the safety features of therapeutic TNF vaccination in chronic inflammatory diseases. Pathogenic levels of TNF-α might be neutralized efficiently while maintaining the minimal levels needed to control host resistance to bacterial infection.In summary, arthritis improvement when targeting TNF-α does not result in a constant and uniform alteration of the host defense against infection. This alteration is variable depending on the strategies used for targeting TNF-α. TNFKi may allow for maintenance of the host defense against All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. For arthritis models, all procedures were approved by the Animal Care and Use Committee of the University of Paris 13 . Infectious protocols were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans (CCO) (no. CLE CCO 2015-1071). We were particularly concerned by a strict application of the 3R rules.Listeria infection, and immuno-histological analysis and helped draft the manuscript. LS performed statistical analysis, drafted and revised the manuscript. NS performed Mycobacteria infection, bacterial burden, and organ weight evaluations. DD participated in the immuno-histological analysis, analysis of the data, and helped to draft the manuscript. PD helped coordinate the study and helped draft the manuscript. BR and VQ established and validated the experimental infectious models and conceived a part of the Listeria and Mycobacteria infection study, participated in coordination and analysis of the data, and helped draft the manuscript. M-CB conceived of the study, analyzed the data, and helped to draft the manuscript. EA conceived of the study, performed coupling of TNF vaccine, participated in coordination of the study, analyzed the data, and drafted the manuscript.NB performed mice immunization, evaluated clinical arthritis, performed immunoassay, BR, DD, EA, LS, M-CB, NB, NS, PD, and VQ declare no conflict of interest.


B. abortus infection. We show that infection of macrophage with B. abortus induces marked expression and secretion of TNF which subsequently binds to TNF receptor 1 (TNFR-1) and activates a downstream signaling cascade of the innate immunity. Blocking of TNF signaling resulted in a notable increase of B. abortus survival which was associated with an increase of anti-inflammatory cytokine interleukin 10 (IL-10), a beneficial effector of Brucella survival, as well as remarkable decrease of reactive oxygen species (ROS) and nitric oxide (NO), antibrucella molecules. However, surprisingly, the interference of TNF did not show any influence on phagolysosome and cell death events. Furthermore, the transcriptional factor NF-kB was found to be a main mediator of TNF signaling when blocking of NF-kB pathway drastically suppressed the TNF-induced brucellacidal effect. Taken together, these findings clearly indicate that the immune cascade activated by TNF/TNFR-1 is required for the sufficient resistance to B. abortus survival in macrophages.In this study, we explore the regulatory roles of pro-inflammatory cytokine tumor necrosis factor alpha (TNF) in the innate immunity of macrophages against  Brucella spp. are facultative intracellular Gram-negative bacteria that cause brucellosis in a variety of mammalian hosts including humans with more than 500,000 new cases annually  is an important regulator of host responses to microbial challenges  through stimulating NF-kB inhibitor, IkBα polyubiquitination that is subsequently responsible for controlling inflammation and antimicrobial immune processes   and membrane-bound (mTNF) and two receptors: TNF receptor 1 and 2 (TNFR-1 and TNFR-2). Binding of either form of TNF to its receptors leads to activation of different signaling cascades. TNFR-1 is expressed in majority of cells and can be triggered by either sTNF or mTNF while TNFR-2 is restricted to immune cells and only triggered by mTNF , NF-kB p105/50, p100/52, p65, Lysosomal-associated membrane protein 1 (LAMP-1) and cathepsin H (CTSH) antibodies were obtained from Santa Cruz Biotechnology, TX, USA. TNF recombinant mouse proteins and the other TNF siRNA construct were obtained from Thermo Fisher Scientific, MA, USA while Texas red-goat anti-rat IgG antibody and Lipofectamine RNAiMAX were purchased from Life Technologies, CA, USA. FITC-conjugated goat anti-rabbit IgG antibody, thenoyltrifluoroacetone (TTFA) and diphenylene iodinium chloride (DPI) were obtained from Sigma-Aldrich, MO, USA.TNF siRNA, 2-Amino-5,6-dihydro-6-methyl-4B. abortus 544 biovar 1 strain was kindly provided by Animal and Plant Quarantine Agency in Korea. B. abortus was routinely cultured overnight in Brucella broth  at 37°C. The murine macrophage RAW 264.7 cells were grown at 37°C in 5% CO2 atmosphere in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS) with or without 100 U/ml penicillin and 100 μg/ml streptomycin.A smooth, virulent B. abortus at a multiplicity of infection (MOI) of 100. At 15, 30, and 45 min post-infection, the infected cells were washed once with phosphate buffer saline (PBS) and then incubated in RPMI/10% FBS and gentamicin (30 μg/ml) for 30 min to kill extracellular bacteria. Finally, the cells were washed with PBS, lysed with distilled water and plated on Brucella agar for bacterial uptake determination. To monitor intracellular growth, after 1 h of incubation at 37°C, 5% CO2, the medium of B. abortus-infected macrophages were replaced by RPMI/10% FBS and gentamicin (30 μg/ml). At 2, 24, or 48 h post-infection, the cells were then washed with PBS, lysed with distilled water and plated on Brucella agar.These assays were performed as previously reported with few modifications  at different time points using a Qiagen RNeasy kit . DNA was removed before final elution of the RNA sample using the Qiagen “On-Column DNase Digestion” protocol.The total RNA was isolated from RAW 264.7 cells  and then frozen in a dry ice-acetone bath. Afterwards, they were thawed in ice bath and the cytoplasmic extracts were collected as supernatants after centrifugation at 1200 × g at 4°C for 10 min. The pellet containing the nuclei was resuspended in buffer C , supplemented with 0.5 mM phenylmethylsulfonyl fluoride (PMSF) proteases inhibitor . This mixture was incubated under gentle stirring and then centrifuged at 20,000 × g at 4°C for 20 min. The supernatant was collected as the nuclear extract. Buffer D , supplemented with 0.5 mM PMSF proteases inhibitor was added to the cytoplasmic and nuclear extracts which were stored and maintained at −70°C until needed.The cytoplasmic and nuclear protein extracts were obtained as previously described  with these markers was performed as previously reported  and processed using FV10-ASW Viewer 3.1 software. The percentage of these proteins colocalization was determined from 100 random cells.The colocalization of This assay was performed as previously described .B. abortus-infected RAW 264.7 cells were treated with either TNF siRNAs, rTNF, TTFA, or DI. At 24 h post-infection (pi), ROS content was evaluated by fluorescence microscopy  and spectrometry  according to the manufacturer's instructions.B. abortus for 24 h. Cellular NO content was evaluated by fluorescence microscopy . The nitrite concentration from culture supernatant was also assessed by Griess reaction .RAW 264.7 cells were treated with either TNF siRNA or rTNF and then infected with The apoptosis and necrosis were evaluated by flow cytometry using apoptosis and necrosis detection kit  at 48 h post-infection (pi) in accordance with the manufacturer's instructions. Briefly, after 2 days incubation at 37°C, infected macrophages were stained with apoptosis indicator (apopxin green) and necrosis indicator (7-AAD). The apoptosis and necrosis percentages were determined from 10,000 random cells.SD). ANOVA with Tukey's HSD exact test was used to statistically compare the groups. Results with P < 0.05 were considered significantly different.The data are expressed as the mean ± standard deviation  that target differently on Tnf mRNA sequence. As a prevailing logic, the results were also shown to be of the same trend with the other one Figure , indicatB. abortus replication in TNF-suppressive macrophages and the known influence of inflammatory cytokines on macrophage function  or inducible NO synthase (Nos2) were shown to markedly enhance the persistence of Brucella , respectively. As shown in Figure Brucella clearance  which was followed by staining with Apopxin and 7-ADD indicators and finally evaluated at 48 h pi by flow cytometry. Infection with B. abortus induced ~10 and 30% of apoptosis and necrosis, respectively; however TNF signaling did not contribute to these processes when no difference on cell death rate was obtained between TNF-suppressive and normal cells . We next evaluated the expression of 20 representatives by qRT-PCR at 2 h post-infection and cell death by flow cytometry at 48 h post-infection. As predicted, the expression of representatives and the cell death were not influenced by the presence of additional TNF (data not shown).For further validation of these data, we treated Altogether, our results suggest that phagolysosome fusion event and cell death processes are not downstream effectors of TNF-induced brucellacidal effect in macrophages.kB activation, a key transcriptional factor that drives cells to different subsequent signaling . As expected, the inhibition of NF-kB markedly repressed the induction of Brucella clearance by rTNF  did not only suppress the production of ROS, but also led to increase intracellular Brucella, suggesting that this molecule could be an essential effector of host immunity against B. abortus infection. On the other hand, a number of previous works (Simon et al., B. abortus-infected macrophages, leading to the argument of other unknown mechanisms activated by B. abortus to inhibit cell death inductive function of TNF.To date, there are pieces of evidence that ROS, a byproduct of oxygen metabolism, enables to inhibit the propagation of a variety of intracellular bacteria through incompletely understood mechanisms (Paiva and Bozza, kB activation in controlling antibrucella immunity. The transcriptional activity of NF-kB is tightly regulated at multiple steps in immune signaling pathways in which one mechanism to prevent NF-kB activation is mediated by IkB family proteins that traps NF-kB in the cytoplasm (Yamamoto and Takeda, kB family proteins, IkBα is known as the most powerful regulator for cRel-p50, p65-p50 and p65-p52 heterodimers (Nishikori, B. abortus infection, binding of TNF to TNFR-1 stimulated IkBα phosphorylation that led to the activation of classical NF-kB pathway. In addition, the inhibition of NF-kB significantly decrease the induction of brucellacidal effect by rTNF which paralleled with the decrease of NO production and pro-inflammatory cytokine IL-6 whereas increase of IL-10 production, suggesting an undescribed role of NF-kB in controlling antimicrobial effectors induced by TNF signaling. Although we have not explored the interaction between TNF/TNFR-1-activated NF-kB and ROS production, it is possible that NF-kB could also cooperate with ROS accumulation during B. abortus infection.A key question in this study is how TNF suppresses anti-inflammation and induces production of ROS and NO. In this report, we have demonstrated the direct link between TNF and NF-B. abortus infection by macrophages. We proved that shifting cells to inflammation and enhancing chemically-radical effectors are two main manners of TNF/TNFR-1 signaling. We also demonstrated the essential roles of NF-kB in antibrucella effect of TNF. Therefore, taken together, our results provide new insights into how a proinflammatory cytokine develops protective immunity against Brucella infection in macrophages.In summary, our study uncovers the predominant role of TNF in the development of protective immunity against HH, LA, AR and TH carried out all experiments, contributed to data collection and analysis, and participated in drafting the manuscript. WM, HL, MR and HC participated in the design of the study and contributed to revise the manuscript. SK participated in the design of the study, carried out the data analysis, conceived the experiment and prepared the manuscript. All authors read and approved the final 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.


Over the last two decades, important advances have been made in explaining some pathophysiological aspects of heart failure with preserved ejection fraction (HFpEF) with repercussions for the successful clinical management of the syndrome. Despite these gains, our knowledge for the natural history of clinical progression from the pre-clinical diastolic dysfunction (PDD) until the final clinical stages is significantly limited. The subclinical progression of PDD to the clinical phenotype of HFpEF and the further clinical progression to some more complex clinical models with multi-organ involvement, similar to heart failure with reduced ejection fraction (HFrEF), continue to be poorly understood. Prospective studies are needed to elucidate the natural history of clinical progression in patients with HFpEF and to identify the exact left ventricular remodeling mechanism that underlies this progression. The clinical phenotype of patients with reduced ejection fraction (HFrEF) is extensively studied and understood, but the clinical phenotype and natural history of patients with preserved ejection fraction (HFpEF) remain poorly defined. There is incomplete knowledge and understanding of HFpEF pathophysiology. The relevant clinical trials are suboptimal in their design, and the treatment to a great extent is empiric . The numDuring the last two decades emerged a new nomenclature of the clinical congestive HF syndrome that is based on the ejection fraction. This classification categorizes the patients with HF into those with HFrEF EF < 50%) and those with HFpEF (EF > 50%). In the community, the prevalence of both phenotypes of HF, HFpEF and HFrEF, is equal . HFrEF i0% and thTo a number of clinicians, in view of the fact that the majority of the patients with HFrEF display to some extent diastolic dysfunction, the clinical distinction between the two syndromes is considered unnecessary . AccordiIn exploring the fundamental molecular mechanisms of HFpEF, it is important to define the exact molecular processes responsible for the slowing of myocardial relaxation and for the increase of elastic stiffness in both the myocardium and the arterial wall of the peripheral arteries. In hypertension, the increased left ventricular afterload is a stimulus for ventricular structural remodeling and stiffening. These structural changes are leading to higher systolic left ventricular wall stress and slowing of ventricular relaxation . The norIt is important to differentiate the name ‘diastolic dysfunction’ from the term of ‘diastolic heart failure’ syndrome. The age-dependent diastolic dysfunction does not necessarily indicate people with an increased risk of diastolic HF . The preThe rising levels of biomarkers, brain natriuretic peptide (BNP) and the N-terminal pro-B-type natriuretic peptide (NT-proBNP), reinforce the clinical diagnosis of HFpEF. Their high negative predictive value excludes the diagnosis in some patients with suspected HF . HoweverIn general, very few original papers are addressed to the natural history of PDD and to the clinical progression from PDD to overt symptomatic HF . FurtherSome publications provided useful epidemiological facts to the preclinical prevalence of diastolic dysfunction in the general population ,13. The In OCHFS (Olmsted County Heart Function Study), for a period of four years, changes were recorded in diastolic function in randomly-selected individuals, 45 years or older . The triIn a cohort of PDD subjects, progression to clinical HF according to Framingham criteria was expected to be low with a two-year cumulative probability of 1.9% . MoreoveThe development of left ventricular diastolic dysfunction is linked to left ventricular hypertrophy (LVH). Yet, different studies in animals and humans indicate that the diastolic dysfunction starts before the appearance of LVH. Dupont et al.  demonstrIn a population-based study, 388 patients, mean age 67 ± years, with a prevalence of renal insufficiency (glomerular filtration rate <60 mL/min) of 34% and with a Grade 2–4 diastolic dysfunction in echocardiography, were followed-up over three years . The objDiabetic cardiomyopathy is linked to the presence of myocardial hypertrophy and stiffness that induce diastolic dysfunction. In diabetic patients, poor glycemic control is related to an increased risk of HF, but it remains unclear whether treating hyperglycemia will reduce progression or reverse the presence of HFpEF syndrome . From etRen et al.  evaluateIn the I-PRESERVE (Irbesartan in Heart Failure with Preserved Ejection Fraction Study) study, the mode of death in patients with HFpEF and the role of irbesartan to alter this possibility were examined . It was The present therapy of HFpEF has not been effective in reducing morbidity and mortality. Angiotensin-converting enzyme inhibitors (ACEI), angiotensin-II receptor antagonists (ARA), diuretics and beta blockers were proven ineffective to decrease the composite product of hospital readmission and death in patients with HFpEF ,26,27,28The HOPE  study revealed that in patients having high cardiovascular risk, particularly patients with borderline hypertension, the risk of developing HF was reduced after therapy with ACEIs . In the A new HFpEF paradigm was presented recently with the emphasis moved from the left ventricular afterload increase to coronary microvascular inflammation. Paulus and Tschope , suggestThe previously-recorded studies were limited in focus and clinical follow-up of patients with PDD and HFpEF. The subclinical progression of the PDD to the clinical entity of HFpEF and the further clinical advance to more complex clinical models of HF, analogous to HFrEF, continue to be poorly understood. Few large animal models that imitate the human HFpEF syndrome are described. Conceicao et al. in a recent article are suggesting that “although most of the heart failure animal models currently available represent heart failure with reduced ejection fraction, several HFpEF animal models have been proposed. However, few of these fulfill all the features present in human disease” . The relHFrEF syndrome has the characteristics of an entity with a progressive clinical deterioration with a relentless course toward end-stage myocardial failure with periods of clinical stabilization and periods of clinical instability . At presThus, in answer to the above questions, the availability of a more suitable model paradigm of clinical HFpEF progression is required. Furthermore, well-designed future longitudinal clinical trials are needed in order to validate specific subgroups of HFpEF phenotypes from the PDD phase up to the terminal stages. This approach will address better the complexity of HFpEF syndrome and will speed the development of novel diagnostic and therapeutic procedures.There is limited information of the natural history and clinical progression of PDD and HFpEF syndromes. The HFpEF syndrome is a term that involves many distinct mechanisms of disease. Thus, diastolic function abnormalities may be driven more by calcium handling in one patient and tissue fibrosis in another and ventricular-aortic coupling in another. This has further complicated our ability to define the natural history and clinical progression of this heart failure syndrome. The insufficient clinical and epidemiological data prohibit the comparison of their natural history with this of HFrEF syndrome. It is also speculated that the HFpEF patients have a different LV remodeling mechanism than that of HFrEF patients. As a consequence of that, they have developed different clinical progression rates. Prospective studies are needed to describe the progressive nature of PDD and HFpEF syndromes. We need to be better at disease phenotyping in HFpEF in order to make progress in this area. LVEF alone will not be adequate.


A considerable body of experimental evidence suggests that tumor necrosis factor (TNF)-α plays a major role in several aspects of inflammation and shock. In particular, it is pivotal in many detrimental effects of acute pancreatitis, and it represents a major determinant of the systemic progression and end-organ damage (such as acute lung injury and liver failure) of this pathologic condition. Given the importance of TNF-α in the pathogenesis of acute pancreatitis, investigators have regarded blocking the action of this mediator as an attractive treatment option. Different specific and nonspecific inhibitors have been developed with promising results in animal models, but, on the other hand, no clinical trials have been designed so far. Difficulties in clinical applications may be multifactorial; experimental models are not fully reliable and reproduce at least some aspects of human disease, timing of intervention should be related to changes in TNF-α serum levels, and inclusion criteria should be accurately selected to better define the population most likely to benefit.